3-anilino-5-methyl-5-(4-phenoxyphenyl)-1,3-oxazolidine-2,4-dione;famoxadone (ISO)

Repeated dose toxicity: inhalation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5-methyl-5-(4-phenoxyphenyl)-3-(phenylamino)-1,3-oxazolidine-2,4-dione

EC Number:

603-520-1

Cas Number:

131807-57-3

Molecular formula:

C22H18N2O4

IUPAC Name:

5-methyl-5-(4-phenoxyphenyl)-3-(phenylamino)-1,3-oxazolidine-2,4-dione

Test material form:

solid

Specific details on test material used for the study:

Substance ID: Famoxadone TGAI
Lot #: ENBK-171342-006
Purity: 98.4%

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

3 mg/m³ air (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/m³ air (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/m³ air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

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

Applicant's summary and conclusion

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.