Registration Dossier
Registration Dossier
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EC number: 603-520-1 | CAS number: 131807-57-3
Endpoint summary
Administrative data
Description of key information
90-Day rat feeding study NOAEL: 50 ppm (3.34 mg/kg bw) for males and 50 ppm (4.24 mg/kg bw) in females; OECD 408, OPP-82-1, JMAFF 59-NohSan-4200; Reliability = 1
90-Day mouse feeding study NOAEL: 350 ppm (equivalent to 62.4 and 79.7 mg/kg bw in males and females respectively); OECD 408, OPP-82-1, JMAFF 59-NohSan-4200; Reliability = 1
90-Day dog feeding study LOAEL (females): 40 ppm (1.4 mg/kg bw), NOAEL (males): 40 ppm (1.3 mg/kg bw); OECD 409, OPP 82-1, JMAFF 59-NohSan-4200; Reliability = 1
1-Year dog feeding study NOAEL: 40 ppm (equivalent to 1.2 mg/kg/day in both male and females); OECD 452, OPP 83-1, JMAFF 59-NohSan-4200; Reliability = 1
1-Year monkey gavage study NOAEL 100 mg/kg/day, not a guideline study, Reliability = 2
28-Day rat inhalation study NOAEC: 15 mg/m³; EPA OPPTS 870.3465; Reliability = 1
28-Day rat dermal study NOAEL: 250 mg/kg/day for males and females; OECD 410, EU Method B.9, OPP 82-2, JMAFF 59-NohSan-4200; Reliability = 1
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- The study was conducted to assess the toxicity of the test material when administered by oral gavage to four groups of cynomolgus monkeys for at least 52 weeks at 0, 1，100, or 1000 mg/kg bw.
- GLP compliance:
- yes
- Specific details on test material used for the study:
- Substance ID: DPX-JE874-221 Technical
Lot #: DPX-JE874-221
Purity: 97.4% - Species:
- monkey
- Strain:
- other: cynomolgus
- Details on species / strain selection:
- The monkey is frequently used in safety evaluation studies as a representative of a nonrodent species
- Sex:
- male/female
- Route of administration:
- oral: gavage
- Details on route of administration:
- Gavage was used because a possible route of exposure in humans is oral.
- Vehicle:
- other: 0.5% Tween® 80 in reverse osmosis (RO) water
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Homogeneity of the test material in the original mixture was assessed before initiation of treatment by taking duplicate samples (5.0 mL) from the top, middle, and bottom of the low- and high-dose preparations. Duplicate samples (5.0 mL) from a daily aliquot of each dose preparation were collected each week for the first 4 weeks and once every 4 weeks thereafter.
Mean values of the homogeneity analyses ranged from 93.2% to 99.3% and 86.1% to 100% of the theoretical concentrations of 0.1 and 100 mg/mL, respectively. These results indicate that the mixing procedure produced a homogeneous distribution in the dose preparations.
The mean concentrations of the dose confirmation analyses for all levels were between 91.8% and 108% of the theoretical concentrations, except for Week 40. It was noted that the mean values for Week 40 for the low- and high-dose preparations were 82.5% and 86.3% of the respective theoretical concentrations. The results of analysis of the retention samples from these preparations were 90.2% and 98.2% of the respective theoretical concentrations. These data indicate that the levels of DPX-JE874-221 were acceptable for all dose preparations. - Duration of treatment / exposure:
- 52 weeks
- Frequency of treatment:
- Once daily
- Dose / conc.:
- 1 mg/kg bw/day (nominal)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 4
- Control animals:
- yes, plain diet
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Some animals treated at 1000 mg/kg/day had intermittent occurrences of feces that were white in color, and except for males treated at 100 mg/kg/day, all animals vomited sometime during the study.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Except for one male given 1 mg/kg/day and one female given 1000 mg/kg/day, all animals survived to the scheduled sacrifice. The male given 1 mg/kg/day (Animal No. 103643) was sacrificed due to poor health on Day 14. Observations for this animal included hypoactivity, low or no food consumption, low or no fecal output, and labored respiration. The female given 1000 mg/kg/day (Animal No. 103669) died during examination by a laboratory animal veterinarian on Day 77. Before death, the animal had labored breathing and cloudy red nasal discharge and was hypoactive and recumbent in the cage. Clinical, macroscopic, and microscopic findings for these animals indicated the cause of death was not test material-related.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no consistent changes in body weight at any level tested. Covariate-adjusted mean body weights were significantly increased at Week 2 for males treated with 1, 100, or 1000 mg/kg/day and significantly decreased for females treated with 1 mg/kg/day at Week 16; no biological significance was attributed to these statistical findings.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Administration of the test substance was associated with mildly lower red blood cell count, hemoglobin, and hematocrit for males and females given 1000 mg/kg/day.
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test material-related gross abnormalities noted at necropsy. A few macroscopic lesions, such as pulmonary adhesions, were observed. These lesions were considered to be either spontaneously occurring or common to cynomolgus monkeys.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- The test material produced microscopic changes which correlated with the mild hemolytic anemia reported at the high dose (1000 mg/kg/day). Mild sinus dilatation of the spleen as well as minimal to mild increases in blood breakdown pigments in the spleen, liver, and kidney were observed in both sexes at this dose. The microscopic hemolytic effects, while secondary and non-adverse in themselves, were indicative of an adverse, albeit mild, hemolysis observed in both sexes at 1000 mg/kg/day.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- haematology
- histopathology: non-neoplastic
- Conclusions:
- 52 Week Monkey NOAEL: 100 mg/kg/day
- Executive summary:
The purpose of the study was to assess the toxicity of the test material when administered by oral gavage to cynomolgus monkeys for at least 52 weeks.
Male and female cynomolgus monkeys were assigned to four groups (four/sex/group). Each group received dose preparations by oral gavage containing the vehicle (0,5% Tween® 80 in reverse osmosis water) or 1，100, or 1000 mg/kg of body weight/day (mg/kg/day) at a dose volume of 10 ml/kg as a single daily dose for at least 52 weeks.
The animals were observed twice daily (a.m and p.m.) for mortality and moribundity. At least once daily, animals were examined for abnormalities and signs of toxicity. Body weights were recorded weekly before initiation of treatment, on the first day of treatment, weekly for Weeks 2 through 16, and once every 2 weeks thereafter. Food consumption was confirmed daily by visual inspection. Ophthalmic examinations were done before initiation of treatment and during Weeks 5, 13, 26, 39, and 52. Blood and urine samples were collected for hematology, clinical chemistry, and urinalysis tests twice before initiation of treatment and during Weeks 5,13, 26, 39, and 53. Blood samples were also collected from the animal sacrificed at an unscheduled interval. Samples for plasma and red blood cell levels of the test substance were collected before initiation of treatment, and on Days 1, 8,15, and 22. After at least 52 weeks of treatment, the animals were anesthetized, weighed, exsanguinated, and necropsied. At necropsy, macroscopic observations were recorded, selected organs were weighed, and selected tissues were collected and preserved. Eyes were maintained in 10% phosphate-buffered formalin and were transferred to the Sponsor's designee (Comparative Ophthalmic Research Laboratories) for evaluation. Remaining tissues (as appropriate) were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and the slides were sent to Haskell Laboratory for microscopic examination.
Except for one male given 1 mg/kg/day and one female given 1000 mg/kg/day, all animals survived to the scheduled sacrifice. The male given 1 mg/kg/day (Animal No. 103643) was sacrificed due to poor health on Day 14. Observations for this animal included hypoactivity, low or no food consumption, low or no fecal output, and labored respiration. The female given 1000 mg/kg/day (Animal No. 103669) died during examination by a laboratory animal veterinarian on Day 77. Before death, the animal had labored breathing and cloudy red nasal discharge and was hypoactive and recumbent in the cage. Clinical, macroscopic, and microscopic findings for these animals indicated the cause of death was not test material-related.
Some animals treated at 1000 mg/kg/day had intermittent occurrences of feces that were white in color, and except for males treated at 100 mg/kg/day, all animals vomited sometime during the study. No consistent changes in body weight were found, and there were no other apparent test material-related effects on antemortem observations or food consumption. Additionally, no test material-related ophthalmic changes or changes in absolute organ weights, organ-to-body weight percentages, and organ-to-brain weight ratios were found.
Daily oral gavage administration of the test substance was associated with mildly lower red blood cell count, hemoglobin, and hematocrit with secondary microscopic changes in the spleen, liver, and kidney for males and females given 1000 mg/kg/day. No other apparent test material-related adverse findings were noted in antemortem observations; body weights; food consumption; ophthalmic examinations; or hematology, clinical chemistry, or urinalysis tests. Based on the hematological changes in males and females given 1000 mg/kg/day, the no-observed-effect level for the test substance is considered to be 100 mg/kg/day when administered daily to cynomolgus monkeys for up to 52 weeks.
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 452 (Chronic Toxicity Studies)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 83-1 (Chronic Toxicity)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: MAFF Japan NohSan No. 4200
- Deviations:
- no
- GLP compliance:
- yes
- Specific details on test material used for the study:
- Substance ID: DPX-JE874-221 Technical
Lot #: DPX-JE874-221
Purity: 97.4% - Species:
- dog
- Strain:
- Beagle
- Details on species / strain selection:
- The animal model, the outbred beagle, is recognized as appropriate for chronic studies and is a widely used strain for which significant historical control data are available.
- Sex:
- male/female
- Route of administration:
- oral: feed
- Details on route of administration:
- The oral route of administration was selected because it is the possible route of human exposure.
- Vehicle:
- other: Feed
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Prior to the initiation of dosing, three collections from each of the control, 10, 20, 40, 300 and 300 ppm recovery group preparations (one from the top, middle and bottom of each) were taken and used for the determination of homogeneity. Triplicate samples from the middle portion of each of these preparations were taken prior to the initiation of dosing and analyzed for stability (7- and 14-day stability under laboratory conditions and 14-day stability under refrigeration).
The test substance was blended homogeneously with canine meal, and the test substance/diet admixtures were stable when fresh frozen, stored at room temperature for 7 or 14 days, or stored refrigerated for 14 days. The diet preparations were considered to contain the amount of test substance specified in the protocol.
Based on analysis of separate purity samples obtained near the beginning and near the end of the study, neat test substance was found to be stable under the protocol-specified storage conditions during the course of the study. - Duration of treatment / exposure:
- 52 weeks
- Frequency of treatment:
- Approximately one hour per day
- Dose / conc.:
- 10 ppm
- Remarks:
- Equivalent to 0.3 mg/kg/day in males and 0.3 mg/kg/day in females
- Dose / conc.:
- 20 ppm
- Remarks:
- Equivalent to 0.6 mg/kg/day in males and 0.6 mg/kg/day in females
- Dose / conc.:
- 40 ppm
- Remarks:
- Equivalent to 1.2 mg/kg/day in males and 1.2 mg/kg/day in females
- Dose / conc.:
- 300 ppm
- Remarks:
- Equivalent to 8.8 mg/kg/day in males and 9.3 mg/kg/day in females
- Dose / conc.:
- 300 ppm
- Remarks:
- Equivalent to 10.1 mg/kg/day in males and 9.9 mg/kg/day in females
This recovery group received test substance for first 3 months only - No. of animals per sex per dose:
- 4
- Control animals:
- yes, plain diet
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test substance-related clinical signs were observed. Clinical signs observed in the treated groups occurred at a similar frequency in the control group (such as soft stool, mucoid feces, emesis, etc.), were findings common in laboratory dogs (such as lacrimation, enlarged nictitating membrane, etc.) or occurred at a low incidence, generally in single animals (such as injected sclera, closed cyst on rear paw, hindpaw swollen, etc.).
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No adverse test substance-related effects were apparent in body weights. However, some differences were apparent when compared to the control group. Sporadic increases and decreases (statistically significant at p < 0.05 or p < 0.01) in mean body weight gains were observed in all treated groups throughout the study. However, no trends were apparent to suggest a test substance-related effect.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- No adverse effects on food consumption.
The average amounts of test article consumed in the 10, 20, 40, and 300 ppm groups and 300 ppm recovery group were 0.3, 0.6, 1.2, 8.8-9.3 and 9.9-10.1 mg/kg/day in both males and females. - Food efficiency:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Since no effects were observed in mean food consumption values, food efficiency values in the treated groups were influenced by body weight change values. All groups had negative food efficiency values for those weeks in which body weight losses occurred and, in general, low food efficiency values for those weeks in which reduced body weight gains occurred.
- Ophthalmological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Clinical ophthalmoscopic examination revealed posterior subcapsular lens
opacities in male and female dogs in the 300 ppm and the 300 ppm-recovery groups. The extent and progression of the posterior subcapsular lesions were
variable. In no animal did the entire lens become opaque and no animal became clinically blind during the course of the study. Regression of the lesion did not occur in any dog exposed to the test substance for the entire year; however, partial, and in a few instances complete, regression was noted in some of the lesions in dogs in the 300 ppm-recovery group.
Equatorial lens opacities, occasionally extending into the cortical regions of the lens, were also observed in male and female dogs in the 300 ppm group. This lesion was not observed in any dog in the 300 ppm-recovery group. Equatorial lesions developed later than the posterior subcapsular lesions and development of this lesion was not dependent on the previous development of a posterior subcapsular lesion. - Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test substance-related changes were observed in haematology parameters. When compared to the control group, some statistically significant differences noted. However, these differences were slight, were not consistent between sexes or evaluations, were not observed in a dose-related manner, were similar to pretest values, and/or were within the historical control ranges.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test substance-related changes were observed in serum chemistry parameters. When compared to the control group, some statistically significant differences noted. However, these differences were slight, were not consistent between sexes or evaluations, were not observed in a dose-related manner, were similar to pretest values, and/or were within the historical control ranges.
- Urinalysis findings:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No adverse test substance-related changes were observed in organ weight data. When compared to the control group, some statistically significant differences noted. However, these differences were not consistent between sexes or evaluations, were not observed in a dose-related manner.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test substance-related gross lesions were observed at the scheduled necropsy. Observations in the treated groups were present at a similar incidence in the control group, were limited to single animals in various dose groups or were those findings commonly observed in laboratory beagle dogs.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test substance-related microscopic findings were limited to degenerative changes in the lens of the 300 ppm group males and females, including those in the 300 ppm-recovery group. These lesions were characterized by fiber swelling with formation of Morgagnian globules and clefts within the lens cortex. Lesions were most common in the posterior subcapsular cortex with variable extensions into the deeper cortex. Changes in equatorial fibers, which in some cases were associated with anterior extensions, were less common than the posterior cortical changes and were not observed in dogs receiving the 300 ppm diet for three months followed by the basal diet. Microscopic findings in the lenses of the 300 ppm group dogs were highly correlated, both in incidence and lesion location, to findings noted on the clinical ophthalmoscopic examination. Such correlations were also present in the 300 ppm-recovery group, although this correlation was less consistent in male dogs. The histomorphology of the lenticular changes diagnosed as "lenticular degeneration" was similar in all affected animals and represented part of the typical spectrum of morphologic responses of lens fibers to injury.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 40 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- ophthalmological examination
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 300 ppm
- System:
- eye
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- 1 Year Dog NOAEL: 40 ppm (equivalent to 1.2 mg/kg/day in both male and females)
- Executive summary:
The toxicologic potential and recovery from the test substance were evaluated in this 52-week dietary chronic toxicity study in outbred beagles according to the guidelines OECD 452 and EPA 83-1. Concentrations of 10, 20, 40 and 300 ppm (parts per million) were selected for the study. The test substance was administered in the diet for 52 weeks. A concurrent control group received the basal diet on a comparable regimen. To evaluate potential recovery from test substance-related effects, an additional group was administered the test substance at a concentration of 300 ppm for 13 weeks. Thereafter, these animals received the basal diet. Each study group consisted of four males and four females. All animals were observed twice daily for mortality and signs of overt toxicity. Individual body weights were recorded weekly. Food consumption was recorded daily and reported weekly. Clinical pathology evaluations were conducted once prior to the initiation of dosing and during the 13th, 26th and 52nd weeks of dosing (study weeks 12, 25 and 51, respectively). Ocular examinations were conducted prior to the initiation of dosing and during study weeks 2, 8, 12, 16, 20, 25, 40 and 50. Neurological examinations were conducted prior to the initiation of dosing and during study week 51. A complete necropsy performed on all dogs euthanized at study termination. Selected organs were weighed at the scheduled necropsy. Tissues were examined microscopically from all dogs.
Test substance administration had no adverse effect on survival, the clinical condition of the animals, mean body weights, food consumption, clinical pathology parameters (hematology, serum chemistry and urinalysis), neurological evaluations, organ weights or macroscopic examination of selected tissues.
Clinical ophthalmoscopic examination revealed posterior subcapsular lens opacities in male and female dogs in the 300 ppm and the 300 ppm-recovery groups. The extent and progression of the posterior subcapsular lesions were variable. In no animal did the entire lens become opaque and no animal became clinically blind during the course of the study. Regression of the lesion did not occur in any dog exposed to the test substance for the entire year; however, partial, and in a few instances complete, regression was noted in some of the lesions in dogs in the 300 ppm-recovery group.
Equatorial lens opacities, occasionally extending into the cortical regions of the lens, were also observed in male and female dogs in the 300 ppm group. This lesion was not observed in any dog in the 300 ppm-recovery group. Equatorial lesions developed later than the posterior subcapsular lesions and development of this lesion was not dependent on the previous development of a posterior subcapsular lesion.
Test substance-related microscopic findings were limited to degenerative changes in the lens of the 300 ppm group males and females, including those in the 300 ppm-recovery group. These lesions were characterized by fiber swelling with formation of Morgagnian globules and clefts within the lens cortex. Lesions were most common in the posterior subcapsular cortex with variable extensions into the deeper cortex. Changes in equatorial fibers, which in some cases were associated with anterior extensions, were less common than the posterior cortical changes and were not observed in dogs receiving the 300 ppm diet for three months followed by the basal diet. Microscopic findings in the lenses of the 300 ppm group dogs were highly correlated, both in incidence and lesion location, to findings noted on the clinical ophthalmoscopic examination. Such correlations were also present in the 300 ppm-recovery group, although this correlation was less consistent in male dogs. The histomorphology of the lenticular changes diagnosed as "lenticular degeneration" was similar in all affected animals and represented part of the typical spectrum of morphologic responses of lens fibers to injury.
In conclusion, dietary exposure of the test substance to dogs produced ocular lesions at a concentration of 300 ppm; the ocular lesions were observed clinically as equatorial and posterior subcapsular lens opacities and microscopically as lenticular degeneration. Partial regression of the visible lesions was seen in some dogs when exposure to the test substance was ended. Statistically significant changes were observed in a few hematology and organ weight parameters, but were not considered to be adverse test substance-related effects as they were not observed at each time point, were not consistently seen in both sexes, were within historical control ranges and/or were not associated with any correlative microscopic changes. The no-observed-effect-level (NOEL) is equivalent to the NOEL as defined by the United States Environmental Protection Agency (1985) and the no-observed-adverse-effect-Ievel (NOAEL) as defined by the European Union (1994). Under the conditions of this study, the NOEL was 40 ppm (equivalent to 1.2 mg/kg/day) for both male and female dogs, based on ocular lesions observed at 300 ppm.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-1 (90-Day Oral Toxicity)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: MAFF Japan NohSan No. 4200
- Deviations:
- no
- GLP compliance:
- yes
- Specific details on test material used for the study:
- Substance ID: DPX-JE874-65 Technical
Lot #: DPX-JE874-65
Purity: 97.4% - Species:
- mouse
- Strain:
- other: Crl:CD-1®BR
- Details on species / strain selection:
- The Crl:CD-1®BR rat was selected on the bases of extensive experience with this strain and its suitability with respect to hardiness, longevity, sensitivity, and low incidence of spontaneous diseases.
- Sex:
- male/female
- Route of administration:
- oral: feed
- Details on route of administration:
- The oral route of administration was selected because it is the potential route of human exposure.
- Vehicle:
- other: Feed
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Samples were collected from all diets prepared on test day -6 to verify concentration, homogeneity, and stability of the test substance in diets. Samples used to verify concentration and homogeneity were collected from the top, middle, and bottom of the diet in the diet mixer and frozen until analyzed.
Samples used to confirm concentration and stability were collected from feeders stored at room temperature for 7 and 14 days. Samples collected from the diet mixer were refrigerated for 14 days and were used to assess stability. After the designated storage period, stability samples were frozen until analyzed.
Two samples were collected from separate feeders at each dietary level to verify concentration of the test substance in the feeders on test days 55 and 83. Feeders were stored at room temperature for 7 days; samples were collected and frozen until analyzed.
The test substance was judged to be stable during the course of the study. Diets prepared on test day -6 contained a homogeneous distribution of the test substance and were stable when fresh frozen, stored refrigerated for 14 days, or stored at room temperature for 7 or 14 days. Test substance was not present in control diet.
Samples from diets prepared on test days 55 and 83 were taken from feeders. Averaged results of original and backup analyses of the 35 ppm cage-site feeder samples were reported due to lower than expected results from the initial analysis, resulting in percent recoveries of 76.6% of nominal (test day 55 samples) and 80.0% of nominal (test day 83). All the other feeder samples met the targeted concentrations (≥80% of nominal). Test substance was not present in control diet.
Based on these results, it was concluded that targeted concentrations in the diet had been met throughout the study. - Duration of treatment / exposure:
- Approximately 90 days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 35 ppm
- Remarks:
- Equivalent to 5.89 mg/kg bw in males and 8.21 mg/kg bw in females
- Dose / conc.:
- 350 ppm
- Remarks:
- Equivalent to 62.4 mg/kg bw in males and 79.7 mg/kg bw in females
- Dose / conc.:
- 3 500 ppm
- Remarks:
- Equivalent to 534 mg/kg bw in males and 757 mg/kg bw in females
- Dose / conc.:
- 7 000 ppm
- Remarks:
- Equivalent to 1149 mg/kg bw in males and 1552 mg/kg bw in females
- No. of animals per sex per dose:
- 20 (The first 10 rats assigned to each group were designated for evaluation of subchronic toxicity; the next 5 rats in each group were designated for cell proliferation evaluation; the remaining 5 rats in each group were designated for biochemical measurements)
- Control animals:
- yes, plain diet
- Other examinations:
- Cell Proliferation Evaluation & Biochemical Measurements: After approximately 2 weeks on study, mice designated for hepatic cell proliferation evaluation and biochemical evaluation were sacrificed and livers were processed for either cell proliferation or biochemical evaluation. The hepatic cell proliferation labeling indices, a rate of peroxisomal beta-oxidation, and the total hepatic cytochrome P-450 content were determined.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were occasional occurrences of clinical observations; however, clinical observations of these types are commonly observed in laboratory mice and occurred in both control and treated groups at similar frequencies or were noted sporadically.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Mortality was observed in both males and females either accidental or cause of death is undetermined. The test substance administration had no effect on the survival of male and female mice.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The observed effects were not considered to be adverse or compound related, due to the lack of a dose-response and because they occurred sporadically.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- no effects observed
- Ophthalmological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The test substance administration did not produce any ophthalmological lesions in male or female mice. The major lesion of the 2 types of lesions found during the end-of-study examination was "phthisis bulbi" or " shrunken globe" (left eye). This Lesion can result from trauma likely to have occurred in mice subjected to post-orbital sinus puncture for the collection of blood for the 45-day hematology evaluation on test day 55.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Male and female mice fed 3500 and 7000 ppm had mild Heinz body-associated hemolysis. Mice fed 7000 ppm had principal erythrocytic alterations which included decreased erythrocyte count, reticulocytosis, increased hemoglobin concentration, MCV, MCH, and MCHC, Mice fed 3500 ppm had similar erythrocytic alterations except erythrocyte counts were not decreased. The hematologic alterations were suggestive of Heinz body-associated hemolysis and morphologic evaluation of selected blood smears from these groups confirmed the presence of Heinz bodies in erythrocytes. Heinz body-associated hemolysis was considered to be compound related and toxicologically important.
Male and female mice fed 7000 ppm had mild, statistically significant and biologically important leukocytosis, characterized by neutrophilia and lymphocytosis. The leukocytic alterations indicated inf lamination which was considered secondary to hemolysis. - Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The following compound-related adverse effects were observed in mice fed 3500 and 7000 ppm:
- liver weights were increased in male and female mice - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The following compound-related adverse effects were observed in mice fed 3500 and 7000 ppm:
- liver lesions were present which included centrilobular hypertrophy, diffuse fatty change, increased bile pigment, and necrosis in male and female mice - Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- The following compound-related adverse effects were observed in mice fed 3500 and 7000 ppm:
- liver lesions were present which included centrilobular hypertrophy, diffuse fatty change, increased bile pigment, and necrosis in male and female mice - Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Cell Proliferation Evaluation & Biochemical Measurements: The following compound-related adverse effects were observed in mice fed 3500 and 7000 ppm
- increased hepatic beta oxidation activity and total cytochrome P-450 content in male and female mice
- hepatic cell proliferation indices were increased in female mice - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 350 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- gross pathology
- haematology
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Remarks on result:
- other: (equivalent to 62.4 and 79.7 mg/kg bw in males and females respectively)
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 3 500 ppm
- System:
- other: hepatobiliary and blood
- Organ:
- blood
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- 13 week Mice NOAEL (male/female): 350 ppm (equivalent to 62.4 and 79.7 mg/kg bw in males and females respectively)
- Executive summary:
A 90-day study was performed to evaluate the subchronic toxicity of the test substance in mice when administered in the diet according to the guidelines OECD 408 and EPA 82-1. Five groups of 20 male and 5 groups of 20 female mice were fed diets that contained either 0, 35, 350, 3500, or 7000 ppm for approximately 90 days. The test substance was analyzed near the beginning and near the end of the study to assess the stability of the test substance. Samples of diet were analyzed to verify concentration, homogeneity, and stability of the test substance in the test diets near the beginning of the study. Samples were collected from feeders to verify the concentration of the test substance in the feed twice during the study, at 55 and 83 days. Body weights, clinical observations, and group food consumption data for the mice were collected weekly. Cage-site inspections of the mice to assess viability were conducted daily throughout the study. Ophthalmological evaluations were conducted on all mice during the pretest period and prior to the final sacrifice. Hematology evaluations were performed on all mice after 55 days on study and on male mice after 91 days on study and on female mice after 92 days on study. After approximately 2 weeks on study, mice designated for hepatic cell proliferation evaluation and biochemical evaluation were sacrificed and livers were processed for either cell proliferation or biochemical evaluation. The hepatic cell proliferation labeling indices, a rate of peroxisomal beta-oxidation, and the total hepatic cytochrome P-450 content were determined. All surviving mice were sacrificed and necropsied after approximately 90 days. Gross examinations were performed and select tissues were examined microscopically.
The test substance was stable based on the 2 analyses conducted during the study, -JE874-65 appeared stable and was distributed homogeneously in the diet over the test period. The overall (test days 0-91) mean daily intake in male mice fed 0, 35, 350, 3500, and 7000 ppm was 0, 5.89, 62.4, 534, and 1149 mg/kg body weight/day, respectively. The overall (test days 0-91) mean daily intake in female mice fed 0, 35, 350, 3500, and 7000 ppm was 0, 8.21, 79.7, 757, and 1552 mg/kg body weight/day, respectively.
Dietary administration of the test substance to mice resulted in primary adverse liver and hematological effects. The following compound-related adverse effects were observed in mice fed 7000 ppm:
Hematological findings:
- mild Heinz body-associated hemolysis was observed in male and female mice
- decreased erythrocyte counts and. erythrocytic alterations were present which included increased hemoglobin concentrations, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH)， and mean mean corpuscular hemoglobin concentration (MCHC), and increased reticulocyte counts in male and female mice
- increased spleen weights, splenic red pulp, and hemosiderin in male and female mice
Hepatic findings:
- liver weights were increased in male and female mice
- liver lesions were present which included centrilobular hypertrophy, diffuse fatty change, increased bile pigment, and necrosis in male and female mice
- increased hepatic beta oxidation activity and total cytochrome P-450 content in male and female mice
- hepatic cell proliferation indices were increased in female mice
The following compound-related adverse effects were observed in mice fed 3500 ppm
Hematological findings:
- mild Heinz body-associated hemolysis was observed in male and female mice
- erythrocytic alterations were present which included increased hemoglobin concentrations, MCV, MCH, and MCHC, as well as increased reticulocyte counts in male and female mice
- increased, spleen weights and hemosiderin in male and female mice
- increased splenic red pulp in female mice
Hepatic findings:
- liver weights were increased in male and female mice
- liver lesions were present which included centrilobular hypertrophy, diffuse fatty change, increased bile pigment, and necrosis in male and female mice
- increased hepatic beta oxidation activity and total cytochrome P-450 content in male and female mice
- hepatic cell proliferation indices were increased in female mice
No compound-related adverse effects were observed in male or female mice fed 350 or 35 ppm. Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for subchronic toxicity of the test substance was 350 ppm for male and female mice.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-1 (90-Day Oral Toxicity)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: MAFF Japan NohSan No. 4200
- Deviations:
- no
- GLP compliance:
- yes
- Specific details on test material used for the study:
- Substance ID: DPX-JE874-221 Technical
Lot #: DPX-JE874-221
Purity: 97.4% - Species:
- dog
- Strain:
- Beagle
- Details on species / strain selection:
- The animal model, the outbred beagle, is recognized as appropriate for toxicity studies and is a widely used strain for which significant historical control data are available.
- Sex:
- male/female
- Route of administration:
- oral: feed
- Details on route of administration:
- The oral route of administration was selected because it is the possible route of human exposure.
- Vehicle:
- other: Feed
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Results from analysis of diet samples submitted for homogeneity indicated a homogeneous distribution of the test article in the diets. The measured concentrations of the test substance in homogeneity diet samples were within ±13% of nominal. The test diets were stable when stored at room temperature for 7 and 14 days, or refrigerated for 14 days. The measured concentrations of test substance in stability diet samples were within ±12% of nominal. Diet samples (days 45 and 90) were within targeted concentrations (±15% of nominal), ranging from 85.8% to 100% of nominal.
Based on analyses of stock test substance samples collected near the beginning and end of the dosing period, the test article was judged to be stable during this study. - Duration of treatment / exposure:
- Approximately 90 days
- Frequency of treatment:
- The test diets were offered for approximately one hour per day for 13 weeks (91 or 92 consecutive days), until the day prior to the scheduled necropsy.
- Dose / conc.:
- 40 ppm
- Remarks:
- Equivalent to 1.3 mg/kg bw in males and 1.4 mg/kg bw in females
- Dose / conc.:
- 300 ppm
- Remarks:
- Equivalent to 10.0 mg/kg bw in males and 10.1 mg/kg bw in females
- Dose / conc.:
- 600 ppm
- Remarks:
- The test article was offered at 1000 ppm during weeks 0-4 and the first two days of week 5 (*equivalent to 23.8 mg/kg bw in males and 23.3 mg/kg bw in females) and 600 ppm during the last five days of week 5 and weeks 6-13 (*equivalent to 21.2 mg/kg bw in males and 20.1 mg/kg bw in females).
- No. of animals per sex per dose:
- 4
- Control animals:
- yes, plain diet
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Test article related clinical signs were limited to the 1000-600 ppm group males and females. The predominant clinical sign was myotonic twitches. This finding was initially observed for all males and females in the 1000-600 ppm group during the fourth week of administration at 1000 ppm (week 3). Ataxia and convulsions were observed for a single female on one occasion. Even after the dietary concentration was lowered to 600 ppm during week 5, myotonic twitches continued to be observed in this group generally through to study termination (week 13). These clinical signs were more frequently observed and were more severe approximately 4-hours after feeding (rarely persisted to the following day). Since the dogs received all exposure to the test article within an approximate 1-hour time interval each day, this 4-hour time of peak effect may correspond to the approximate time of peak blood levels of the test substance. An increased incidence of soft stool in the 1000-600 ppm group, especially during the period of administration at 600 ppm, was attributed to the test article.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean body weight losses and/or reduced mean body weight gains were observed in the 1000-600 ppm group males and females for weeks 0 to 1 through 4 to 5. By the end of this period (week 5)，mean body weights in the 1000-600 ppm group males and females were 17% and 19% lower, respectively, than the control group values. At study termination (week 13)，mean body weights in the 1000-600 ppm group males and females were 11% and 14% lower, respectively, than the control group values.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Food consumption, evaluated as g/animal/day, was moderately reduced in the 1000-600 ppm group males and females during the first six weeks of test article administration (weeks 0 to 1 through 5 to 6).
The average amounts of test article consumed in the 40, 300 and 1000-600 ppm groups were 1.3, 10.0 and 23.8-21.2 mg/kg/day for males and 1.4, 10.1 and 23.3-20.1 mg/kg/day for females, respectively. - Food efficiency:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Food efficiency values in the 1000-600 ppm group were negative for weeks 0 to 1, 1 to 2 and 2 to 3 for the males and for weeks 0 to 1 and 1 to 2 for the females. In addition, the weeks 2 to 3 and 3 to 4 values for females were markedly lower than the control group values.
- Ophthalmological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- At the week 12 ocular examination, bilateral posterior cortical cataracts (graded as slight) were present in two of four males and one of four females in the 300 ppm group and two of four males and two of four females in the 1000-600 ppm group. However, none of these dogs exhibited clinical signs of visual impairment.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- A test article related effect on red blood cell parameters was apparent in the 1000-600 ppm group males and females at the week 5 and 12 evaluations. Mean red blood cell count, hemoglobin, hematocrit and MCHC (mean corpuscular hemoglobin concentration) were reduced and mean MCV (mean corpuscular volume), reticulocyte count, Heinz bodies count and MCH (mean corpuscular hemoglobin) were increased. The differences from the control group were more pronounced at week 5. In addition, mean platelet counts were increased in the 1000-600 ppm group males and females at weeks 5 and 12. The increases in platelet and reticulocyte counts and MCV were indicative of a physiological response to the lower red blood cell counts.
A few statistically significant changes in red blood cell parameters were observed in the 40 ppm group females and 300 ppm group males and females. In the 300 ppm males and females, these changes were associated with increased pigment (hemosiderin) in hepatic Kupffer cells and/or bone marrow macrophages. However, most values were within the historical control ranges and/or were similar to pretreatment values. Moreover, the reticulocyte counts in the 40 and 300 ppm group males and females were not elevated. Therefore, the differences in red blood cell parameters in the 40 ppm group females and 300 ppm group males and females were not considered to be toxicologically significant since there was no reticulocyte response. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The only apparent test article related effect on serum chemistry parameters was increased mean potassium in the 1000-600 ppm group males and females at weeks 5 (fasted and nonfasted) and/or 12. This hyperkalemia is the possible cause of the myotonic twitches seen in the 1000-600 ppm group dogs.
- Urinalysis findings:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Mean absolute and relative testicular and epididymal weights in the 1000-600 group males were lower than the control group values and minimal to mild bilateral seminiferous tubule immaturity was observed microscopically. This is not an uncommon occurrence when body weight gains have been suppressed in dogs as they are reaching sexual maturity. The dogs in this group had statistically significant weight losses early in the study and body weights remained below the control group means for the entire 13-week exposure period. The effect on the testicular and epididymal weights is considered to be secondary to the body weight effect and not a direct toxic effect of the test article.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- An increase in the incidence and severity of dilated renal tubules containing eosinophilic crystalline structures was observed in the 1000-600 ppm female group. This lesion can occasionally be seen in control dogs; therefore, it was considered a test article exacerbation of a background lesion of uncertain biological significance.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Histopathological examination revealed microscopic lesions associated with the lenses of the eyes (7/8 animals), increased pigment (hemosiderin) in the liver and bone marrow (related to the red blood cell reduction) and bilateral seminiferous tubule immaturity (associated with the lower body, testes and epididymal weights). At a concentration of 300 ppm, bilateral posterior cortical cataracts (graded as slight) were observed in 3 of 8 animals. Microscopic changes in the lenses of the eyes were observed for all 8 dogs in the 300 ppm group. In addition, one female in the 40 ppm group had minimal lens changes.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 40 ppm
- Sex:
- male
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- food consumption and compound intake
- haematology
- histopathology: non-neoplastic
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 40 ppm
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- clinical signs
- food consumption and compound intake
- haematology
- histopathology: non-neoplastic
- Remarks on result:
- other: A NOAEL for female dogs could not be ascertained since one female at 40 ppm exhibited minimal lesions in the lens of the eyes.
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 40 ppm
- System:
- eye
- Organ:
- lens
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- 13 week Dog NOAEL (male): 40 ppm (1.3 mg/kg bw)
13 week Dog NOAEL (female): Could not be ascertained - Executive summary:
The toxicologic potential of the test substance was evaluated in this 13-week dietary subchronic toxicity study in outbred beagles according to the guideline OECD 409 and EPA 82-1. Concentrations of 40, 300 and 1000 ppm were selected for the study. The test article was administered in the diet for approximately 1-hour per day for 13 weeks (91 or 92 consecutive days). A concurrent control group received the basal diet on a comparable regimen. The 1000 ppm concentration was lowered to 600 ppm during the sixth week of administration (week 5, day 37) due to myotonic twitches of extended duration and a convulsion observed at the 1000 ppm concentration. Each study group consisted of four males and four females. All animals were observed twice daily for mortality and moribundity. Detailed physical examinations were performed weekly on all dogs. Clinical observations were recorded two to three times daily. Individual body weights were recorded weekly. Food consumption was recorded daily and reported weekly. Clinical pathologic evaluations (hematology, serum chemistry and urinalysis) were conducted once prior to dosing initiation and during the 6th and 13th weeks of dosing (study week numbers 5 and 12, respectively) on fasted animals. In addition, blood was collected from all animals in all groups approximately 4- hours following the feeding period during week 5 (day 36, last day of offering the test article in the diet at 1000 ppm to the high dose group). These samples were analyzed for glucose, phosphorus, chloride, sodium and potassium. Ocular examinations were conducted prior to dosing initiation and during study week 12. A complete necropsy was performed on all dogs euthanized at study termination and selected organs were weighed. Tissues were examined microscopically from all dogs.
All animals survived to the scheduled euthanization. Test article related clinical signs were limited to the 1000-600 ppm group males and females. The predominant clinical sign was myotonic twitches. This finding was initially observed for all males and females in the 1000-600 ppm group during the fourth week of administration at 1000 ppm (week 3). Ataxia and convulsions were observed for a single female on one occasion. Even after the dietary concentration was lowered to 600 ppm during week 5, myotonic twitches continued to be observed in this group generally through to study termination (week 13). These clinical signs were more frequently observed and were more severe approximately 4-hours after feeding (rarely persisted to the following day). Since the dogs received all exposure to the test article within an approximate 1-hour time interval each day, this 4-hour time of peak effect may correspond to the approximate time of peak blood levels of the test substance. An increased incidence of soft stool in the 1000-600 ppm group, especially during the period of administration at 600 ppm, was attributed to the test article.
Mean body weight losses and/or reduced mean body weight gains were observed in the 1000-600 ppm group males and females for weeks 0 to 1 through 4 to 5. By the end of this period (week 5)，mean body weights in the 1000-600 ppm group males and females were 17% and 19% lower, respectively, than the control group values. At study termination (week 13)，mean body weights in the 1000-600 ppm group males and females were 11% and 14% lower, respectively, than the control group values. Food consumption, evaluated as g/animal/day, was moderately reduced in the 1000-600 ppm group males and females during the first six weeks of test article administration (weeks 0 to 1 through 5 to 6). Food efficiency values in the 1000-600 ppm group were negative for weeks 0 to 1, 1 to 2 and 2 to 3 for the males and for weeks 0 to 1 and 1 to 2 for the females. In addition, the weeks 2 to 3 and 3 to 4 values for females were markedly lower than the control group values. The average amounts of test article consumed in the 40, 300 and 1000-600 ppm groups were 1.3, 10.0 and 23.8-21.2 mg/kg/day for males and 1.4, 10.1 and 23.3-20.1 mg/kg/day for females, respectively.
A test article related effect on red blood cell parameters was apparent in the 1000-600 ppm group males and females at the week 5 and 12 evaluations. Mean red blood cell count, hemoglobin, hematocrit and MCHC (mean corpuscular hemoglobin concentration) were reduced and mean MCV (mean corpuscular volume), reticulocyte count, Heinz bodies count and MCH (mean corpuscular hemoglobin) were increased. The differences from the control group were more pronounced at week 5. In addition, mean platelet counts were increased in the 1000-600 ppm group males and females at weeks 5 and 12. The increases in platelet and reticulocyte counts and MCV were indicative of a physiological response to the lower red blood cell counts.
A few statistically significant changes in red blood cell parameters were observed in the 40 ppm group females and 300 ppm group males and females. In the 300 ppm males and females, these changes were associated with increased pigment (hemosiderin) in hepatic Kupffer cells and/or bone marrow macrophages. However, most values were within the WIL Research Laboratories, Inc. historical control ranges and/or were similar to pretreatment values. Moreover, the reticulocyte counts in the 40 and 300 ppm group males and females were not elevated. Therefore, the differences in red blood cell parameters in the 40 ppm group females and 300 ppm group males and females were not considered to be toxicologically significant since there was no reticulocyte response.
The only apparent test article related effect on serum chemistry parameters was increased mean potassium in the 1000-600 ppm group males and females at weeks 5 (fasted and nonfasted) and/or 12. This hyperkalemia is the possible cause of the myotonic twitches seen in the 1000-600 ppm group dogs.
No test article related effects were apparent on urinalysis parameters or at the gross necropsy.
At the week 12 ocular examination, bilateral posterior cortical cataracts (graded as slight) were present in two of four males and one of four females in the 300 ppm group and two of four males and two of four females in the 1000-600 ppm group. However, none of these dogs exhibited clinical signs of visual impairment. At histopathologic examination, these cataracts predominantly consisted of swollen fibers at the Y suture of the posterior lens capsule and were observed in the lenses of the eyes in 4/4 and 3/4 males in the 300 and 1000-600 ppm groups, respectively, and in 1/4, 4/4 and 4/4 females in the 40, 300 and 1000-600 ppm groups, respectively.
Mean absolute and relative testicular and epididymal weights in the 1000-600 group males were lower than the control group values and minimal to mild bilateral seminiferous tubule immaturity was observed microscopically. This is not an uncommon occurrence when body weight gains have been suppressed in dogs as they are reaching sexual maturity. The dogs in this group had statistically significant weight losses early in the study and body weights remained below the control group means for the entire 13-week exposure period. The effect on the testicular and epididymal weights is considered to be secondary to the body weight effect and not a direct toxic effect of the test article.
An increase in the incidence and severity of dilated renal tubules containing eosinophilic crystalline structures was observed in the 1000-600 ppm female group. This lesion can occasionally be seen in control dogs; therefore, it was considered a test article exacerbation of a background lesion of uncertain biological significance.
The test diets were homogeneous and stable at room temperature for 7 and 14 days and under refrigeration for 14 days. The test diets contained the amount of test article specified in the protocol. Analysis of bulk test article samples collected near the beginning and end of the dosing period revealed that the test substance was stable during this study.
In conclusion, a concentration of 1000-600 ppm test substance administered to dogs for 13 weeks produced systemic toxicity consisting of clinical signs of toxicity (primarily myotonic twitches)，inhibition of body weight gain and food consumption, decreased circulating erythrocyte mass, increased serum potassium, bilateral posterior cortical cataracts (4/8 dogs; graded as slight) and lower testes and epididymal weights (secondary to decreased body weight and not a direct toxic effect of the test substance). Histopathological examination revealed microscopic lesions associated with the lenses of the eyes (7/8 animals), increased pigment (hemosiderin) in the liver and bone marrow (related to the red blood cell reduction) and bilateral seminiferous tubule immaturity (associated with the lower body, testes and epididymal weights). At a concentration of 300 ppm, bilateral posterior cortical cataracts (graded as slight) were observed in 3 of 8 animals. Microscopic changes in the lenses of the eyes were observed for all 8 dogs in the 300 ppm group. In addition, one female in the 40 ppm group had minimal lens changes. Mild changes in some red blood cell parameters and increased pigment (hemosiderin) in the liver and/or bone marrow were observed in the 300 ppm group males and females and reductions in red blood cell parameters were seen in the 40 ppm group female dogs. However, the hematology and pigment effects in the 40 and 300 ppm group dogs were not considered to be toxicologically significant as they were of small magnitude, many changes were not statistically significant, most values were within the historical control ranges, and the effects at these concentrations were not associated with elevation in Heinz bodies or reticulocytes.
The no-observed-effect level (NOEL) for this study is defined as the highest dose at which toxicologically important effects attributable to the test substance were not detected. Thus, for this study, the NOEL is equivalent to the NOEL as defined by the United States Environmental Protection Agency (1985)2 and the no-observed-adverse-effect level (NOAEL) as defined by the European Union (1994)3. Under the conditions of this study, the NOEL for this study was 40 ppm for male dogs, based on clinical and microscopic evidence of slight posterior subcortical cataracts observed at 300 ppm. A NOEL for female dogs could not be ascertained since one female at 40 ppm exhibited minimal lesions in the lens of the eyes. Based on the results of this study, concentrations of 10, 20, 40 and 300 ppm were selected for the one year dietary study in dogs with the test substance.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-1 (90-Day Oral Toxicity)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: MAFF Japan NohSan No. 4200
- Deviations:
- no
- GLP compliance:
- yes
- Specific details on test material used for the study:
- Substance ID: DPX-JE874-65 Technical
Lot #: DPX-JE874-65
Purity: 97.4% - Species:
- rat
- Strain:
- other: Crl:CD®BR
- Details on species / strain selection:
- The Crl:CD®BR rat was selected on the bases of extensive experience with this strain and its suitability with respect to hardiness, longevity, sensitivity, and low incidence of spontaneous diseases.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Inc. Kingston, New York
- Age at study initiation: Approximately 45 days (at day 0)
- Weight at study initiation: Approximately 224-229 g for males and 157-160 g for females
- Fasting period before study: No
- Housing: All rats were housed individually in stainless steel, wire-mesh cages suspended above cage boards (three per cage, sexes separate in quarantine)
- Diet: Irradiated Purina® Certified Rodent Chow® #5002 meal
- Water: Tap water, ad libitum
- Acclimation period: 22 days
ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 2°C
- Humidity: 50 ± 10%
- Photoperiod: 12 hrs dark /12 hrs light - Route of administration:
- oral: feed
- Details on route of administration:
- The oral route of administration was selected because it is the potential route of human exposure.
- Vehicle:
- other: Feed
- Details on oral exposure:
- DIET PREPARATION
The test substance was added to Purina® Certified Rodent Chow® #5002 meal (PCRC) and thoroughly mixed for 3 minutes in a high-speed mixer. Control diets were mixed for the same period of time. All diets were prepared every other week and refrigerated until used. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- On test day -21, samples (approximately 50 grams each) were collected from each concentration of diet prepared with the test substance. These samples were analyzed to verify concentration, homogeneity, and stability of the test substance in the test diets. Samples used to verify concentration were collected from the diet mixer and frozen until analyzed. Samples used to verify homogeneity were collected from the top, middle, and bottom of the diet mixer and frozen until analyzed. Samples to verify stability were collected from feed jars and were stored at room temperature for 7 and 14 days. Stability samples from the diet mixer were refrigerated for 14 days.
Samples (approximately 50 grams) were collected from feed jars from diets prepared on test days -1 and 35 to verify the concentration.
Measured concentrations were 99.3% and 101% of nominal at 50 ppm and 93.4% and 95.0% of nominal at 1680 and 1760 ppm, respectively. Based on these data, the analytical method was considered to have performed satisfactorily over the concentration range of the study.
Test day -21: The average measured concentrations of a.i. in the samples taken from the diet mixture to verify concentration and homogeneity ranged from 45.7-46.6 ppm for the 50 ppm level, 185-187 ppm for the 200 ppm level, 737-749 ppm for the 800 ppm level, and 1,390-1,440 ppm for the 1,600 ppm level. Since the measured concentrations were within 14% of nominal at all levels and the recovery efficiency ranged from 93.4-101% of nominal in recovery samples, the test substance was considered to be present at targeted concentration in all diets. The coefficient of variation for the average concentrations measured in samples taken from the top, middle, and bottom regions of the mixer was 1.1%, 0.5%, 0.8%, and 1.9% at the 50, 200, 800, and 1,600 ppm levels, respectively. This indicates that the test substance was distributed homogeneously in the diet.
The stability of the a.i. in test substance was assessed by comparing samples collected from each dietary concentration and held under the following storage conditions: 1) fresh frozen, 2) 7- and 14-day room temperature, and 3) 14-day refrigeration. Fresh frozen samples ranged from 89.0-93.4% of nominal; 7-day room temperature samples ranged from 81.6-91.4% of nominal; 14-day room temperature samples ranged from 86.4-93.4% of nominal; and 14-day refrigerated samples ranged from 86.8-91.7% of nominal. These data demonstrate that the a.i. in test substance was stable in diets under relevant storage conditions.
Samples of diets prepared on test days -1 and 35 were collected from cage site feed jars and analyzed to verify the concentration of test substance delivered to the rats. Measured concentrations were 83.1% to 99.0% of nominal for diets prepared on days -1 and 97.3% to 105% of nominal for diets prepared on days 35. Based on these data, the preparation of diets met the targeted concentrations.
The test substance was not detected in control diets at any sampling time. - Duration of treatment / exposure:
- Approximately 90 days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 50 ppm
- Remarks:
- Equivalent to 3.34 mg/kg bw in males and 4.24 mg/kg bw in females
- Dose / conc.:
- 200 ppm
- Remarks:
- Equivalent to 13.0 mg/kg bw in males and 16.6 mg/kg bw in females
- Dose / conc.:
- 800 ppm
- Remarks:
- Equivalent to 52.1 mg/kg bw in males and 65.7 mg/kg bw in females
- Dose / conc.:
- 1 600 ppm
- Remarks:
- Equivalent to 106.0 mg/kg bw in males and 130.0 mg/kg bw in females
- No. of animals per sex per dose:
- 20 (The first 10 rats assigned to each group were designated for evaluation of subchronic toxicity; the next 5 rats in each group were designated for cell proliferation evaluation; the remaining 5 rats in each group were designated for biochemical measurements)
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: A two-week study was conducted to evaluate the repeated dose oral toxicity of the test substance when administered in the diet. Dietary concentrations of 100, 1000, 6000, and 20000 ppm of the test substance were tested in rats. Mean body weight, body weight gain, and food consumption were significantly decreased in males and females at 6000 and 20000 ppm and in females at 1000 ppm. Compound-related hepatocellular hypertrophy, degeneration, and necrosis of the liver were observed in both sexes receiving 1000 ppm and above of the test substance. Based on the above information, the no-observable-adverse-effect level (NOAEL) for the test substance was 100 ppm for both male and female rats.
- Fasting period before blood sampling for clinical biochemistry: Yes, at least 16 hours - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At every weighing
BODY WEIGHT: Yes
- Time schedule for examinations: Once per week
FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
FOOD EFFICIENCY
- Body weight gain in kg/food consumption in kg per unit time, calculated as time-weighted averages from the consumption and body weight gain data: Yes
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Test day -22 and 85
- Dose groups that were examined: All rats
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Approximately 45 and 90 days after initiation of
the study
- Anaesthetic used for blood collection: Yes (light carbon dioxide anesthesia)
- Animals fasted: Yes
- How many animals: The first 10 rats from each group were selected for the 45-day evaluation. All surviving rats from these same 10 rats per group were used for the 90-day evaluation.
- Parameters checked: Erythrocytes (RBC), leukocytes (WBC), platelets (PLAT), hemoglobin concentration (Hb), hematocrit (Ht), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), neutrophils (Neut), band neutrophils (Band), lymphocytes (Lymph), atypical lymphocytes (Alym), monocytes (Mono), eosinophils (Eosin), basophils (Baso), reticulocytes (Retic)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Approximately 45 and 90 days after initiation of
the study
- Anaesthetic used for blood collection: Yes (light carbon dioxide anesthesia)
- Animals fasted: Yes
- How many animals: The first 10 rats from each group were selected for the 45-day evaluation. All surviving rats from these same 10 rats per group were used for the 90-day evaluation.
- Parameters checked: Alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH) urea nitrogen (BUN), total protein (TPROT), albumin (ALBMN), creatinine (CREAT), bilirubin (BILRN), cholesterol (CHOL), glucose (GLUCO), calcium (CALC), sodium (Na), potassium (K), phosphate (PHOS), chloride (Cl), Serum globulin (GLOBN)
URINALYSIS: Yes
- Time schedule for collection of urine: Approximately 45 and 90 days after initiation of
the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: volume (VOL), osmolality (OSMOL), pH, urobilinogen (UROBL), hemoglobin or occult blood (BLOOD), glucose, protein, bilirubin, ketone (acetoacetic acid) - Sacrifice and pathology:
- GROSS PATHOLOGY: All rats that were found dead or accidentally killed were necropsied. After approximately 90 days on test (test days 95 and 96), all surviving rats were sacrificed by chloroform and exsanguination and necropsied. The order of sacrifice was random among all groups within a sex. Selected organs (adrenals, brain, heart, kidneys, liver, spleen, and testes) were weighed at necropsy for all animals which were sacrificed by design at the study termination.
Representative samples of the following tissues were saved at necropsy:
Integumentary System: Skin
Hematopoietic System: Bone marrow (sternum and femur), lymph nodes (mesenteric and mandibular), spleen, thymus
Circulatory System: Aorta, heart
Respiratory System: Trachea, lungs
Digestive System: Salivary glands, esophagus, stomach, liver, pancreas, small intestine (duodenum, jejunum, and ileum), large intestine (cecum, colon, and rectum)
Urinary System: kidneys, urinary bladder
Endocrine System: Pituitary, thyroid-parathyroid, adrenal glands (cortex and medulla)
Reproductive System: Male (prostate, testes, epididymides, seminal vesicles); Female (mammary glands, ovaries, uterus, vagina)
Nervous System: Brain, spinal cord, peripheral nerve (sciatic)
Musculoskeletal System: Skeletal muscle, femur (including knee joint), sternum
Other: Eyes, exorbital lacrimal glands, harderian glands, and select gross lesions
HISTOPATHOLOGY: Tissues collected from rats in the high-concentration groups, control groups, and the rat that was found dead (tissue integrity permitting) were further processed to slides and examined microscopically. Liver, kidneys, lungs, all organs with gross lesions, and target organs from rats in the low, low intermediate, and high-intermediate-concentration groups were also examined microscopically. - Other examinations:
- Cell Proliferation Evaluation: After approximately 2 weeks of feeding, the 5 rats from each group selected for cell proliferation were implanted (subcutaneously) with Alzet® osmotic pumps. The pumps were loaded with 20 mg/ml 5-bromo-2-deoxyuridine (BrdU) dissolved in a 0.5 N sodium bicarbonate buffer. Three days after implantation, the rats were sacrificed by chloroform anesthesia and exsanguination. Tissues were collected. Pathological Evaluation and fixed in neutral buffered formalin, but were not weighed. Liver and duodenum were processed for immunohistochemical analysis of BrdU incorporation into DNA. The labeling indices were determined from the livers of rats in all groups.
Biochemical Measurements: After approximately 2 weeks of feeding, the 5 rats from each group selected for biochemical measurements were sacrificed by chloroform anesthesia followed by exsanguination. The livers were removed, weighed, and homogenized (1 g tissue/4 mL buffer) with a polytron. The protein content of the microsomes and peroxisomes determined was before and after analysis by the Biorad method.
The microsomal cytochrome P-450 contents were measured according to the method of Omura and Sato. The microsomal suspensions were diluted so that the final protein concentration was approximately 1 mg/mL. The spectra were recorded at room temperature with a Beckman DU7500 spectrophotometer. Peroxisomal beta-oxidation activity was determined using [14C] palmitoyl CoA the substrate. The peroxisomal suspensions were diluted so that the protein concentration was approximately 0.25 mg/mL. - Statistics:
- Body weights, body weight gains, clinical pathology measurements, organ weights, labeling indices, and biochemical measurements were analyzed by a one-way analysis of variance. Pairwise comparisons between test and control groups were made with the Dunnett’s test. Survival among groups on the final food consumption day (test day 91) for all groups of rats was evaluated with the Cochran- Armitage test for trend, increases in the incidences of clinical observations were evaluated by the Fisher's exact test with a Bonferroni correction.
The Bartlett’s test for homogeneity of variances was performed on the pathology weights and clinical pathology data and, if significant, was followed by nonparametric procedures. The Kruskal- Wallis test for equal medians, the Mann-Whitney U test, and Jonckheere test (nonparametric procedures) were employed.
The Cochran-Armi tage trend test was applied to all gross and microscopic observations in tissues or organs where all of these tissues or organs (subject to availability) were evaluated in all groups. When all tissues or organs were not microscopically evaluated in all groups, the Fisher's exact test was applied to compare the control and high dose group lesion incidences. If the Fisher’s exact test generated a p value < 0.05, the tissue or organ was microscopically evaluated in all animals in all groups unless additional evaluation was not warranted in the scientific opinion of the pathologist. The incidences of each lesion in question were further evaluated using the Cochran- Armitage trend test.
For any parameter evaluated by the Cochran-Armitage trend test, the following procedures were applicable: When the trend test was positive for the high- and intermediate-concentration groups, the incidence observed in the lowest concentration group was compared to its respective control group lesion incidence by using the Fisher's exact test.
Except for the Bartlett’s test, (p < 0.005), all significance was judged at p < 0.05. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No compound-related clinical signs of toxicity were observed. A few clinical signs commonly observed in laboratory rats were seen in all test groups, including controls. These signs included alopecia, ocular discharge, hyperreactivity, sores, swollen eye(s), and stained inguen or perineum.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Two male rats in the 200 ppm group died prior to the end of the study. One rat was accidentally killed on test day 11. The other rat was found dead on test day 42; the cause of death was septicemia and was unrelated to compound administration. Survival was 100% in all other groups.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean final body weights were significantly decreased in male rats at 1600 ppm (85% of control) and in female rats at 1600 ppm (85% of control) and 800 ppm (87% of control). A non-statistically significant reduction in body weight was also seen in males at 800 ppm (91% of control) and in females at 200 ppm (91% of control). Although these differences were not statistically significant, they associated with statistically significant decreases in body weight gain.
Overall (test days 0-91) body weight gain was 85% of control in 800 ppm males and 81% of control in 200 ppm females. Overall body weight gains were also depressed in a dose-dependent manner at higher dietary concentrations. Much of the differences in body weight gain in these groups occurred during the first 2-3 weeks of the study; however, body weight gain in these groups were generally below that of controls throughout most of the 91 days on test The body weight effects are considered to be compound related and toxicologically significant at 800 ppm and above in male rats and at 200 ppm and above in female rats. Mean body weight and body weight gain were comparable to control values in male and female rats at 50 ppm and in male rats at 200 ppm. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- The observed body weight effects were associated with comparable decreases in food consumption. Overall (test day 0-91) mean food consumption as a percentage of control means was 86% in males and 88% in females at 1600 ppm, 92% in males and 90% in females at 800 ppm, and 95% in females at 200 ppm. Reductions in food efficiency in male rats were comparable to the reductions in food consumption, while female rats displayed somewhat greater effects on food efficiency. Food consumption in male rats at 50 and 200 ppm and in female rats at 50 ppm was comparable to control values.
The overall mean daily intake of the test substance in male rats in the 0, 50, 200, 800, and 1600 ppm groups was 0, 3.34, 13.0, 52.1, and 106 mg/kg body weight, respectively. Overall mean daily intake in female rats at these concentrations was 0, 4.24, 16.6, 65.7, and 130 mg/kg body weight, respectively. - Food efficiency:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Food efficiency was lower than in controls in all groups exposed to the test substance; however, the differences in both sexes were due primarily to a large decrease in food efficiency in the 800 and 1600 ppm groups during the first week of dietary exposure. In subsequent weeks, a clear-cut dose response was not evident for food efficiency. The food consumption and food efficiency data suggest that part of the body weight effects may reflect reduced food consumption, possibly due to poor palatability.
- Ophthalmological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No compound-related ocular effects were produced by exposure to DPX-JE874-65. At the final ophthalmological evaluation (test day 85), a phthisical left eye was observed in one male rat at 50 ppm and unilateral radial linear retinal atrophy was observed in one female rat at 200 ppm. Orbital sinus puncture of the left eye was performed at the 45-day clinical pathology evaluation; therefore, the phthisical eye may have been the result of trauma during this procedure. Based on the low number of ocular lesions and the lack of a dose-response, neither lesion was considered to be compound related.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Mild anemia was observed in male and female rats at 800 and 1600 ppm and was considered to be compound related and toxicologically significant. The anemia was characterized by reductions in numerous indicators of circulating red blood cell mass and was classified as regenerative, based on evidence of reticulocytosis. The anemia was considered to be hemolytic in origin as there was no evidence of hemorrhage and because increases in congestion, hemosiderin, and extramedullary hematopoiesis were observed in the spleens of rats in these test groups.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Clinical chemistry abnormalities indicative of hepatocellular injury were detected in male and female rats at 800 and 1600 ppm. In male rats, mild increases were measured in the levels of bilirubin and moderate increases were measured in the activity of all evaluated serum liver enzymes (alkaline phosphatase [ALP], alanine aminotransferase [ALT], aspartate aminotransferase [AST], and sorbitol dehydrogenase [SDH]). In female rats, mild but significant increases were observed only in the activity of SDH, the most sensitive indicator of hepatocellular injury, and bilirubin. The sex-specific difference in the pattern of clinical pathologic effects correlates with similar differences observed in histopathologic evaluation of livers.
In male and female rats at 800 and 1600 ppm, a few other statistically significant differences in clinical chemistry parameters were observed which were considered secondary to the hepatocellular injury, rather than due to direct effects of exposure to the test substance. These differences included increased serum cholesterol in females, minimally decreased serum glucose in males and females, minimally decreased serum globulin in males and females, and decreased total protein in males. The cholesterol effect in female rats may have been due to alterations in lipid metabolism, rather than to hepatotoxicity, as the same cholesterol effect was not seen in male rats despite evidence of more severe hepatocellular injury in males. - Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Urine urobilinogen concentration was increased in male rats at 1600 ppm. This increase was attributed to the hepatocellular injury in these animals. Evidence of mild dehydration (decreased urine volume and increased urine osmolality and serum albumin) were noted in 800 and 1600 ppm females at the 45-day sampling time. No other compound-related abnormalities in urinalysis were observed.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Organ weight effects attributed to test substance exposure were observed in male and female rats in the 800 and 1600 ppm groups. Mean absolute and relative liver weights were increased in female rats at these concentrations, while mean absolute liver weights were decreased in male rats in-these groups. Mean absolute and relative spleen weights were increased in both males and females at these concentrations. The organ weight effects correlate with observed histopathologic observations.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Small, scattered white foci were observed at necropsy in the livers of 4 of 10 male rats at 1600 ppm. These gross lesions correlated with prominent microscopic focal degeneration in this group. No other compound-related gross lesions were observed.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Compound-related microscopic lesions were observed in liver, spleen, and bone marrow of male and female rats at 800 and 1600 ppm. In liver, the most common observation in both sexes was hepatocellular hypertrophy. This observation was likely due to induction of smooth endoplasmic reticulum (SER) and SER-associated enzymes and/or peroxisome proliferation. The latter process is supported by an increase in beta-oxidation (an indicator of peroxisome proliferation) which was detected in male and female rats at 800 and 1600 ppm and in female rats at 200 ppm. No increase in total cytochrome P-450 was measured in any group.
Focal degeneration (particularly in periportal and midzonal regions), single cell necrosis (apoptosis), and an associated increase in mitotic figures were other microscopic lesions noted m the livers of male and female rats at 800 and 1600 ppm, with the greatest incidence and severity occurring in the male 1600 ppm group. In male rats, the focal degeneration seen in the 1600 ppm group was associated with an increase in grossly observed small, scattered white foci. Bile duct hyperplasia was also observed in male rats at 800 and 1600 ppm. The greater severity of focal degeneration in male rats compared to female rats is a likely explanation for the difference in compound-related effect on absolute liver weights (decreased in males; increased in females).
The bile duct hyperplasia and increase in mitotic figures indicate a cellular proliferative response, perhaps to the focal degeneration and single cell necrosis. These lesions were generally more prominent in male rats than in female rats and correlated with a marked increase in BrdU labeling in hepatocytes (a measurement of cell proliferation) in male rats at 800 and 1600 ppm. Female rats also demonstrated histopathologic evidence of cell proliferation (although to a lesser degree than male rats), and this correlated with a smaller increase in the level of BrdU labeling of hepatocytes in females. The sex-specific pattern of hepatocellular lesions correlated with clinical pathology, in which male rats demonstrated evidence of more severe hepatotoxicity. The significance of apoptosis is not clear but this process may represent a mechanism by which the liver eliminates excess cells resulting from the hypertrophy occurring in this organ. - Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Cell Proliferation Evaluation: An increase in the BrdU labeling index of hepatocytes was observed in male rats at 800 and 1,600 ppm (approximately 18-19 times control level). In female rats, smaller increases in labeling indices were also seen at 800 and 1,600 ppm (approximately 2 and 5 times control level, respectively). These results correlated with the increased mitotic activity seen in livers at both levels and with the sex-specific differences in sensitivity to liver effects.
Biochemical Measurements: The test substance exposure produced no effects on total cytochrome P-450 levels in either sex. The microscopic liver alterations seen in this study (e.g., hepatocellular hypertrophy) are often associated with increases in smooth endoplasmic reticulum and cytochrome P-450. The discrepancy between liver microscopic observations and the lack of a measurable induction of cytochrome P-450 is not clear.
Hepatic beta-oxidation activity was elevated approximately 2-fold in male and female rats at 800 and 1600 ppm and approximately 1.5-fold in female rats at 200 ppm. This increased activity is a marker for peroxisomal proliferation. It is considered to be a pharmacologic response and is not considered to be adverse. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 50 ppm
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- haematology
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 50 ppm
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- clinical biochemistry
- food consumption and compound intake
- food efficiency
- gross pathology
- haematology
- histopathology: non-neoplastic
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 200 ppm
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- 13 week Rat NOAEL (male): 200 ppm (13.0 mg/kg bw)
13 week Rat NOAEL (female): 50 ppm (4.24 mg/kg bw) - Executive summary:
The objective of this study was to assess the potential subchronic toxicity of the test substance in rats according to the guidelines OECD 408 and EPA 82-1. Five groups of 20 male rats and 5 groups of 20 female rats were fed diets that contained 0, 50, 200, 800, or 1600 ppm test substance for approximately 90 days. Samples of the prepared diets were collected and analyzed to assess the homogeneity, stability, and concentration of the test substance in each diet. The rats were weighed once a week during the study. Group food consumption and clinical signs of toxicity were also assessed throughout the study.
Cell proliferation was evaluated in the livers of 5 rats from each group after approximately 2 weeks of feeding the test substance. At the same time point, 5 additional rats from each group were sacrificed and evaluated for hepatic peroxisomal beta-oxidation and hepatic cytochrome P-450 content. An ophthalmological examination was conducted on all surviving rats on test day 85. Clinical pathology evaluations were conducted on all surviving rats after approximately 45 and 90 days of dietaiy exposure to the test substance. After approximately 90 days on test, all surviving rats were sacrificed and necropsied. Selected organs were weighed and tissues were examined for the presence of any gross and/or microscopic lesions.
The overall mean daily intake of the test substance in the 0, 50, 200, 800, and 1600 ppm groups was 0, 3.34, 13.0, 52.1, and 106 mg/kg body weight, respectively, for male rats and 0, 4.24, 16.6, 65.7, and 130 mg/kg body weight, respectively, for female rats.
Decreases in mean final body weight occurred in male rats at 800 ppm (91% of control) and 1600 ppm (85% of control) and in female rats at 200 ppm (91% of control), 800 ppm (87% of control), and 1600 ppm (85% of control). Although the differences in mean final body weight were not statistically significant in 800 ppm male rats and in 200 ppm female rats, they were considered to be biologically significant and compound related as the differences were dose-dependent and associated with statistically significant decreases in overall mean body weight gain (test day 0-91). Decreases in food consumption of comparable magnitude were observed in these same test groups. Overall food efficiency (test day 0-91) was lower in all treated groups than in controls, but a clear dose-response for this effect was not evident after the first week of exposure.
There were no mortalities, clinical signs of toxicity, or ophthalmologic observations attributed to dietary exposure to the test substance in any test group.
Mild anemia was observed in male and female rats at 800 and 1600 ppm and was considered to be compound related and toxicologically significant. The anemia was characterized by reductions in numerous indicators of circulating red blood cell mass and was classified as regenerative, based on evidence of reticulocytosis. The anemia was considered to be hemolytic in origin as there was no evidence of hemorrhage and because increases in congestion, hemosiderin, and extramedullary hematopoiesis were observed in the spleens of rats in these test groups.
Clinical chemistry abnormalities indicative of hepatocellular injury were detected in male and female rats at 800 and 1600 ppm. In male rats, mild increases were measured in the levels of bilirubin and moderate increases were measured in the activity of all evaluated serum liver enzymes (alkaline phosphatase [ALP], alanine aminotransferase [ALT], aspartate aminotransferase [AST], and sorbitol dehydrogenase [SDH]). In female rats, mild but significant increases were observed only in the activity of SDH, the most sensitive indicator of hepatocellular injury, and bilirubin. The sex-specific difference in the pattern of clinical pathologic effects correlates with similar differences observed in histopathologic evaluation of livers.
In male and female rats at 800 and 1600 ppm, a few other statistically significant differences in clinical chemistry parameters were observed which were considered secondary to the hepatocellular injury, rather than due to direct effects of exposure to the test substance. These differences included increased serum cholesterol in females, minimally decreased serum glucose in males and females, minimally decreased serum globulin in males and females, and decreased total protein in males. The cholesterol effect in female rats may have been due to alterations in lipid metabolism, rather than to hepatotoxicity, as the same cholesterol effect was not seen in male rats despite evidence of more severe hepatocellular injury in males.
Urine urobilinogen concentration was increased in male rats at 1600 ppm. This increase was attributed to the hepatocellular injury in these animals. Evidence of mild dehydration (decreased urine volume and increased urine osmolality and serum albumin) were noted in 800 and 1600 ppm females at the 45-day sampling time. No other compound-related abnormalities in urinalysis were observed.
A large increase in the BrdU labeling index of hepatocytes (approximately 18-19 times control level), indicative of cell proliferation, was observed in male rats at 800 and 1600 ppm. Smaller increases were seen in female rats at 800 and 1600 ppm (approximately 2 and 5 times control level, respectively). Hepatic beta-oxidation, a marker for peroxisome proliferation, was elevated approximately 2-fold in male and female rats at 800 and 1600 ppm and approximately 1.5-fold in female rats at 200 ppm. Dietary exposure to the test substance produced no significant, measurable differences in hepatic cytochrome P-450 content in either male or female rats.
Organ weight effects attributed to test substance exposure were observed in male and female rats in the 800 and 1600 ppm groups. Mean absolute and relative liver weights were increased in female rats at these concentrations, while mean absolute liver weights were decreased in male rats in-these groups. Mean absolute and relative spleen weights were increased in both males and females at these concentrations. The organ weight effects correlate with observed histopathologic observations.
Compound-related microscopic lesions were observed in liver, spleen, and bone marrow of male and female rats at 800 and 1600 ppm. In liver, the most common observation in both sexes was hepatocellular hypertrophy. This observation was likely due to induction of smooth endoplasmic reticulum (SER) and SER-associated enzymes and/or peroxisome proliferation. The latter process is supported by an increase in beta-oxidation (an indicator of peroxisome proliferation) which was detected in male and female rats at 800 and 1600 ppm and in female rats at 200 ppm. No increase in total cytochrome P-450 was measured in any group.
Focal degeneration (particularly in periportal and midzonal regions), single cell necrosis (apoptosis), and an associated increase in mitotic figures were other microscopic lesions noted m the livers of male and female rats at 800 and 1600 ppm, with the greatest incidence and severity occurring in the male 1600 ppm group. In male rats, the focal degeneration seen in the 1600 ppm group was associated with an increase in grossly observed small, scattered white foci. Bile duct hyperplasia was also observed in male rats at 800 and 1600 ppm. The greater severity of focal degeneration in male rats compared to female rats is a likely explanation for the difference in compound-related effect on absolute liver weights (decreased in males; increased in females).
The bile duct hyperplasia and increase in mitotic figures indicate a cellular proliferative response, perhaps to the focal degeneration and single cell necrosis. These lesions were generally more prominent in male rats than in female rats and correlated with a marked increase in BrdU labeling in hepatocytes (a measurement of cell proliferation) in male rats at 800 and 1600 ppm. Female rats also demonstrated histopathologic evidence of cell proliferation (although to a lesser degree than male rats), and this correlated with a smaller increase in the level of BrdU labeling of hepatocytes in females. The sex-specific pattern of hepatocellular lesions correlated with clinical pathology, in which male rats demonstrated evidence of more severe hepatotoxicity. The significance of apoptosis is not clear but this process may represent a mechanism by which the liver eliminates excess cells resulting from the hypertrophy occurring in this organ.
Microscopic lesions were observed in the spleens of male and female rats at 800 and 1600 ppm and included increases in congestion, hemosiderin, and extramedullary hematopoiesis. These same treatment groups also demonstrated increases in bone marrow cellularity. The lesions in the spleen were associated with increased absolute and relative spleen weights. The spleen and bone marrow lesions correlated with the hematological effects seen in these groups. The relevance of these lesions to the liver effects is not clear.
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was judged to be 200 ppm in male rats. This level is based upon decreases in body weight, body weight gain, and food consumption and/or food efficiency; clinical and microscopic pathologic evidence of mild, regenerative, hemolytic anemia; and clinical, macroscopic, and microscopic pathologic evidence of hepatotoxicity at 800 and 1600 ppm.
Under the conditions of this study, the NOAEL was judged to be 50 ppm in female rats. This level is based upon decreased body weight and body weight gain at 200 ppm. Other compound-related, toxicologically significant effects were observed at 800 and 1600 ppm. These effects include decreases in body weight, body weight gain, and food consumption and/or food efficiency; clinical and microscopic pathologic evidence of mild, regenerative, hemolytic anemia; and clinical, macroscopic, and microscopic pathologic evidence of hepatotoxicity.
Although the NOAEL for hepatic effects was the same in both sexes, male rats developed more severe hepatotoxicity than female rats exposed to comparable levels of the test substance.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- LOAEL
- 1.4 mg/kg bw/day
- Study duration:
- subchronic
- Experimental exposure time per week (hours/week):
- 168
- Species:
- dog
- Quality of whole database:
- 90-day guideline studies for were available in rat, mice and dogs. In addition a 1-year guideline study was available in dogs, a non-guideline 1-year study was available in monkeys, and a 2-year guideline study was available in rats.
- System:
- eye
- Organ:
- lens
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
- Deviations:
- no
- GLP compliance:
- yes
- Specific details on test material used for the study:
- Substance ID: Famoxadone TGAI
Lot #: ENBK-171342-006
Purity: 98.4% - Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Details on species / strain selection:
- Rats have historically been used in safety evaluation studies for inhalation toxicity testing. The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with the strain at the test facility.
- Sex:
- male/female
- Route of administration:
- inhalation: aerosol
- Type of inhalation exposure:
- nose only
- Vehicle:
- air
- Mass median aerodynamic diameter (MMAD):
- 3.4 - 4 µm
- Geometric standard deviation (GSD):
- 1.9
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Samples taken from the faceplate and the reference port of the 100 mg/m³ exposure chamber demonstrated differences that were less than 20% from the overall mean aerosol concentration; therefore, the test substance atmospheres were considered to be homogenously distributed in the breathing zones of the animals; therefore, the use of the sampling ports for air sampling was considered adequate.
- Duration of treatment / exposure:
- 4 weeks
- Frequency of treatment:
- Each group of animals was exposed for 6 hours/day, 5 days/week, for 4 consecutive weeks for a total of 20 exposures
- Dose / conc.:
- 3 mg/m³ air (nominal)
- Dose / conc.:
- 15 mg/m³ air (nominal)
- Dose / conc.:
- 100 mg/m³ air (nominal)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- There were no toxicologically significant clinical observations made during the study.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no statistically significant differences in mean body weights or mean body weight gains in this study.
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Test substance-related hematology changes included decreased red cell mass parameters (red blood cell concentration (RBC), hematocrit (HCT) and hemoglobin concentration (HGB)) in females at 100 mg/m³. Associated with these changes, females at 100 mg/m³ also had increased mean corpuscular volume (MCV), red cell distribution width (RDW), and absolute reticulocyte count (ARET), indicative of a regenerative response. Males at 100 mg/m³ also exhibited an increased RDW. Of these hematology findings, the mild decreases in red cell mass parameters in females at 100 mg/m³ were considered adverse.
Additionally, females at 100 mg/m³ also had increased absolute monocyte (AMON), absolute large unstained cell (ALUC), and secondarily, total white blood cell (WBC) concentrations. These changes were considered non-adverse, and were considered secondary to hemolysis. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Test substance-related clinical chemistry changes included increased sorbitol dehydrogenase concentration (SDH) in females at 15 and 100 mg/m³, which was considered secondary to hepatocellular injury, although microsomal enzyme induction may have contributed. Changes considered attributable to microsomal enzyme induction included minimal to mild increases in SDH, alanine aminotransferase concentration (ALT) and alkaline phosphatase concentration (ALKP) in males at 100 mg/m³ (with hepatocellular injury possibly contributing to the increased SDH and ALT), increased cholesterol concentration (CHOL) in males and females at 100 mg/m³, and possibly the increased albumin (ALB) and total protein (TP) concentrations in males and females at 100 mg/m³ (and females at 15 mg/m³). None of these changes in clinical chemistry parameters were considered adverse themselves, although the increased SDH in females at 100 mg/m³ was considered reflective of the adverse single cell necrosis observed microscopically in the liver.
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Urine volume was statistically significantly lower in males exposed to 15 mg/m³ and in females exposed to 3 or 15 mg/m³. pH was also statistically significantly lower in females exposed to 15 mg/m³. These changes were considered likely unrelated to treatment because they did not occur in an exposure-related pattern.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Test substance-related organ weight changes included increased liver weights in males and females at 100 mg/m³, and increased spleen weights in females at 100 mg/m³, compared to controls.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test substance-related gross observations. All gross observations at necropsy were interpreted to be spontaneous changes typical of background findings in rats of this age and strain.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased liver weights in both sexes were observed at 100 mg/m³ and correlated with the microscopic finding of centrilobular hepatocellular hypertrophy observed in both sexes at ≥15 mg/m³. The hypertrophy was compatible with the non-adverse induction of hepatic microsomal enzymes. Additional test substance-related microscopic findings in the liver included increased hepatocellular single cell necrosis and mitoses in both sexes at 100 mg/m³, which correlated with the increased hepatocellular enzyme concentrations. These microscopic changes were indicative of increased hepatocyte turnover, and the hepatocellular single cell necrosis was considered adverse.
Test substance-related microscopic findings in the nose consisted of minimal goblet cell hypertrophy/hyperplasia in the septum and nasopharyngeal duct of both sexes at 100 mg/m³. This finding was consistent with inhalation exposure to a slight irritant, and was considered an adaptive response and non-adverse. Test substance-related microscopic findings in hematopoietic tissue included increased splenic extramedullary hematopoiesis and increased splenic pigment in males at 100 mg/m³ and females at ≥15 mg/m³, splenic sinusoidal dilatation in both sexes at 100 mg/m³, and bone marrow erythroid hyperplasia in females at 100 mg/m³. These findings correlated with increased spleen weights in females at 100 mg/m³ compared to controls. Additionally, individual female rats at 100 mg/m³ exhibited minimal hepatic extramedullary hematopoiesis and increased Kupffer cells. All of these findings were considered secondary to test substance-related adverse effects on red cell mass parameters as described for females at 100 mg/m³. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 15 mg/m³ air (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical biochemistry
- haematology
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 15 mg/m³ air (nominal)
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Rat 4 week Inhalation NOAEL: 15 mg/m³
- Executive summary:
The objective of this study was to evaluate the potential toxicity of the test substance (according to the guideline EPA OPPTS 870.3465) when administered by inhalation as a dust aerosol to male and female (nulliparous and non-pregnant) Crl:CD(SD) albino rats for 4 weeks. Four groups of 10 male and 10 female rats were exposed nose-only for 6 hours per day for 5 days per week over a period of 28 days to design concentrations of 0 (air control), 3.0, 15, and 100 mg/m³ test substance. The chamber aerosol atmosphere was generated by suspension of the test substance in air. The test substance was metered to an air source using K-tron twin screw volumetric feeders. High pressure air, metered to the air source by Brooks mass flow controllers, carried the resulting atmosphere into the exposure chamber. Chamber aerosol concentrations of the test substance were determined by gravimetric analysis for the 15 and 100 mg/m³ chambers. The air control and 3.0 mg/m³ concentrations were determined by desorbing the gravimetric filters in acetonitrile and analyzing the test substance concentration by HPLC/DAD analysis.
Animals were observed for clinical signs of toxicity at least once per day and weighed at least once per week. The approximate amount of food consumed by each animal over a weekly weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount of spillage from the feeder during the interval from the initial weight. From these measurements, mean daily food consumption over the interval was determined.
Ophthalmology evaluations were conducted by a veterinary ophthalmologist 2 days prior to the initiation of exposures and on test day 25. The baseline examination was performed on all animals received for the study, prior to assignment to groups. Where possible, any animals with preexisting ophthalmology abnormalities were excluded from assignment to the study.
Following the final exposure, rats were fasted overnight, blood and urine samples were collected for clinical pathology evaluations, and the animals were euthanized for anatomic pathology assessment including gross examinations and histopathology. The mean determined airborne aerosol concentration by HPLC/DAD in the 0 mg/m³ (air control and the 3.0 mg/m³) exposure chambers were 0.0 ± 0.0 and 3.60 ± 1.4 mg/m³ (mean ± standard deviation of every sample collected). The 15 and 100 mg/m³ groups were exposed to gravimetrically determined mean concentrations of 16 ± 3.4 and 110 ± 19 mg/m³, respectively. The MMADs from all exposure groups ranged from 3.4 to 4.0 μm and the GSDs ranged from 1.9 to 2.6.
All animals survived to their scheduled necropsy. All rats displayed normal alerting response during all exposures. There were no toxicologically significant clinical signs or test substance related ophthalmologic findings observed in any rats in this study.
There were no statistically significant differences in mean weekly body weights, mean body weight gains, or cumulative food consumption between the air control and test substance exposed groups at the end of the exposure period. Additionally, there were no changes in coagulation or urinalysis parameters, and no gross observations at necropsy considered related to test substance exposure.
Test substance-related hematology changes included decreased red cell mass parameters (red blood cell concentration (RBC), hematocrit (HCT) and hemoglobin concentration (HGB)) in females at 100 mg/m³. Associated with these changes, females at 100 mg/m³ also had increased mean corpuscular volume (MCV), red cell distribution width (RDW), and absolute reticulocyte count (ARET), indicative of a regenerative response. Males at 100 mg/m³ also exhibited an increased RDW. In both sexes at 100 mg/m³, these changes were associated with an increased bilirubin concentration (BILI), and microscopic findings of increased splenic extramedullary hematopoiesis, pigment and sinusoidal dilatation (as well as increased bone marrow erythropoiesis in females), suggestive of hemolysis. Of these hematology findings, the mild decreases in red cell mass parameters in females at 100 mg/m³ were considered adverse.
Additionally, females at 100 mg/m³ also had increased absolute monocyte (AMON), absolute large unstained cell (ALUC), and secondarily, total white blood cell (WBC) concentrations. These changes were considered non-adverse, and were considered secondary to hemolysis. Test substance-related clinical chemistry changes included increased sorbitol dehydrogenase concentration (SDH) in females at 15 and 100 mg/m³, which was considered secondary to hepatocellular injury, although microsomal enzyme induction may have contributed. Changes considered attributable to microsomal enzyme induction included minimal to mild increases in SDH, alanine aminotransferase concentration (ALT) and alkaline phosphatase concentration (ALKP) in males at 100 mg/m³ (with hepatocellular injury possibly contributing to the increased SDH and ALT), increased cholesterol concentration (CHOL) in males and females at 100 mg/m³, and possibly the increased albumin (ALB) and total protein (TP) concentrations in males and females at 100 mg/m³ (and females at 15 mg/m³). None of these changes in clinical chemistry parameters were considered adverse themselves, although the increased SDH in females at 100 mg/m³ was considered reflective of the adverse single cell necrosis observed microscopically in the liver.
Increased liver weights in both sexes were observed at 100 mg/m³ and correlated with the microscopic finding of centrilobular hepatocellular hypertrophy observed in both sexes at ≥15 mg/m³. The hypertrophy was compatible with the non-adverse induction of hepatic microsomal enzymes. Additional test substance-related microscopic findings in the liver included increased hepatocellular single cell necrosis and mitoses in both sexes at 100 mg/m³, which correlated with the increased hepatocellular enzyme concentrations. These microscopic changes were indicative of increased hepatocyte turnover, and the hepatocellular single cell necrosis was considered adverse.
Test substance-related microscopic findings in the nose consisted of minimal goblet cell hypertrophy/hyperplasia in the septum and nasopharyngeal duct of both sexes at 100 mg/m³. This finding was consistent with inhalation exposure to a slight irritant, and was considered an adaptive response and non-adverse. Test substance-related microscopic findings in hematopoietic tissue included increased splenic extramedullary hematopoiesis and increased splenic pigment in males at 100 mg/m³ and females at ≥15 mg/m³, splenic sinusoidal dilatation in both sexes at 100 mg/m³, and bone marrow erythroid hyperplasia in females at 100 mg/m³. These findings correlated with increased spleen weights in females at 100 mg/m³ compared to controls. Additionally, individual female rats at 100 mg/m³ exhibited minimal hepatic extramedullary hematopoiesis and increased Kupffer cells. All of these findings were considered secondary to test substance-related adverse effects on red cell mass parameters as described for females at 100 mg/m³.
No test substance-related effects were observed at 3 mg/m³ in either sex.
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for the test substance is considered to be 15 mg/m³ for male and female rats based on increased single cell necrosis in the liver of both sexes at 100 mg/m³ and decreased red cell mass parameters in female rats at 100 mg/m³.
Reference
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEC
- 15 mg/m³
- Study duration:
- subacute
- Experimental exposure time per week (hours/week):
- 30
- Species:
- rat
- Quality of whole database:
- A 5-day range-finder study and a 28-day inhalation study were available in rats.
- System:
- hepatobiliary
- Organ:
- erythrocyte development
- liver
Repeated dose toxicity: inhalation - local effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
- Deviations:
- no
- GLP compliance:
- yes
- Specific details on test material used for the study:
- Substance ID: Famoxadone TGAI
Lot #: ENBK-171342-006
Purity: 98.4% - Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Details on species / strain selection:
- Rats have historically been used in safety evaluation studies for inhalation toxicity testing. The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with the strain at the test facility.
- Sex:
- male/female
- Route of administration:
- inhalation: aerosol
- Type of inhalation exposure:
- nose only
- Vehicle:
- air
- Mass median aerodynamic diameter (MMAD):
- 3.4 - 4 µm
- Geometric standard deviation (GSD):
- 1.9
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Samples taken from the faceplate and the reference port of the 100 mg/m³ exposure chamber demonstrated differences that were less than 20% from the overall mean aerosol concentration; therefore, the test substance atmospheres were considered to be homogenously distributed in the breathing zones of the animals; therefore, the use of the sampling ports for air sampling was considered adequate.
- Duration of treatment / exposure:
- 4 weeks
- Frequency of treatment:
- Each group of animals was exposed for 6 hours/day, 5 days/week, for 4 consecutive weeks for a total of 20 exposures
- Dose / conc.:
- 3 mg/m³ air (nominal)
- Dose / conc.:
- 15 mg/m³ air (nominal)
- Dose / conc.:
- 100 mg/m³ air (nominal)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- There were no toxicologically significant clinical observations made during the study.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no statistically significant differences in mean body weights or mean body weight gains in this study.
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Test substance-related hematology changes included decreased red cell mass parameters (red blood cell concentration (RBC), hematocrit (HCT) and hemoglobin concentration (HGB)) in females at 100 mg/m³. Associated with these changes, females at 100 mg/m³ also had increased mean corpuscular volume (MCV), red cell distribution width (RDW), and absolute reticulocyte count (ARET), indicative of a regenerative response. Males at 100 mg/m³ also exhibited an increased RDW. Of these hematology findings, the mild decreases in red cell mass parameters in females at 100 mg/m³ were considered adverse.
Additionally, females at 100 mg/m³ also had increased absolute monocyte (AMON), absolute large unstained cell (ALUC), and secondarily, total white blood cell (WBC) concentrations. These changes were considered non-adverse, and were considered secondary to hemolysis. - Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Test substance-related clinical chemistry changes included increased sorbitol dehydrogenase concentration (SDH) in females at 15 and 100 mg/m³, which was considered secondary to hepatocellular injury, although microsomal enzyme induction may have contributed. Changes considered attributable to microsomal enzyme induction included minimal to mild increases in SDH, alanine aminotransferase concentration (ALT) and alkaline phosphatase concentration (ALKP) in males at 100 mg/m³ (with hepatocellular injury possibly contributing to the increased SDH and ALT), increased cholesterol concentration (CHOL) in males and females at 100 mg/m³, and possibly the increased albumin (ALB) and total protein (TP) concentrations in males and females at 100 mg/m³ (and females at 15 mg/m³). None of these changes in clinical chemistry parameters were considered adverse themselves, although the increased SDH in females at 100 mg/m³ was considered reflective of the adverse single cell necrosis observed microscopically in the liver.
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Urine volume was statistically significantly lower in males exposed to 15 mg/m³ and in females exposed to 3 or 15 mg/m³. pH was also statistically significantly lower in females exposed to 15 mg/m³. These changes were considered likely unrelated to treatment because they did not occur in an exposure-related pattern.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Test substance-related organ weight changes included increased liver weights in males and females at 100 mg/m³, and increased spleen weights in females at 100 mg/m³, compared to controls.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test substance-related gross observations. All gross observations at necropsy were interpreted to be spontaneous changes typical of background findings in rats of this age and strain.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Increased liver weights in both sexes were observed at 100 mg/m³ and correlated with the microscopic finding of centrilobular hepatocellular hypertrophy observed in both sexes at ≥15 mg/m³. The hypertrophy was compatible with the non-adverse induction of hepatic microsomal enzymes. Additional test substance-related microscopic findings in the liver included increased hepatocellular single cell necrosis and mitoses in both sexes at 100 mg/m³, which correlated with the increased hepatocellular enzyme concentrations. These microscopic changes were indicative of increased hepatocyte turnover, and the hepatocellular single cell necrosis was considered adverse.
Test substance-related microscopic findings in the nose consisted of minimal goblet cell hypertrophy/hyperplasia in the septum and nasopharyngeal duct of both sexes at 100 mg/m³. This finding was consistent with inhalation exposure to a slight irritant, and was considered an adaptive response and non-adverse. Test substance-related microscopic findings in hematopoietic tissue included increased splenic extramedullary hematopoiesis and increased splenic pigment in males at 100 mg/m³ and females at ≥15 mg/m³, splenic sinusoidal dilatation in both sexes at 100 mg/m³, and bone marrow erythroid hyperplasia in females at 100 mg/m³. These findings correlated with increased spleen weights in females at 100 mg/m³ compared to controls. Additionally, individual female rats at 100 mg/m³ exhibited minimal hepatic extramedullary hematopoiesis and increased Kupffer cells. All of these findings were considered secondary to test substance-related adverse effects on red cell mass parameters as described for females at 100 mg/m³. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 15 mg/m³ air (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical biochemistry
- haematology
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 15 mg/m³ air (nominal)
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Rat 4 week Inhalation NOAEL: 15 mg/m³
- Executive summary:
The objective of this study was to evaluate the potential toxicity of the test substance (according to the guideline EPA OPPTS 870.3465) when administered by inhalation as a dust aerosol to male and female (nulliparous and non-pregnant) Crl:CD(SD) albino rats for 4 weeks. Four groups of 10 male and 10 female rats were exposed nose-only for 6 hours per day for 5 days per week over a period of 28 days to design concentrations of 0 (air control), 3.0, 15, and 100 mg/m³ test substance. The chamber aerosol atmosphere was generated by suspension of the test substance in air. The test substance was metered to an air source using K-tron twin screw volumetric feeders. High pressure air, metered to the air source by Brooks mass flow controllers, carried the resulting atmosphere into the exposure chamber. Chamber aerosol concentrations of the test substance were determined by gravimetric analysis for the 15 and 100 mg/m³ chambers. The air control and 3.0 mg/m³ concentrations were determined by desorbing the gravimetric filters in acetonitrile and analyzing the test substance concentration by HPLC/DAD analysis.
Animals were observed for clinical signs of toxicity at least once per day and weighed at least once per week. The approximate amount of food consumed by each animal over a weekly weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount of spillage from the feeder during the interval from the initial weight. From these measurements, mean daily food consumption over the interval was determined.
Ophthalmology evaluations were conducted by a veterinary ophthalmologist 2 days prior to the initiation of exposures and on test day 25. The baseline examination was performed on all animals received for the study, prior to assignment to groups. Where possible, any animals with preexisting ophthalmology abnormalities were excluded from assignment to the study.
Following the final exposure, rats were fasted overnight, blood and urine samples were collected for clinical pathology evaluations, and the animals were euthanized for anatomic pathology assessment including gross examinations and histopathology. The mean determined airborne aerosol concentration by HPLC/DAD in the 0 mg/m³ (air control and the 3.0 mg/m³) exposure chambers were 0.0 ± 0.0 and 3.60 ± 1.4 mg/m³ (mean ± standard deviation of every sample collected). The 15 and 100 mg/m³ groups were exposed to gravimetrically determined mean concentrations of 16 ± 3.4 and 110 ± 19 mg/m³, respectively. The MMADs from all exposure groups ranged from 3.4 to 4.0 μm and the GSDs ranged from 1.9 to 2.6.
All animals survived to their scheduled necropsy. All rats displayed normal alerting response during all exposures. There were no toxicologically significant clinical signs or test substance related ophthalmologic findings observed in any rats in this study.
There were no statistically significant differences in mean weekly body weights, mean body weight gains, or cumulative food consumption between the air control and test substance exposed groups at the end of the exposure period. Additionally, there were no changes in coagulation or urinalysis parameters, and no gross observations at necropsy considered related to test substance exposure.
Test substance-related hematology changes included decreased red cell mass parameters (red blood cell concentration (RBC), hematocrit (HCT) and hemoglobin concentration (HGB)) in females at 100 mg/m³. Associated with these changes, females at 100 mg/m³ also had increased mean corpuscular volume (MCV), red cell distribution width (RDW), and absolute reticulocyte count (ARET), indicative of a regenerative response. Males at 100 mg/m³ also exhibited an increased RDW. In both sexes at 100 mg/m³, these changes were associated with an increased bilirubin concentration (BILI), and microscopic findings of increased splenic extramedullary hematopoiesis, pigment and sinusoidal dilatation (as well as increased bone marrow erythropoiesis in females), suggestive of hemolysis. Of these hematology findings, the mild decreases in red cell mass parameters in females at 100 mg/m³ were considered adverse.
Additionally, females at 100 mg/m³ also had increased absolute monocyte (AMON), absolute large unstained cell (ALUC), and secondarily, total white blood cell (WBC) concentrations. These changes were considered non-adverse, and were considered secondary to hemolysis. Test substance-related clinical chemistry changes included increased sorbitol dehydrogenase concentration (SDH) in females at 15 and 100 mg/m³, which was considered secondary to hepatocellular injury, although microsomal enzyme induction may have contributed. Changes considered attributable to microsomal enzyme induction included minimal to mild increases in SDH, alanine aminotransferase concentration (ALT) and alkaline phosphatase concentration (ALKP) in males at 100 mg/m³ (with hepatocellular injury possibly contributing to the increased SDH and ALT), increased cholesterol concentration (CHOL) in males and females at 100 mg/m³, and possibly the increased albumin (ALB) and total protein (TP) concentrations in males and females at 100 mg/m³ (and females at 15 mg/m³). None of these changes in clinical chemistry parameters were considered adverse themselves, although the increased SDH in females at 100 mg/m³ was considered reflective of the adverse single cell necrosis observed microscopically in the liver.
Increased liver weights in both sexes were observed at 100 mg/m³ and correlated with the microscopic finding of centrilobular hepatocellular hypertrophy observed in both sexes at ≥15 mg/m³. The hypertrophy was compatible with the non-adverse induction of hepatic microsomal enzymes. Additional test substance-related microscopic findings in the liver included increased hepatocellular single cell necrosis and mitoses in both sexes at 100 mg/m³, which correlated with the increased hepatocellular enzyme concentrations. These microscopic changes were indicative of increased hepatocyte turnover, and the hepatocellular single cell necrosis was considered adverse.
Test substance-related microscopic findings in the nose consisted of minimal goblet cell hypertrophy/hyperplasia in the septum and nasopharyngeal duct of both sexes at 100 mg/m³. This finding was consistent with inhalation exposure to a slight irritant, and was considered an adaptive response and non-adverse. Test substance-related microscopic findings in hematopoietic tissue included increased splenic extramedullary hematopoiesis and increased splenic pigment in males at 100 mg/m³ and females at ≥15 mg/m³, splenic sinusoidal dilatation in both sexes at 100 mg/m³, and bone marrow erythroid hyperplasia in females at 100 mg/m³. These findings correlated with increased spleen weights in females at 100 mg/m³ compared to controls. Additionally, individual female rats at 100 mg/m³ exhibited minimal hepatic extramedullary hematopoiesis and increased Kupffer cells. All of these findings were considered secondary to test substance-related adverse effects on red cell mass parameters as described for females at 100 mg/m³.
No test substance-related effects were observed at 3 mg/m³ in either sex.
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for the test substance is considered to be 15 mg/m³ for male and female rats based on increased single cell necrosis in the liver of both sexes at 100 mg/m³ and decreased red cell mass parameters in female rats at 100 mg/m³.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
Repeated dose toxicity: dermal - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: MAFF Japan 59 NohSan No, 4200 Testing Guidelines for Toxicology Studies (1985)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- Substance ID: Famoxadone Technical (DPX-JE874)
Lot #: JE874-221
Purity: 97.28% - Species:
- rat
- Strain:
- other: Crl:CD®(SD)IGS BR
- Details on species / strain selection:
- Rats are considered an acceptable species suited for conducting dermal absorption studies. Their use is designated as appropriate according to the testing guidelines for toxicological evaluation of pesticides by the dermal route of exposure. The Crl:CD®(SD)IGS BR rat was selected because extensive background information is available from the literature, the supplier, and previous studies conducted at the test facility. This strain is also considered suitable relative to hardiness and low incidence of spontaneous disease.
- Sex:
- male/female
- Type of coverage:
- semiocclusive
- Vehicle:
- water
- Remarks:
- deionized
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- approximately 6 hr
- Frequency of treatment:
- daily for 28 consecutive days
- Dose / conc.:
- 250 mg/kg bw/day
- Dose / conc.:
- 500 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test substance-related clinical observations in either males or females at any dosage during the clinical signs inventory conducted at the time of body weight determination or prior to the daily treatment. In addition, there were no test substance-related clinical signs of toxicity observed following the daily applications in males or females at any dosage. There was an increase, albeit not significant, in the incidence of desquamation and neck sores in the 500 and 1000 mg/kg/day male dose groups, and epidermal scaling in the 250, 500, and 1000 mg/kg/day female dose groups. The desquamation observed in males was not dose-responsive in nature, occurred with similar frequency among all female dose groups, and therefore was not considered to be compound related. The neck sores in the 500 and 1000 mg/kg/day male dose groups were attributed to the use of plastic collars and were not considered compound related. The epidermal scaling in females was not dose-responsive in nature, did not occur in the males, and therefore was not considered biologically significant. A variety of other dermal changes was noted following both application of either the vehicle control or the test substance including erythema, scratches, scabs, and sores; however, these occurred with similar frequency among all groups.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no adverse, test substance-related effects on body weight or body weight gain at any dosage in either males or females. Body weight gain of males in the 250 mg/kg/day group was statistically significantly lower for test days 1-4. The lower weight gain value for the 250 mg/kg/day males was not consistent with a dose-response relationship and therefore, is not considered to be compound related. For test days 21-24, a loss in mean body weight for all of the male dose groups was observed. The reason(s) for these decreases is not known, but is not considered to be compound related because all dose groups, including the control, were affected.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- no effects observed
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistically significant, but very slight, decreases in red blood cell count (RBC) and hemoglobin (Hb) were present in female rats in the 500 and 1000 mg/kg/day groups. These changes may indicate compound related increased red blood cell turnover (hemolysis), as mild hemolysis was produced in previous studies with the test substance. However, the decreases in RBC and Hb were very small (6-7% decreases relative to controls) and were not of sufficient magnitude to elicit a bone marrow regenerative response (based on the absence of a reticulocytosis in the affected groups). In addition, the decreases were not associated with statistically significant changes in hematocrit (Ht) or with histological evidence of increased red cell turnover. Based on these considerations, the slight changes in RBC and Hb in 500 and 1000 mg/kg/day females, though possibly compound related, were considered not to be biologically adverse.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Statistically significant increases in alkaline phosphatase (ALP), alanine aminotransferase (ALT), and sorbitol dehydrogenase (SDH) were present in the 500 and 1000 mg/kg/day male groups. The biological significance of these liver enzyme changes is equivocal. In all cases, increases in enzyme activities were small (less than two-fold relative to controls), the increases were not associated with definitive microscopic evidence of hepatotoxicity, and no effects on liver enzymes occurred in female rats.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Liver weight parameters were increased in all treated male and female groups. Means for liver weight relative to body weight in male rats were increased 10%, 12%, and 21% relative to controls for the 250, 500, and 1000 mg/kg/day groups, respectively; in females, increases were11%, 14%, and 9%, respectively. Liver weight increases were statistically significant for all parameters in 1000 mg/kg/day males and for the liver:body weight ratio in 500 mg/kg/day males. In the 500 and 1000 mg/kg/day males and females, these liver weight increases were associated with minimal hepatocellular hypertrophy microscopically.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test substance-related gross observations. Gross observations occurred in low incidences across groups and were typical of spontaneous lesions seen in rats of this strain and age.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Compound-related microscopic changes were limited to the livers of male and female rats in the 500 and 1000 mg/kg groups. The principal change was minimal hypertrophy of centrilobular hepatocytes, which was consistent with the increase in liver weights noted above. In the 500 and 1000 mg/kg/day male groups, hypertrophy was associated with low incidences of slight apoptosis in the liver. Apoptosis has been reported to occur following administration of compounds that induce liver enzymes, and may occur as a controlled event to eliminate excess cells rather than as a result of primary cytotoxicity. Such is likely the case for the apoptosis observed in conjunction with hypertrophy in the present study, as no overt microscopic evidence of hepatotoxicity, such as zonal or locally extensive necrosis, was present. At any rate, the low incidence and subtle nature of the apoptosis observed in the affected groups likely does not account for the changes in liver enzymes noted in these groups.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- clinical biochemistry
- histopathology: non-neoplastic
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- haematology
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 500 mg/kg bw/day
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Dermal 28 day NOAEL (Male rat): 250 mg/kg/day
Dermal 28 day NOAEL (Female rat): 1000 mg/kg/day (highest dose tested) - Executive summary:
The objective of this study was to determine the potential toxicity from repeated dermal exposure to the test substance according to the guidelines OECD 410 and EPA 82-2. The test substance was applied to the shaved, intact skin of male and female Crl:CD®(SD)IGS BR rats for 28 consecutive days. Three groups of 10 male and 10 female rats were treated dermally with 250, 500, or 1000 mg/kg/day. A control group of 10 male and 10 female rats was similarly treated with deionized water. Body weights were measured at 3-4 day intervals, and clinical signs were recorded at least once daily. Food consumption was determined weekly. Blood samples were collected prior to sacrifice on test day 29. All rats underwent necropsy for gross and microscopic pathological evaluation.
There were no test substance-related effects on body weight, food consumption, clinical signs of toxicity, or mortality at any dosage in either males or females. There were no test substance related effects on hematology in males at any dosage. Females in the 500 and 1000 mg/kg/day groups had slight decreases in red blood cell count and hemoglobin. In the absence of other hematologic effects, these very small changes were not considered to be biologically significant. There were no test substance-related effects on clinical chemistry parameters in females at any dosage. Statistically significant increases in alkaline phosphatase, alanine aminotransferase, and sorbitol dehydrogenase were present in the 500 and 1000 mg/kg/day male groups. These increases in the aforementioned enzymes were considered to be indicative of minimal hepatocellular toxicity.
Liver weight parameters were increased in all treated male and female groups. Males in the 1000 mg/kg/day group had statistically significant increases in absolute and relative liver weights (relative to body weight and brain weight), while males in the 500 mg/kg/day group had statistically significant increases in relative liver weights (relative to body weight). In the 500 and 1000 mg/kg/day males and females, the liver weight increases were associated with minimal hepatocellular hypertrophy microscopically. Additionally, in the 500 and 1000 mg/kg/day male groups, hypertrophy was associated with low incidences of slight apoptosis in the liver. These changes were considered adaptive, physiologic responses of the liver to administration of the test substance.
The NOAEL for this study was 250 mg/kg/day in males based on the slight increases in liver enzymes suggestive of minimal hepatotoxicity. The NOAEL in females was 1000 mg/kg/day, the highest dose tested.
Reference
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 250 mg/kg bw/day
- Study duration:
- subacute
- Experimental exposure time per week (hours/week):
- 42
- Species:
- rat
- Quality of whole database:
- One 28-day dermal study was available in rats.
- System:
- hepatobiliary
- Organ:
- liver
Repeated dose toxicity: dermal - local effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: MAFF Japan 59 NohSan No, 4200 Testing Guidelines for Toxicology Studies (1985)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- Substance ID: Famoxadone Technical (DPX-JE874)
Lot #: JE874-221
Purity: 97.28% - Species:
- rat
- Strain:
- other: Crl:CD®(SD)IGS BR
- Details on species / strain selection:
- Rats are considered an acceptable species suited for conducting dermal absorption studies. Their use is designated as appropriate according to the testing guidelines for toxicological evaluation of pesticides by the dermal route of exposure. The Crl:CD®(SD)IGS BR rat was selected because extensive background information is available from the literature, the supplier, and previous studies conducted at the test facility. This strain is also considered suitable relative to hardiness and low incidence of spontaneous disease.
- Sex:
- male/female
- Type of coverage:
- semiocclusive
- Vehicle:
- water
- Remarks:
- deionized
- Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- approximately 6 hr
- Frequency of treatment:
- daily for 28 consecutive days
- Dose / conc.:
- 250 mg/kg bw/day
- Dose / conc.:
- 500 mg/kg bw/day
- Dose / conc.:
- 1 000 mg/kg bw/day
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test substance-related clinical observations in either males or females at any dosage during the clinical signs inventory conducted at the time of body weight determination or prior to the daily treatment. In addition, there were no test substance-related clinical signs of toxicity observed following the daily applications in males or females at any dosage. There was an increase, albeit not significant, in the incidence of desquamation and neck sores in the 500 and 1000 mg/kg/day male dose groups, and epidermal scaling in the 250, 500, and 1000 mg/kg/day female dose groups. The desquamation observed in males was not dose-responsive in nature, occurred with similar frequency among all female dose groups, and therefore was not considered to be compound related. The neck sores in the 500 and 1000 mg/kg/day male dose groups were attributed to the use of plastic collars and were not considered compound related. The epidermal scaling in females was not dose-responsive in nature, did not occur in the males, and therefore was not considered biologically significant. A variety of other dermal changes was noted following both application of either the vehicle control or the test substance including erythema, scratches, scabs, and sores; however, these occurred with similar frequency among all groups.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no adverse, test substance-related effects on body weight or body weight gain at any dosage in either males or females. Body weight gain of males in the 250 mg/kg/day group was statistically significantly lower for test days 1-4. The lower weight gain value for the 250 mg/kg/day males was not consistent with a dose-response relationship and therefore, is not considered to be compound related. For test days 21-24, a loss in mean body weight for all of the male dose groups was observed. The reason(s) for these decreases is not known, but is not considered to be compound related because all dose groups, including the control, were affected.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- no effects observed
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistically significant, but very slight, decreases in red blood cell count (RBC) and hemoglobin (Hb) were present in female rats in the 500 and 1000 mg/kg/day groups. These changes may indicate compound related increased red blood cell turnover (hemolysis), as mild hemolysis was produced in previous studies with the test substance. However, the decreases in RBC and Hb were very small (6-7% decreases relative to controls) and were not of sufficient magnitude to elicit a bone marrow regenerative response (based on the absence of a reticulocytosis in the affected groups). In addition, the decreases were not associated with statistically significant changes in hematocrit (Ht) or with histological evidence of increased red cell turnover. Based on these considerations, the slight changes in RBC and Hb in 500 and 1000 mg/kg/day females, though possibly compound related, were considered not to be biologically adverse.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Statistically significant increases in alkaline phosphatase (ALP), alanine aminotransferase (ALT), and sorbitol dehydrogenase (SDH) were present in the 500 and 1000 mg/kg/day male groups. The biological significance of these liver enzyme changes is equivocal. In all cases, increases in enzyme activities were small (less than two-fold relative to controls), the increases were not associated with definitive microscopic evidence of hepatotoxicity, and no effects on liver enzymes occurred in female rats.
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Liver weight parameters were increased in all treated male and female groups. Means for liver weight relative to body weight in male rats were increased 10%, 12%, and 21% relative to controls for the 250, 500, and 1000 mg/kg/day groups, respectively; in females, increases were11%, 14%, and 9%, respectively. Liver weight increases were statistically significant for all parameters in 1000 mg/kg/day males and for the liver:body weight ratio in 500 mg/kg/day males. In the 500 and 1000 mg/kg/day males and females, these liver weight increases were associated with minimal hepatocellular hypertrophy microscopically.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no test substance-related gross observations. Gross observations occurred in low incidences across groups and were typical of spontaneous lesions seen in rats of this strain and age.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Compound-related microscopic changes were limited to the livers of male and female rats in the 500 and 1000 mg/kg groups. The principal change was minimal hypertrophy of centrilobular hepatocytes, which was consistent with the increase in liver weights noted above. In the 500 and 1000 mg/kg/day male groups, hypertrophy was associated with low incidences of slight apoptosis in the liver. Apoptosis has been reported to occur following administration of compounds that induce liver enzymes, and may occur as a controlled event to eliminate excess cells rather than as a result of primary cytotoxicity. Such is likely the case for the apoptosis observed in conjunction with hypertrophy in the present study, as no overt microscopic evidence of hepatotoxicity, such as zonal or locally extensive necrosis, was present. At any rate, the low incidence and subtle nature of the apoptosis observed in the affected groups likely does not account for the changes in liver enzymes noted in these groups.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- clinical biochemistry
- histopathology: non-neoplastic
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- haematology
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 500 mg/kg bw/day
- System:
- hepatobiliary
- Organ:
- liver
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Dermal 28 day NOAEL (Male rat): 250 mg/kg/day
Dermal 28 day NOAEL (Female rat): 1000 mg/kg/day (highest dose tested) - Executive summary:
The objective of this study was to determine the potential toxicity from repeated dermal exposure to the test substance according to the guidelines OECD 410 and EPA 82-2. The test substance was applied to the shaved, intact skin of male and female Crl:CD®(SD)IGS BR rats for 28 consecutive days. Three groups of 10 male and 10 female rats were treated dermally with 250, 500, or 1000 mg/kg/day. A control group of 10 male and 10 female rats was similarly treated with deionized water. Body weights were measured at 3-4 day intervals, and clinical signs were recorded at least once daily. Food consumption was determined weekly. Blood samples were collected prior to sacrifice on test day 29. All rats underwent necropsy for gross and microscopic pathological evaluation.
There were no test substance-related effects on body weight, food consumption, clinical signs of toxicity, or mortality at any dosage in either males or females. There were no test substance related effects on hematology in males at any dosage. Females in the 500 and 1000 mg/kg/day groups had slight decreases in red blood cell count and hemoglobin. In the absence of other hematologic effects, these very small changes were not considered to be biologically significant. There were no test substance-related effects on clinical chemistry parameters in females at any dosage. Statistically significant increases in alkaline phosphatase, alanine aminotransferase, and sorbitol dehydrogenase were present in the 500 and 1000 mg/kg/day male groups. These increases in the aforementioned enzymes were considered to be indicative of minimal hepatocellular toxicity.
Liver weight parameters were increased in all treated male and female groups. Males in the 1000 mg/kg/day group had statistically significant increases in absolute and relative liver weights (relative to body weight and brain weight), while males in the 500 mg/kg/day group had statistically significant increases in relative liver weights (relative to body weight). In the 500 and 1000 mg/kg/day males and females, the liver weight increases were associated with minimal hepatocellular hypertrophy microscopically. Additionally, in the 500 and 1000 mg/kg/day male groups, hypertrophy was associated with low incidences of slight apoptosis in the liver. These changes were considered adaptive, physiologic responses of the liver to administration of the test substance.
The NOAEL for this study was 250 mg/kg/day in males based on the slight increases in liver enzymes suggestive of minimal hepatotoxicity. The NOAEL in females was 1000 mg/kg/day, the highest dose tested.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
Additional information
Short-term (14-90 days) feeding studies in rats and mice revealed evidence of weight loss, mild hepatotoxicity, and mild regenerative anaemia at toxic doses. Reversibility of the haematology effects were demonstrated in rats where partial reversal was noted within 9 days and full reversal within 23 days of exposure to control diet. The lowest NOAEL (taking dose spacing into consideration) observed in short-term rodent studies was 50 ppm (3.34 and 4.24 mg/kg bw/day) in males and females from the 90-day study in rats based on decreased body weight, mild haemolytic anaemia, and clinical chemistry and microscopic changes indicative of hepatocellular injury at 800 ppm and haematological effects at 200 ppm.
In dogs, weight loss and mild regenerative anaemia at toxic doses were observed following 90 days of dietary exposure; however, hepatotoxicity was not observed. Hyperkalemia and muscle twitches were also observed at the high dose level (1000/600 ppm). In addition, compound-related lens opacities were observed in males and females at 300 (10.0 and 10.1 mg/kg bw/day, respectively) and 1000/600 pm (23.8-21.1 and 23.3-20.1 mg/kg bw/day, respectively). A minimal microscopic lens lesion in an individual low dose (40 ppm, 1.4 mg/kg bw/day) female dog made this a possible lowest observed effect level (LOAEL). In males, 40 ppm (1.3 mg/kg bw/day was the NOAEL). The NOAELs for the 90-day feeding study in dogs, excluding lens effects, were 300 ppm in both males and females (10.0 and 10.1 mg/kg bw/day, respectively).
The 1-year feeding study in dogs, with dietary levels of 0, 20, 40, and 300 ppm, produced lens opacities only at the 300 ppm level in both sexes (10.1 and 9.9 mg/kg bw/day, respectively). The 40 ppm males and females (1.2 mg/kg bw/day in both sexes) had no compound-related effects. In this study, an additional 300 ppm recovery group demonstrated that the lens opacities did not progress, or completely regress, when dogs were returned to basal diet after 3 months exposure at the high dose. The NOAELs for the 1-year feeding study in dogs, excluding lens effects, were 300 ppm in both males and females (8.8 and 9.3 mg/kg bw/day, respectively).
A 1-year gavage study in cynomolgus monkeys, conducted at dose levels of 0, 1, 100, 1000 mg/kg bw/day, showed no clinical or microscopic evidence of lens opacities nor was there any evidence of hepatotoxicity. Mild haemolytic anaemia was noted in both sexes at the high dose. The NOAEL was 100 mg/kg bw/day in both male and female primates. These results indicate that primates either do not develop cataracts induced by famoxadone or that they are significantly less sensitive than dogs.
The NOAEL in a 28-day dermal study in rats was 250 mg/kg bw/day in males and females, based on slight increases in liver serum enzymes suggestive of minimal hepatotoxicity observed in males exposed to 500 and 1000 mg/kg bw/day and increased cholesterol and hepatocellular hypertrophy.
The NOAEL in a 28-day inhalation study in rats was 15 mg/m³ for male and female rats based on increased single cell necrosis in the liver of both sexes at 100 mg/m³ and decreased red cell mass parameters in female rats at 100 mg/m³.
Justification for classification or non-classification
Significant ocular lesions were seen in male and female dogs from a dose of 10 mg/kg bw/d in the 90-day study. Although these were observed just at the guidance value for STOT-RE Category 1, given the uncertainties about their relevance to humans (not seen in rats, mice, and monkeys) and considering that the guidance values are not strict cut-off figures, classification with STOT-RE Category 2 (H373) remains justified.
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