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Repeated dose toxicity: oral

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sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Not specified in report
according to
other: Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food (FDA, PB83-170696, 1982).
Not specified in report
according to
other: Japanese Toxicity Test Guideline of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, February 15, 1984
Not specified in report
Principles of method if other than guideline:
Not applicable
GLP compliance:
Limit test:

Test material

Details on test material:
Received: July 7, 1989
Reference: 10820-113-1
Appearance: colorless liquid at 28C and a waxy solid at 22C

Test animals

Details on test animals and environmental conditions:
Animals, Housing, and Diet: Sprague-Dawley rats (Charles River Breeding Laboratories, Inc., Portage, MI) were used for this study. The rats were
received August 16, 1989 and weighed 51-118 g at approximately 4 weeks of age. The rats were individually housed in stainless steel cages measuring 24.0 x 17.8 x 17.6 cm during the quarantine, feeding and recovery periods. The cages were suspended over excrement pans fitted with deotized cage boards (Shepherd Specialty Papers, Kalamazoo, MI). Each rat was identified by means of a permanent metal tag inserted through the pinna of the right ear, bearing a number unique within the animal room. A card containing the study number, test article number, animal number, and group was also attached to each cage.

Air conditioned animal rooms were maintained at approximately 72F (66-77F) and 40% (26-78%) relative humidity. Fluorescent lighting was
provided for 12 hours followed by 12 hours of darkness.

Untreated or appropriate test article-treated Certified Ground Purina Rodent Chow 5002 (Ralston Purina Co., St. Louis, MO) and reverse osmosispurified water, supplied by an automatic watering system, were provided ad libitum.

Administration / exposure

Route of administration:
oral: feed
unchanged (no vehicle)
Details on oral exposure:
The test article was administered orally admixed to the diet (pulverized) at the levels indicated previously. Aliquots of the test article were warmed slightly (approximately 28C) to convert it to a liquid immediately prior to mixing it with the diet. All three levels of test diet were obtained by first preparing a premix (10,000 ppm) and then diluting aliquots of the premix with control feed to achieve the desired concentration of test diet. The premix and all diets were mixed using a 16-quart (11 kg) capacity Patterson-Kelly V-blender containing a high speed intensifier bar. Diets were stored frozen (-20C) in sealed plastic bags prior to and during use.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Prior to initiation of the study, diets were prepared in order to assess the homogeneity of the mixed diets and the stability of the test article in the diet. Batches of test diet at each of the dietary concentrations were mixed. Samples were taken from the left side, right side and bottom of the V-blender and analyzed for the concentration of Diphenyl Ether.
Additional samples from each dietary level were stored at room temperature and under freezing conditions (approximately -20°C) for 14 days and analyzed after 7 and 14 days to verify the stability of the test article in the diet. Test diets were mixed weekly during the study and fresh diet provided to the animals on a weekly basis. Samples were obtained from each dietary level, as well as the control diet, at weeks 1, 3, 5, 7, 9, 11 and 13 and analyzed for test article concentration.

The method of analysis for Diphenyl Ether (TA625) in Purina Rodent Chow 5002 was developed in house by Paula Ignat, M.S., Associate Toxicologist.

An accurately weighed diet sample (nearest 0.1 mg) was placed in a 100 ml round bottom flask to which 35 ml of methanol was added. In order for the concentration of analyte (i.e., Diphenyl Ether) extracted from a sample to remain within the limits of the calibration curve for the analysis, the weight of sample extracted was dependent upon the expected concentration of the diet be., 4.00 g for the 200 ppm diet, 2.00 g for the 1,000 ppm diet, and 1.00 g for the 5,000 ppm diet and the 10,000 ppm premix). A condenser was attached to the flask and the diet was extracted by refluxing for 30 minutes. The flask was allowed to cool to room temperature and the extract was filtered through a Whatman No. 41 filter paper into a 50 ml volumetric flask. The round bottom flask was washed 2-3 times with 3-5 ml aliquots of methanol and the washings filtered. The sample was diluted with methanol to the final volume. Prior to HPLC analysis, a small amount of the sample was filtered a second time through a Nylon membrane syringe filter (0.45 um exclusion size, 13 mm diameter).

The samples were analyzed using a Varian 5560 High Performance Liquid Chromatograph (HPLC) with a UV200 detector, a Varian 402 Data Station and a Varian Star 9095 Autosampler, which were calibrated against a standard calibration curve covering the range of concentrations expected in the sample analyses. The HPLC conditions were as follows:
Column: Vydac, C18, 201TP, 4.6 mm diameter x 250 mm length, 5 um particle size
Mobile Phase: Methano1:Water (70:30)
Injection Volume: 10 ul sample loop, filled with 2.5 times volume (i.e., 25 ul injection)
Flow: 1.5 ml/min
Retention Time: approximately 5.2 minutes
Zero Offset: 5%
Chart Speed: 0.5 cm/min.
Stop Time: 6.5 minutes
Plot Attenuation: 32
Detector: Varian W 200 at 277 nm
Time Constant: 0.5 seconds
Absorbance Range: 0.05 au/mv
The analytical method employed was validated by the range of results obtained in determining the linearity, precision and recovery of the analyte through the method. The homogeneity of the diet mixtures and the stability of each analyte level in the diet were also determined. During the study, the diet concentrations of Diphenyl Ether were determined on a bi-weekly basis beginning with Week 1.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 200, 1000 or 5000 ppm
nominal in diet
Doses / Concentrations:
Males- 0, 11.7, 60.7 and 301.1 mg/kg/day, Females- 0, 14.5, 73.9 and 334.8 mg/kg/day
actual ingested
No. of animals per sex per dose:
10/sex/dose (main study) and 10/sex/dose (recovery groups at control and all treatment groups)
Control animals:
yes, plain diet
Details on study design:
Four groups of rats (20/sex, 160 total) were randomly selected from a pool of rats whose body weights did not deviate more than three standard deviation units from the population mean. Three of the groups were exposed to graded concentrations of the test article in the diet for 13 weeks, the fourth group (20 rats/sex) was administered untreated rodent chow and served as a control group. Ten rats/sex/group were designated as recovery rats, which were retained for 4 weeks after the 13-week feeding period. All recovery rats received untreated rodent chow during the recovery period. The other 10 rats/sex, not designated as recovery rats, were sacrificed and necropsied approximately 18 hours after the termination of the 13-week feeding period.

To facilitate necropsy, the four study groups were divided into two series (I and II) which were exposed to the appropriate control or test diet on
successive days; each series contained 10 rats/sedgroup. Animals were assigned to groups using a constrained random process.The study was initiated on August 30 (Series I) or 31 (Series II, 1989 and the final day of feeding was November 28 (Series I) or 29 (Series II), 1989. The non-recovery rats were sacrificed approximately 18 hours following the final treatment day for each series. The recovery rats were sacrificed 4 weeks following the final treatment day (i.e., December 28 and 29, 1989).
Positive control:


Observations and examinations performed and frequency:
Mortality/Morbidity Observations: Rats were observed once daily for morbidity and mortality during the 2-week quarantine period. Following initiation of feeding, all rats were observed twice daily on weekdays and once daily on weekends and holidays.

Physical Examinations and Clinical Observations: Physical examinations were performed on each animal once prior to study initiation to ensure suitability for use as a test animal and observations for adverse clinical symptoms were made daily during the 13-week feeding period. Recovery rats were examined daily during the recovery period.

Body Weights: Body weights were measured prior to initiation of the study, weekly during the feeding period and at the termination of the study immediately prior to sacrifice (fasted weight). Recovery rats were weighed weekly during the recovery period and immediately prior to sacrifice (fasted weight). The fasted body weights were used to calculate organ-to-body weight ratios.

Food Consumption: Individual animal food consumption was measured weekly during the study, including the recovery period. Food conversion
ratios were calculated weekly from body weight and food consumption data [Food Conversion = Body Weight Gain (g)/Food Consumption (g)].
Sacrifice and pathology:
Clinical Laboratory Procedures: At their scheduled time of sacrifice, the rats were fasted for approximately 16-20 hours prior to necropsy and anesthetized with sodium pentobarbital. Blood samples were obtained from the abdominal aorta for the following serum chemistry analyses: glucose
(GLU), creatime kinase (CK), alanine aminotransferase (ALT/SGPT), aspartate aminotransferase (AST/SGOT), alkaline phosphatase (ALKP),
gamma glutamyl transpeptidase (GGT), urea nitrogen (BUN), creatinine (CREA), sodium (Na), potassium (K), calcium (Ca), chloride (Cl), phosphorus (PHOS), total protein (TPRO), albumin (ALB), total bilirubin (TBIL) and cholesterol (CHOL). Hematological parameters measured consisted of total erythrocyte count (RBC), hemoglobin (HGB), mean corpuscular volume (MCV), total leukocyte count (WBC), differential leukocyte count and platelet count (PLAT). The following values were calculated from the data. globulin (GLOB), albumin/globulin ratio (A/G ratio), hematocrit (HCT), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC).

It was concluded that one female from each of the mid- and high-dose
groups (non-recovery) had died prior to the blood sample collection; therefore, the data from these two rats were subsequently omitted from all
summaries and comparisons.

Urine analysis was performed on 10 rats/sex/group at the end of the 13-week feeding period. Urine specimens were obtained from fasted
animals overnight (approximately 16 hours) when they were housed in stainless steel metabolic cages. The following parameters were measured or evaluated: appearance (color), volume, specific gravity, occult blood, protein, pH, ketones, urobilinogen, glucose, bilirubin and microscopic
examination of sediment.

Postmortem Examination Procedures: Necropsies were performed on all rats and the following tissues were collected and fixed in 10% neutral
buffered formalin: adrenals, brain, epididymides, eyes, esophagus, femur and bone marrow (smear), gonads, heart, duodenum, jejunum, ileum, cecum, colon, rectum, kidneys, liver, lungs, mesenteric lymph node, mammary gland, nasal turbinates, exorbital lachrymal glands, Harderian glands,
pancreas, parathyroids, aorta, pituitary, prostate and seminal vesicles, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord, spleen,
sternum (bone marrow), stomach, thymus, thyroid, tongue, trachea, urinary bladder, uterus, vagina, gross lesions and ear with attached tag. The
adrenal glands, brain, gonads, heart, kidneys, liver and spleen were weighed at necropsy and the organ weights relative to body weights were calculated. Only one adrenal gland was recovered from a low dose recovery group male, which was subsequently omitted from all organ weight summaries and comparisons.

Microscopic examination was performed on the complete set of collected tissues (with the exception of the femur, ear, nasal turbinates,
exorbital lachyrmal glands and Harderian glands) from the control and high dose (5000 ppm) animals sacrificed at the end of the 13-week feeding
period. In addition, the lungs, liver, kidneys and gross lesions from the low and medium dose animals sacrificed after 13 weeks were also examined
microscopically. The remaining tissues from the low and medium dose rats and tissues from the recovery animals were collected but were not
processed and examined microscopically.
Other examinations:
Ophthalmic Examination: Ocular examinations were performed by a veterinary ophthalmologist on all rats prior to initiation of the study and
after 13 weeks of feeding. Eyes were examined by indirect ophthalmoscopy after pupil dilation with 1% Mydriacy(Tropicamide).
Statistical Procedures: Means and standard deviations (SD) were calculated for all quantitative parameters. The data were log-transformed (except
body weight gains, food consumption and focd conversion ratios) and statistically analyzed using both multivariate and univariate two-factor
fixed-effects analyses of variance (ANOVA). The body weights, body weight gains, food consumption and food conversion ratio data were evaluated using multivariate repeated-measure analyses of variance to determine the shape of the dose-response relationship over time (Bock, R.D., Multivariate Statistical Methods in Behavioral Research, Chapter 7, McGraw Hill, NY, 1975). In the presence of significant main effects or interactions, all possible post-hoc comparisons for combined data of sexes were conducted using the Dunnett's test (C.W. Dunnett, A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Am. Stat. Assn., 50: 1096 - 1121, 1955). In the presence of a significant sex interaction, post-hoc comparisons were performed separately for male and female rats. These statistical methods were applied using SYSTAT software (SYSTAT: The System f r Statistics Systat, Inc., Evanston, IL, vers. 4.1, 1988) on a Zenith 2-200 PC/AT. A minimum significance level of p

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Diet Analysis: Results of the homogeneity analysis prior to initiation of feeding showed no apparent location effects. The relative standard deviation of test article concentrations in the diet ranged from 1.36% (5000 ppm) to 5.53% (200 ppm). The stability analysis showed Diphenyl Ether to be stable in the feed under both room temperature and freezing storage conditions for up to 14 days.

Diet analyses during the study indicated that the diets were prepared accurately and homogeneously. Animals received the following average dietary
concentrations of Diphenyl Ether over the course of the study: 207 +/- 3.15 ppm (low dose), 1002 +/- 17.7 ppm (mid dose), and 4906 +/- 82.7 ppm (high dose).

Mortality/Morbidity: None of the rats died during the 13-week feeding or 4-week recovery periods.

Clinical Observations: No signs of test article-related toxicity were seen in any animal during the 13-week treatment period. The majority of animals in all groups showed no clinical signs during the 13 weeks of treatment. The most common observations noted were redness around the eyes and/or nose and alopecia, which were incidental in occurrence and evenly distributed among all groups. Other observations in the treated and treatewrecovery groups were of an incidental nature and unrelated to treatment with Diphenyl Ether.

Body Weights, Food Consumption, and Food Conversion: Mean weekly body weight and food consumption were significantly decreased in the 5000 ppm males and females during the entire 13-week treatment period. Statistically significant decreases in mean body weight and food consumption were also noted in the 1000 ppm females during most of the 13-week treatment period. These changes were attributable to decreased palatability of the test diet in both sexes at the 5000 ppm level and in the 1000 ppm females, as evidenced by statistically significant increases in food consumption and/or body weight gain during one or more weeks of the recovery period when these rats were fed untreated basal diet. Mean food consumption during the 13-week treatment period was decreased 16 and 23% from control values for the 5000 ppm males and females, respectively, and 7% from control levels for the 1000 ppm females.

Food conversion ratios in the 5000 ppm males were significantly decreased during weeks 1-4 of the 13-week treatment period, but were significantly
increased when compared to the control males during weeks 14-16 of the recovery period. Food conversion ratios were significantly reduced in the 5000 ppm females at weeks 1, 2, 4, 10 and 13, but significantly increased compared to the control value at the end of the first week of recovery (week 14). Food conversion ratios in the 1000 ppm females were significantly decreased from control values after 1, 2 and 4 weeks of treatment, but significantly increased from the control value after one week of recovery. These changes were again compatible with decreased palatability of the test diet, rather than an indication of test article toxicity. No test article or persistent palatability related decreases in body weight, body weight gain or food consumption were seen in the 1000 ppm males or in either sex at the 200 ppm level.

Test Article Intake (Mg/Kg/Day): After each week of treatment, part per million dosage levels were converted to mg/kg/day doses from the weekly food intake and body weight data as follows:
mg/kg/day = [ppm (mg/kg) x food consumption (kg) /7] /body weight (kg)

Male rats at the 200, 1000 and 5000 ppm levels received an average weekly dose of 11.7, 60.7 and 301.1 mg/kg/day, respectively, over the course of the 13-week treatment period, while females at the 200, 1000 and 5000 ppm levels received an average weekly dose of 14.5, 73.9 and 334.8 mg/kg/day of Diphenyl Ether during the study.

Clinical Chemistry: Statistically significant differences noted between the test groups and the respective control group after 13 weeks of treatment consisted of: glucose -significantly decreased in mid dose males; phosphorus - significantly increased in high dose males; albumin - significantly decreased in mid-dose females. Statistically significant differences observed at the end of the 4-week recovery period consisted of: total protein and globulin - significantly decreased in mid dose males; potassium - significantly decreased in low and high dose females. Since these changes were either not dose-related (glucose, albumin), within the range of in-house historical control values for rats of this strain and age (phosphorus; range: 4.8 - 8 6 mg/dl), or occurred only in the recovery animals (total protein, globulin, potassium), they were not considered test article related.

Hematology: No statistically significant differences were noted between any test article-treated group and the respective control group for any of the hematology or differential white blood cell parameters examined after 13 weeks of treatment and/or 4 weeks of recovery.

Urine Analysis: No effects related to administration of Diphenyl Ether were noted on any of the parameters examined after 13 weeks of treatment.

Ophthalmology: Ocular examinations revealed the presence of chorioretinal degeneration in one low and two high dose animals. These changes are typical post-inflammatory lesions and not indicative of a toxic effect. Thus, no ocular manifestations of a toxic nature could be attributed to
feeding of Diphenyl Ether.

Organ Weights: Absolute heart weights were significantly reduced in high dose males and females and in mid dose females after 13 weeks of feeding. Absolute adrenal weight was also significantly reduced in the high dose females after 13 weeks. No statistically significant differences were observed in the absolute weight of any organs at the end of the recovery period. Since no microscopic changes were seen in the heart or adrenal glands of the high dose animals after 13 weeks of treatment which could have accounted for the decreased absolute weight of these organs, the weight changes were not considered toxicologically significant.

Statistically significant differences in the relative weight of several organs were detected after 13 weeks of treatment and 4 weeks of recovery. Fasted body weights were significantly decreased in both sexes at the high dose level and in the mid dose females after 13 weeks of treatment. This difference in fasted body weights resulted in significant increases in the relative weight of the brain, liver, spleen, kidneys and gonads in both sexes at the high dose level after 13 weeks of treatment, as well as significantly increased relative ovary weight in the low and mid dose females. Relative kidney weight was significantly increased in the high dose males, while relative brain and heart weights were significantly increased in the high dose females at the end of the recovery period. Since the absolute weight of these organs was not increased in these animals, the statistically significant increases observed were attributable to the statistically significant decreases in the fasted body weights of the animals which were still present at the end of the recovery period. Thus, the increases in relative organ weights were ultimately attributable to the decreased palatability of the test diet, rather than to direct toxic effects of the test article itself.

Gross Pathology: The most common gross lesions seen in animals sacrificed after the 13-week treatment and/or 4-week recovery periods included red andfor enlarged mandibular lymph nodes, lung foci and urinary bladder calculi (males only). Since there was essentially no difference in the incidence of these observations'between the treated and control groups, they were not considered to be treatment-related. Other gross observations were of a minor, incidental nature.

Histopathology: No test article-related microscopic abnormalities were seen in any organ or tissue from any animal examined at the end of the treatment period. Due to the lack of test article-related abnormalities in the high dose animals after 13 weeks of treatment, the tissues collected from the recovery rats were not processed or examined.

Effect levels

open allclose all
Dose descriptor:
Effect level:
301 mg/kg bw/day (nominal)
Basis for effect level:
other: Based on food consumption (palatability) at the 5000 ppm dose level.
Dose descriptor:
Effect level:
335 mg/kg bw/day (nominal)
Basis for effect level:
other: Based on food consumption (palatability) at the 5000 ppm dose level.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables


Applicant's summary and conclusion

Dietary administration of Diphenyl Ether to Sprague-Dawley rats for 13 consecutive weeks at levels of 200, 1000 and 5000 ppm resulted in no significant toxicological or pathological effects which were related to the test compound. Based on food consumption (palatability) at the 5000 ppm dose level, the no observed- effect level (NOEL) for this study was 301 mg/kg/day for males, and 335 mg/kg/day for females.
Executive summary:

Sprague-Dawley rats (20/sex/group) were fed Diphenyl Ether at levels of 200, 1000 and 5000 ppm in the diet for 13 weeks to evaluate its potential toxicity. A control group of similar size was fed basal diet alone. Animals were observed daily and their body weight and food consumption were measured weekly. Half of the rats/sex/group were sacrificed and subjected to a gross necropsy after 13 weeks of treatment, while the remaining animals were held for 4 weeks to assess the recovery potential from any adverse effects, then sacrificed and necropsied. Hematology, serum chemistry and urinalysis parameters were evaluated after 13 weeks and again at the end of the recovery period (except urinalysis). Selected organs from all animals were weighed at necropsy. Detailed histological examination was performed on tissues from the control and high dose groups of the 13-week animals; lung, liver, kidneys and gross lesions from the remaining groups were also examined microscopically.

No signs of overt systemic toxicity were seen at any dose level after 13 weeks of treatment and/or 4 weeks of recovery. Mean body weight, body weight gain, food

consumption and food conversion ratios were significantly decreased in the 5000 ppm males and females and in the 1000 ppm females during the 13-week feeding period when compared to the controls; these changes were attributed to decreased palatability of the test diet. Otherwise, clinical pathology parameters, ophthalmic examinations, gross necropsy observations, organ weight determinations and microscopic tissue evaluation revealed no treatment-related effects at any dose level. Thus, a no-observed-effect level (NOEL) of 301 mg/kg/day (males) and 335 mg/kg/day (females) in the diet was established for Diphenyl Ether, based upon food consumption (palatability) at the 5000 ppm level.