Registration Dossier

Administrative data

Description of key information

Oral: 13-week dietary study  in rats was conducted according to OECD Guideline 408
Dermal: 13-week dermal study was conducted in rats. No information on guidelines; however, the study was published in a peer reviewed journal in 2003.
Inhalation: Repeated inhalation exposures were conducted in rats, rabbits and dogs 7 hours per day, 5 days per week for a total of 20 exposures. Study was conducted prior to guidelines and published in a peer reviewed journal.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Remarks:
Not specified in report
Qualifier:
according to
Guideline:
other: Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food (FDA, PB83-170696, 1982).
Deviations:
no
Remarks:
Not specified in report
Qualifier:
according to
Guideline:
other: Japanese Toxicity Test Guideline of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, February 15, 1984
Deviations:
no
Remarks:
Not specified in report
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
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.
Route of administration:
oral: feed
Vehicle:
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:
yes
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.

METHOD DESCRIPTION
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:
daily
Remarks:
Doses / Concentrations:
0, 200, 1000 or 5000 ppm
Basis:
nominal in diet
Remarks:
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
Basis:
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:
None
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).
Statistics:
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
Clinical signs:
no effects observed
Mortality:
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.
Dose descriptor:
NOEL
Effect level:
301 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: Based on food consumption (palatability) at the 5000 ppm dose level.
Dose descriptor:
NOEL
Effect level:
335 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: Based on food consumption (palatability) at the 5000 ppm dose level.
Critical effects observed:
not specified

None

Conclusions:
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
301 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
acceptable quality

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1974
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was conducted prior to GLP and test guidelines, but sufficient data is available for interpretation of results.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline available
Principles of method if other than guideline:
Repeated inhalation studies using rats, rabbits, and dogs were conducted at mean exposure concentrations of 0, 4.9 and 10.0 ppm DPO vapor. Exposures were 7 hours per day, 5 days per week for a total of 20 exposures. An additional group of rats w ere exposed to 0 or 20 ppm DPO vapor for atotal of 20 exposures.
GLP compliance:
no
Species:
other: Rat, rabbit, dog
Strain:
other: Sprague-Dawley rats, New Zealand albino rabbits and beagle dogs
Sex:
male/female
Details on test animals and environmental conditions:
rats- female (control and 20 ppm, only) and males
rabbits- males only
dogs- males only

Food and water were not available to control or exposed animals during the daily exposures. Between exposures, food and water were provided ad libitum to all animals.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No data
Details on inhalation exposure:
Exposures to 5 or 10 ppm DPO vapor were conducted under dynamic conditions in two one cubic meter glass-walled exposure chambers. DPO vapor was generated by metering the liquid at a controlled rate into a temperature regulated vaporization flask. Nitrogen was used to sweep the vapor from the vaporization flask into the air inlet where mixing and dilution with filtered room air occurred. The ratio of nitrogen flow to total chamber airflow was 0.005, so that the oxygen level in the chamber atmosphere was not significantly reduced. The nominal concentration of the vapor was calculated from the ratio of the rate of compound dissemination to the rate of total chamber airflow (the volume of nitrogen ejected from the generator plus the volume of make-up air ) .
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical concentration of DPO vapor in the chamber was determined by gas chromatography of samples of the chamber atmosphere collected with a gas syringe. All gas chromatography was done on a 5' long by 1/8" diameter column containing a 2% SE-54 liquid phase on 80-100 mesh Chromsorb W packing. The Aerograph Model 550 chromatograph was operated isothermally at 200°C with a 225°C injection port temperature. A flame ionization detector was employed using helium as the carrier gas. A 10 ppm DPO vapor standard was used in calculating the analytical chamber
concentration. This standard was generated by vaporizing the appropriate amount of DPO into a saran bag containing 100 L of air. A solution of 10 ppm DPO in carbon disulfide gave results comparable to the gaseous standard and was therefore used as the working standard for routine chamber analysis. Both the 5 and 10 ppm nominal concentration exposure chambers were analyzed several times during each daily exposure. Exposures of rats to 20 ppm DPO vapor were conducted as previously described except a 160L glass walled chamber and filtered room air, rather than nitrogen, to sweep the vaporization flask, were used. Only the nominal concentration was determined for the 20 ppm exposures.
Duration of treatment / exposure:
7 hrs/day, 5 days/week for a total of 20 exposures in 31 or 3 days
Frequency of treatment:
5 days/week
Remarks:
Doses / Concentrations:
Males: controls, 5 or 10 ppm (rats, rabbits and dogs)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Males and females: control and 20 ppm (rats only)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4.9 +/- 1.5 ppm and 10.0 +/- 2.0 ppm; 20 ppm (nominal only)
Basis:
analytical conc.
No. of animals per sex per dose:
Rats: 20 male rats (control, 5 or 10 ppm) and 10/sex (control and 20 ppm)
Rabbits: 4 male rabbits (control, 5 or 10 ppm)
Dogs: 2 male dogs (control, 5 or 10 ppm)
Control animals:
yes, concurrent no treatment
Details on study design:
Exposure Regimen:
Two groups of animals, each consisting of 20 male Sprague-Dawley rats of Spartan strain, 4 male New Zealand albino rabbits, and 2 male beagle dogs, were exposed, to nominal concentrations of 5 or 10 ppm DPO vapor for 7 hours per day, 5 days per week for a total of 20 exposures in 31 or 33 days, respectively. A third group of animals were kept under ambient conditions and served as controls. An additional group of 10 male and 10 female Sprague-Dawley rats of Spartan strain were exposed to a nominal concentration of 20 ppm DPO vapor for 7 hours per day, 5 days per week for a total of 20 exposures in 27 days. Equal numbers of male and female rats were kept under ambient conditions and served as controls. Food and water were not available to control or exposed animals during the daily exposures. Between exposures, food and water were provided ad libitum to all animals.

Evaluation of Toxicity
All animals were observed for signs of toxicity and irritation periodically throughout the experiment and in particular during each exposure. Body weights of all animals were recorded at regular intervals. At the termination of the study, basic hematological determinations, including red, white, and differential cell counts, hemaglobin concent rations, and hematocrit, were conducted on samples of blood from 10 rats and all of the rabbits and dogs. Also determined were blood urea nitrogen (BUN), and the activities of serum glutamic pyruvic transaminase (SGPT), and alkaline phosphatase
(AP). Additional hematological determinations were made on all rats exposed to 20 ppm DPO vapor after 1, 4, and 19 days of exposure. All animals were deprived of food, sacrificed and subjected to gross pathological examination within 20 hours after termination of the exposures. Organ weights for brain, heart, liver, kidney, and testes were obtained for all animals exposed to 0, 5 or 10 ppm DPO vapor and from 10 rats (5 per sex)
exposed to 0 or 20 ppm DPO vapor. In addition, spleen and thymus weights were obtained from 10 rats (5 per sex) exposed to 0 or 20 ppm DPO vapor, and adrenal gland weights were obtained for all dogs. All major organs and tissues, including the nasal turbinates and adjacent bone, pituitary gland, brain, trachea, thyroid, parathyroid, aorta, lungs, thoracic lymph nodes, thymus, salivary glands, liver, pancreas, small intestine, stomach, large intestine, mesenteric lymph node, adrenal gland, accessory sex glands, testes, epididymis, peripheral nerve, skeletal muscle, esophagus, urinary bladder, spleen, and any other grossly visible pathologic lesion were prepared and examined histologically. All tissues were routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The nasal turbinates and adjacent bone were decalcified prior to processing. Tissues were
fixed in a 10% buffered formalin fixative. The lungs of all rats and rabbits were distended with 10% buffered formalin fixative following removal. Organ to body weight ratios, as well as hematological and biochemical parameters were analyzed statistically using analysis of variance and Dunnett's test . The level of significance in all cases was p<0.05.
Positive control:
None
Observations and examinations performed and frequency:
All animals were observed for signs of toxicity and irritation periodically throughout the experiment and in particular during each exposure. Body weights of all animals were recorded at regular intervals.

Additional hematological determinations were made on all rats exposed to 20 ppm DPO vapor after 1, 4, 19 or 20 days of exposure.
Sacrifice and pathology:
At the termination of the study, basic hematological determinations, including red, white, and differential cell counts, hemaglobin concent rations, and hematocrit, were conducted on samples of blood from 10 rats and all of the rabbits and dogs. Also determined were blood urea nitrogen (BUN), and the activities of serum glutamic pyruvic transaminase (SGPT), and alkaline phosphatase (AP).

All animals were deprived of food, sacrificed and subjected to gross pathological examination within 20 hours after termination of the exposures. Organ weights for brain, heart, liver, kidney, and testes were obtained for all animals exposed to 0, 5 or 10 ppm DPO vapor and from 10 rats (5 per sex) exposed to 0 or 20 ppm DPO vapor. In addition, spleen and thymus weights were obtained from 10 rats (5 per sex) exposed to 0 or 20 ppm DPO vapor, and adrenal gland weights were obtained for all dogs. All major organs and tissues, including the nasal turbinates and adjacent bone, pituitary gland, brain, trachea, thyroid, parathyroid, aorta, lungs, thoracic lymph nodes, thymus, salivary glands, liver, pancreas, small intestine, stomach, large intestine, mesenteric lymph node, adrenal gland, accessory sex glands, testes, epididymis, peripheral nerve, skeletal muscle, esophagus, urinary bladder, spleen, and any other grossly visible pathologic lesion were prepared and examined histologically.
Other examinations:
None
Statistics:
Organ to body weight ratios, as well as hematological and biochemical parameters were analyzed statistically using analysis of variance and Dunnett's test. The level of significance in all cases was p<0.05.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
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:
Chamber Analysis and Observations During Exposure:
The means and standard deviations of the concentrations of DPO vapor in the two exposure chambers, as determined by analysis during the experiment, were 4.9+/-1 .5 and 10.0+/-2 .0 ppm, respectively. No signs of toxicity or irritation were observed in animals exposed to 4.9 ppm DPO vapor. Rats and rabbits exposed to 10.0 ppm exhibited mild eye and nasal irritation. Dogs exposed to 10.0 ppm exhibited no signs of toxicity or irritation. Both male and female rats exposed to 20 ppm exhibited eye and nasal irritation.

Analysis of Body Weights and Organ Weights
The body weight gains of animals exposed to either 4.9, 10.0 or 20 ppm DPO vapor were comparable to and not statistically different than those of the controls. Analysis of the data indicate statistically significant decreases in the mean absolute organ weight and the organ to body weight ratio for livers of rats exposed to both 4.9 and 10.0, but not 20 ppm-DPO vapor. A statistically significant increase in the brain to body weight ratio was observed in male rats exposed to 20 ppm.

Clinical Chemistry and Pathology
Rats exposed to 4.9 or 10.0 ppm DPO vapor exhibited a statistically significant decrease in the mean white blood cell count. Additionally, there was a statistically significant decrease in the concentration of hemoglobin in the blood of rats exposed to 10.0 ppm. Male rats exposed to 20 ppm DPO vapor exhibited a statistically significant increase in the mean white blood cell count after 19, but not 1, 4, or 20 days of exposure. Female rats exposed to this concentration exhibited statistically significant decreases in the mean white and red blood cell counts, hematocrit, and hemoglobin concent
ration after 4, but not 1, 19, or 20 days of exposure. No statistically significant differences were noted in the hematological parameters of exposed rabbits or dogs, when compared to controls.

Rabbits exposed to 4.9 and 10.0 ppm DPO vapor, as well as dogs exposed to 4.9 ppm, exhibited statistically significant decreases in BUN values. A concurrent decrease in terminal BUN values was not observed in dogs exposed to 10.0 ppm or in rats exposed to 4.9 or 10.0 ppm.

Gross and histopathological examination of all exposed and control animals revealed no discernible lesions related to exposure to 4.9, 10.0, or 20 ppm DPO vapor. Several exposed and control animals exhibited focal reddened areas in the lungs or mild lesions resulting from an occasional parasite. These findings were not considered related to the exposures to DPO vapor, as they were present in control as well as exposed animals.
Dose descriptor:
NOEL
Effect level:
4.9 ppm
Sex:
male
Basis for effect level:
other: NOEL for rats and rabbits based on eye and nasal irritation observed at higher concentrations.
Dose descriptor:
NOEL
Effect level:
10 ppm
Sex:
male
Basis for effect level:
other: NOEL for dogs (highest concentration tested)
Critical effects observed:
not specified

DISCUSSION

Exposure of rats or rabbits to 10.0 ppm or rats to 20 ppm DPO vapor induced primary irritation of the eyes and nares. Hematological changes, including statistically significant decreases in white blood cell counts of rats exposed to 4.9 and 10.0 ppm, and decreases in the hemoglobin concentrations of rats exposed to 10.0 ppm were not observed in rabbits or dogs exposed to these concentrations. Furthermore, exposure of both male and female rats to the higher concentration of 20 ppm produced no consistent reductions in white blood cell counts or hemoglobin concentrations in either sex. For example, in the male control rats used in this investigation, a considerable variation in the mean white blood cell counts was observed. At the inception of the exposures to 20 ppm DPO vapor, the mean control white blood cell count was 20.4 +/-3.2 e3/mm3. Eighteen days later, a 14.7 +/-1.6 e3 / mm3 white blood cell count was observed for these same control rats. This, as well as all other mean hematological parameters are within the normal range of variation observed in this laboratory. Hence, the statistically significant decreases in both the white blood cell counts or hemoglobin concentrations are very likely unrelated to exposure to DPO vapor.

The statistically significant decrease in the liver to body weight ratios observed in rats exposed to either 4.9 or 10.0 ppm DPO vapor are very likely unrelated to the exposures, as there were no differences in the liver to body weight ratios of rats exposed to 20 ppm. Additionally, the liver to body weight ratios of rats exposed to 4.9 or 10.0 ppm were within the normal range of variation when compared to control rats used in previous studies in this laboratory. Further, there were no gross pathological or histopathological lesions in the organs, including liver, of any animals exposed to DPO vapor. The statistically significant increase in the brain to body weight ratios observed in rats exposed to 20 ppm DPO vapor are very likely unrelated to the exposures, because the brain to body weight ratios for the control rats are lower than normally observed in this laboratory. Additionally, the statistically significant decrease in the terminal body weight of rats exposed to 20 ppm DPO vapor was not observed during the exposures, but resulted from the food deprivation prior to sacrifice and is thus very likely unrelated to the exposures.

The statistically significant decrease in mean terminal BUN values for all exposed rabbits, as well as dogs exposed to 4.9 ppm DPO vapor likewise appear to be unrelated to exposure. The mean terminal BUN values are within the normal range of variation observed in this laboratory. For example, the mean BUN of a group of 59 control male albino rabbits was found to be 21.4 mg/%, range 15.0 - 27.0. (Unpublished data - Dow Chemical Co.) In this investigation mean terminal BUN values were 21.3, 15.0, and 16.5 mg/% for rabbits exposed to 0.0, 4.9, and 10.0 ppm DPO vapor, respectively. The decrease in terminal BUN observed in dogs exposed to 4.9 ppm is of doubtful significance, as a concurrent decrease was not observed in dogs exposed to the higher concentration of 10.0 ppm.

The results of this investigation indicate a lack of untoward effects in rats, rabbits and dogs repeatedly exposed to 4.9 ppm DPO vapor. Concentrations of 10.0 ppm DPO vapor appear to be physically irritating to rats and rabbits, but not dogs. Exposure to 20 ppm DPO vapor resulted in physical irritation as well as slight, but not statistically significant, decreases in the rate of body weight gain by male rats and concomitant changes in organ to body weight ratios, as well. Thus, the only untoward effect in animals exposed to 10.0 or 20 ppm DPO vapor for 20 days is physical irritation. Irritation, especially to the eyes and nares, would very likely precede any organic damage caused by exposure to DPO vapor. Therefore, it is suggested that occasional short term exposure of humans to 5 ppm DPO vapor should be without untoward effect. Further studies are necessary to establish the effect of chronic exposure to DPO vapor.

Conclusions:
The results of the study indicate a lack of untoward effects in rats, rabbits and dogs repeatedly exposed to 4.9 ppm diphenyl oxide (DPO) vapor. Only untoward effect in animals exposed to 10 or 20 ppm for 20 days was physical irritation.
Executive summary:

Repeated inhalation studies using rats, rabbits, and dogs were conducted at mean exposure concentrations of 0, 4.9 and 10.0 ppm DPO vapor. Exposures were 7 hours per day, 5 days per week for a total of 20 exposures. An additional group of rats were exposed to 0 or 20 ppm DPO vapor for a total of 20 exposures. No untoward effects were observed in animals exposed to 4.9 ppm. Eye and nasal irritation were observed in rats and rabbits exposed to 10.0 ppm and in rats exposed to 20 ppm. Aside from this irritation no other untoward effects were discerned.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
139 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
acceptable quality

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1974
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was conducted prior to GLP and test guidelines, but sufficient data is available for interpretation of results.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline available
Principles of method if other than guideline:
Repeated inhalation studies using rats, rabbits, and dogs were conducted at mean exposure concentrations of 0, 4.9 and 10.0 ppm DPO vapor. Exposures were 7 hours per day, 5 days per week for a total of 20 exposures. An additional group of rats w ere exposed to 0 or 20 ppm DPO vapor for atotal of 20 exposures.
GLP compliance:
no
Species:
other: Rat, rabbit, dog
Strain:
other: Sprague-Dawley rats, New Zealand albino rabbits and beagle dogs
Sex:
male/female
Details on test animals and environmental conditions:
rats- female (control and 20 ppm, only) and males
rabbits- males only
dogs- males only

Food and water were not available to control or exposed animals during the daily exposures. Between exposures, food and water were provided ad libitum to all animals.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: No data
Details on inhalation exposure:
Exposures to 5 or 10 ppm DPO vapor were conducted under dynamic conditions in two one cubic meter glass-walled exposure chambers. DPO vapor was generated by metering the liquid at a controlled rate into a temperature regulated vaporization flask. Nitrogen was used to sweep the vapor from the vaporization flask into the air inlet where mixing and dilution with filtered room air occurred. The ratio of nitrogen flow to total chamber airflow was 0.005, so that the oxygen level in the chamber atmosphere was not significantly reduced. The nominal concentration of the vapor was calculated from the ratio of the rate of compound dissemination to the rate of total chamber airflow (the volume of nitrogen ejected from the generator plus the volume of make-up air ) .
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical concentration of DPO vapor in the chamber was determined by gas chromatography of samples of the chamber atmosphere collected with a gas syringe. All gas chromatography was done on a 5' long by 1/8" diameter column containing a 2% SE-54 liquid phase on 80-100 mesh Chromsorb W packing. The Aerograph Model 550 chromatograph was operated isothermally at 200°C with a 225°C injection port temperature. A flame ionization detector was employed using helium as the carrier gas. A 10 ppm DPO vapor standard was used in calculating the analytical chamber
concentration. This standard was generated by vaporizing the appropriate amount of DPO into a saran bag containing 100 L of air. A solution of 10 ppm DPO in carbon disulfide gave results comparable to the gaseous standard and was therefore used as the working standard for routine chamber analysis. Both the 5 and 10 ppm nominal concentration exposure chambers were analyzed several times during each daily exposure. Exposures of rats to 20 ppm DPO vapor were conducted as previously described except a 160L glass walled chamber and filtered room air, rather than nitrogen, to sweep the vaporization flask, were used. Only the nominal concentration was determined for the 20 ppm exposures.
Duration of treatment / exposure:
7 hrs/day, 5 days/week for a total of 20 exposures in 31 or 3 days
Frequency of treatment:
5 days/week
Remarks:
Doses / Concentrations:
Males: controls, 5 or 10 ppm (rats, rabbits and dogs)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Males and females: control and 20 ppm (rats only)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4.9 +/- 1.5 ppm and 10.0 +/- 2.0 ppm; 20 ppm (nominal only)
Basis:
analytical conc.
No. of animals per sex per dose:
Rats: 20 male rats (control, 5 or 10 ppm) and 10/sex (control and 20 ppm)
Rabbits: 4 male rabbits (control, 5 or 10 ppm)
Dogs: 2 male dogs (control, 5 or 10 ppm)
Control animals:
yes, concurrent no treatment
Details on study design:
Exposure Regimen:
Two groups of animals, each consisting of 20 male Sprague-Dawley rats of Spartan strain, 4 male New Zealand albino rabbits, and 2 male beagle dogs, were exposed, to nominal concentrations of 5 or 10 ppm DPO vapor for 7 hours per day, 5 days per week for a total of 20 exposures in 31 or 33 days, respectively. A third group of animals were kept under ambient conditions and served as controls. An additional group of 10 male and 10 female Sprague-Dawley rats of Spartan strain were exposed to a nominal concentration of 20 ppm DPO vapor for 7 hours per day, 5 days per week for a total of 20 exposures in 27 days. Equal numbers of male and female rats were kept under ambient conditions and served as controls. Food and water were not available to control or exposed animals during the daily exposures. Between exposures, food and water were provided ad libitum to all animals.

Evaluation of Toxicity
All animals were observed for signs of toxicity and irritation periodically throughout the experiment and in particular during each exposure. Body weights of all animals were recorded at regular intervals. At the termination of the study, basic hematological determinations, including red, white, and differential cell counts, hemaglobin concent rations, and hematocrit, were conducted on samples of blood from 10 rats and all of the rabbits and dogs. Also determined were blood urea nitrogen (BUN), and the activities of serum glutamic pyruvic transaminase (SGPT), and alkaline phosphatase
(AP). Additional hematological determinations were made on all rats exposed to 20 ppm DPO vapor after 1, 4, and 19 days of exposure. All animals were deprived of food, sacrificed and subjected to gross pathological examination within 20 hours after termination of the exposures. Organ weights for brain, heart, liver, kidney, and testes were obtained for all animals exposed to 0, 5 or 10 ppm DPO vapor and from 10 rats (5 per sex)
exposed to 0 or 20 ppm DPO vapor. In addition, spleen and thymus weights were obtained from 10 rats (5 per sex) exposed to 0 or 20 ppm DPO vapor, and adrenal gland weights were obtained for all dogs. All major organs and tissues, including the nasal turbinates and adjacent bone, pituitary gland, brain, trachea, thyroid, parathyroid, aorta, lungs, thoracic lymph nodes, thymus, salivary glands, liver, pancreas, small intestine, stomach, large intestine, mesenteric lymph node, adrenal gland, accessory sex glands, testes, epididymis, peripheral nerve, skeletal muscle, esophagus, urinary bladder, spleen, and any other grossly visible pathologic lesion were prepared and examined histologically. All tissues were routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The nasal turbinates and adjacent bone were decalcified prior to processing. Tissues were
fixed in a 10% buffered formalin fixative. The lungs of all rats and rabbits were distended with 10% buffered formalin fixative following removal. Organ to body weight ratios, as well as hematological and biochemical parameters were analyzed statistically using analysis of variance and Dunnett's test . The level of significance in all cases was p<0.05.
Positive control:
None
Observations and examinations performed and frequency:
All animals were observed for signs of toxicity and irritation periodically throughout the experiment and in particular during each exposure. Body weights of all animals were recorded at regular intervals.

Additional hematological determinations were made on all rats exposed to 20 ppm DPO vapor after 1, 4, 19 or 20 days of exposure.
Sacrifice and pathology:
At the termination of the study, basic hematological determinations, including red, white, and differential cell counts, hemaglobin concent rations, and hematocrit, were conducted on samples of blood from 10 rats and all of the rabbits and dogs. Also determined were blood urea nitrogen (BUN), and the activities of serum glutamic pyruvic transaminase (SGPT), and alkaline phosphatase (AP).

All animals were deprived of food, sacrificed and subjected to gross pathological examination within 20 hours after termination of the exposures. Organ weights for brain, heart, liver, kidney, and testes were obtained for all animals exposed to 0, 5 or 10 ppm DPO vapor and from 10 rats (5 per sex) exposed to 0 or 20 ppm DPO vapor. In addition, spleen and thymus weights were obtained from 10 rats (5 per sex) exposed to 0 or 20 ppm DPO vapor, and adrenal gland weights were obtained for all dogs. All major organs and tissues, including the nasal turbinates and adjacent bone, pituitary gland, brain, trachea, thyroid, parathyroid, aorta, lungs, thoracic lymph nodes, thymus, salivary glands, liver, pancreas, small intestine, stomach, large intestine, mesenteric lymph node, adrenal gland, accessory sex glands, testes, epididymis, peripheral nerve, skeletal muscle, esophagus, urinary bladder, spleen, and any other grossly visible pathologic lesion were prepared and examined histologically.
Other examinations:
None
Statistics:
Organ to body weight ratios, as well as hematological and biochemical parameters were analyzed statistically using analysis of variance and Dunnett's test. The level of significance in all cases was p<0.05.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
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:
Chamber Analysis and Observations During Exposure:
The means and standard deviations of the concentrations of DPO vapor in the two exposure chambers, as determined by analysis during the experiment, were 4.9+/-1 .5 and 10.0+/-2 .0 ppm, respectively. No signs of toxicity or irritation were observed in animals exposed to 4.9 ppm DPO vapor. Rats and rabbits exposed to 10.0 ppm exhibited mild eye and nasal irritation. Dogs exposed to 10.0 ppm exhibited no signs of toxicity or irritation. Both male and female rats exposed to 20 ppm exhibited eye and nasal irritation.

Analysis of Body Weights and Organ Weights
The body weight gains of animals exposed to either 4.9, 10.0 or 20 ppm DPO vapor were comparable to and not statistically different than those of the controls. Analysis of the data indicate statistically significant decreases in the mean absolute organ weight and the organ to body weight ratio for livers of rats exposed to both 4.9 and 10.0, but not 20 ppm-DPO vapor. A statistically significant increase in the brain to body weight ratio was observed in male rats exposed to 20 ppm.

Clinical Chemistry and Pathology
Rats exposed to 4.9 or 10.0 ppm DPO vapor exhibited a statistically significant decrease in the mean white blood cell count. Additionally, there was a statistically significant decrease in the concentration of hemoglobin in the blood of rats exposed to 10.0 ppm. Male rats exposed to 20 ppm DPO vapor exhibited a statistically significant increase in the mean white blood cell count after 19, but not 1, 4, or 20 days of exposure. Female rats exposed to this concentration exhibited statistically significant decreases in the mean white and red blood cell counts, hematocrit, and hemoglobin concent
ration after 4, but not 1, 19, or 20 days of exposure. No statistically significant differences were noted in the hematological parameters of exposed rabbits or dogs, when compared to controls.

Rabbits exposed to 4.9 and 10.0 ppm DPO vapor, as well as dogs exposed to 4.9 ppm, exhibited statistically significant decreases in BUN values. A concurrent decrease in terminal BUN values was not observed in dogs exposed to 10.0 ppm or in rats exposed to 4.9 or 10.0 ppm.

Gross and histopathological examination of all exposed and control animals revealed no discernible lesions related to exposure to 4.9, 10.0, or 20 ppm DPO vapor. Several exposed and control animals exhibited focal reddened areas in the lungs or mild lesions resulting from an occasional parasite. These findings were not considered related to the exposures to DPO vapor, as they were present in control as well as exposed animals.
Dose descriptor:
NOEL
Effect level:
4.9 ppm
Sex:
male
Basis for effect level:
other: NOEL for rats and rabbits based on eye and nasal irritation observed at higher concentrations.
Dose descriptor:
NOEL
Effect level:
10 ppm
Sex:
male
Basis for effect level:
other: NOEL for dogs (highest concentration tested)
Critical effects observed:
not specified

DISCUSSION

Exposure of rats or rabbits to 10.0 ppm or rats to 20 ppm DPO vapor induced primary irritation of the eyes and nares. Hematological changes, including statistically significant decreases in white blood cell counts of rats exposed to 4.9 and 10.0 ppm, and decreases in the hemoglobin concentrations of rats exposed to 10.0 ppm were not observed in rabbits or dogs exposed to these concentrations. Furthermore, exposure of both male and female rats to the higher concentration of 20 ppm produced no consistent reductions in white blood cell counts or hemoglobin concentrations in either sex. For example, in the male control rats used in this investigation, a considerable variation in the mean white blood cell counts was observed. At the inception of the exposures to 20 ppm DPO vapor, the mean control white blood cell count was 20.4 +/-3.2 e3/mm3. Eighteen days later, a 14.7 +/-1.6 e3 / mm3 white blood cell count was observed for these same control rats. This, as well as all other mean hematological parameters are within the normal range of variation observed in this laboratory. Hence, the statistically significant decreases in both the white blood cell counts or hemoglobin concentrations are very likely unrelated to exposure to DPO vapor.

The statistically significant decrease in the liver to body weight ratios observed in rats exposed to either 4.9 or 10.0 ppm DPO vapor are very likely unrelated to the exposures, as there were no differences in the liver to body weight ratios of rats exposed to 20 ppm. Additionally, the liver to body weight ratios of rats exposed to 4.9 or 10.0 ppm were within the normal range of variation when compared to control rats used in previous studies in this laboratory. Further, there were no gross pathological or histopathological lesions in the organs, including liver, of any animals exposed to DPO vapor. The statistically significant increase in the brain to body weight ratios observed in rats exposed to 20 ppm DPO vapor are very likely unrelated to the exposures, because the brain to body weight ratios for the control rats are lower than normally observed in this laboratory. Additionally, the statistically significant decrease in the terminal body weight of rats exposed to 20 ppm DPO vapor was not observed during the exposures, but resulted from the food deprivation prior to sacrifice and is thus very likely unrelated to the exposures.

The statistically significant decrease in mean terminal BUN values for all exposed rabbits, as well as dogs exposed to 4.9 ppm DPO vapor likewise appear to be unrelated to exposure. The mean terminal BUN values are within the normal range of variation observed in this laboratory. For example, the mean BUN of a group of 59 control male albino rabbits was found to be 21.4 mg/%, range 15.0 - 27.0. (Unpublished data - Dow Chemical Co.) In this investigation mean terminal BUN values were 21.3, 15.0, and 16.5 mg/% for rabbits exposed to 0.0, 4.9, and 10.0 ppm DPO vapor, respectively. The decrease in terminal BUN observed in dogs exposed to 4.9 ppm is of doubtful significance, as a concurrent decrease was not observed in dogs exposed to the higher concentration of 10.0 ppm.

The results of this investigation indicate a lack of untoward effects in rats, rabbits and dogs repeatedly exposed to 4.9 ppm DPO vapor. Concentrations of 10.0 ppm DPO vapor appear to be physically irritating to rats and rabbits, but not dogs. Exposure to 20 ppm DPO vapor resulted in physical irritation as well as slight, but not statistically significant, decreases in the rate of body weight gain by male rats and concomitant changes in organ to body weight ratios, as well. Thus, the only untoward effect in animals exposed to 10.0 or 20 ppm DPO vapor for 20 days is physical irritation. Irritation, especially to the eyes and nares, would very likely precede any organic damage caused by exposure to DPO vapor. Therefore, it is suggested that occasional short term exposure of humans to 5 ppm DPO vapor should be without untoward effect. Further studies are necessary to establish the effect of chronic exposure to DPO vapor.

Conclusions:
The results of the study indicate a lack of untoward effects in rats, rabbits and dogs repeatedly exposed to 4.9 ppm diphenyl oxide (DPO) vapor. Only untoward effect in animals exposed to 10 or 20 ppm for 20 days was physical irritation.
Executive summary:

Repeated inhalation studies using rats, rabbits, and dogs were conducted at mean exposure concentrations of 0, 4.9 and 10.0 ppm DPO vapor. Exposures were 7 hours per day, 5 days per week for a total of 20 exposures. An additional group of rats were exposed to 0 or 20 ppm DPO vapor for a total of 20 exposures. No untoward effects were observed in animals exposed to 4.9 ppm. Eye and nasal irritation were observed in rats and rabbits exposed to 10.0 ppm and in rats exposed to 20 ppm. Aside from this irritation no other untoward effects were discerned.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
35 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
acceptable quality

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The publication lists no information on guideline/s and GLP but the report contains sufficient data for interpretation of study results.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The 13-week subchronic toxicity study was performed with groups of 12 Sprague-Dawley rats/sex/dose. Rats received semi-occluded daily dermal applications of DPO for 6 h/day. All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the diethyl phthalate (DEP) vehicle at concentrations to administer 0, 100, 300 or 1000 mg DPO/kg body weight/day.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Sprague-Dawley CD rats were obtained from Charles River (UK) Ltd (Margate, Kent, UK).
Type of coverage:
semiocclusive
Vehicle:
other: diethyl phthalate
Details on exposure:
Four groups of 12 male and 12 female Sprague-Dawley rats were used. The hair on their backs was clipped before dosing and then twice weekly. Three groups were dosed daily with DPO via the dermal route of application using a semiocclusive dressing for a period of 6 h/day for 13 weeks. Doses of 2 ml/kg were applied to gauze squares ( ~ 4 cm x 4 cm) with an aluminium foil back. These patches were placed on the shaved skin on the animals' backs and held in place by a semi-occlusive dressing (50 mm Micropore, 3M) and tape (50 mm Blenderm, 3M). The gauze squares and dressing were removed after 6 h, and the dosed area was washed with DEP to remove the non-absorbed DPO. The rats were dosed at a constant volume of 2 ml/kg body weight and concentrations in DEP were calculated to achieve dose levels of 100, 300 or 1000 mg/kg body weight/day.
One control group (12 males/12 females) received DEP at a dose volume of 2 ml/kg.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day
Remarks:
Doses / Concentrations:
Solvent control, 100, 300 or 1000 mg/kg body weight/day
Basis:
other: nominal
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The 13-week subchronic toxicity study was performed with groups of 12 Sprague-Dawley rats/sex/dose. Rats received semi-occluded daily dermal applications of DPO for 6 h/day. All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the diethyl phthalate (DEP) vehicle at concentrations to administer 0, 100, 300 or 1000 mg/kg bw/day.

During the study, the animals were assessed for general clinical signs, skin irritation (erythema, eschar, edema and thickening), body weight, food consumption and water consumption. During week 13, samples from 10 males and 10 females from all groups were taken for urinalysis, hematology and clinical blood chemistry parameters.

After 13 weeks of dosing, animals were killed by CO2 narcosis, and necropsied. Selected organs were examined and weighed. Histopathologic
examinations were performed on major tissues from all animals in the control and high-dose groups; kidneys from all dose groups were examined histopathologically.

Hematology, clinical chemistry, organ weight and body weight data were statistically analyzed for homogeneity of variance by using the 'F-max' test. If the group variances appeared homogeneous a parametric ANOVA was used, and pair wise conparisons were made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, a non-parametric test such as Kruskal Wallis ANOVA was used. Organ weights were also analyzed conditional on body weight (i.e. covariance and relative analyses). Histology data were analyzed using Fisher's Exact Probability test.
Positive control:
No data
Observations and examinations performed and frequency:
During the study, the animals were assessed for general clinical signs, skin irritation (erythema, eschar, edema and thickening), body weight, food consumption and water consumption.
Sacrifice and pathology:
During week 13, samples from 10 males and 10 females from all groups were taken for urinalysis, hematology and clinical blood chemistry parameters. After 13 weeks of dosing, animals were killed by CO2 narcosis, and necropsied. Selected organs were examined and weighed. Histopathologic
examinations were performed on major tissues from all animals in the control and high-dose groups; kidneys from all dose groups were examined histopathologically.
Other examinations:
No data
Statistics:
Hematology, clinical chemistry, organ weight and body weight data were statistically analyzed for homogeneity of variance by using the 'F-max' test. If the group variances appeared homogeneous a parametric ANOVA was used, and pair wise conparisons were made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, a non-parametric test such as Kruskal Wallis ANOVA was used. Organ weights were also analyzed conditional on body weight (i.e. covariance and relative analyses). Histology data were analyzed using Fisher's Exact Probability test.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the DEP vehicle at concentrations to administer 0, 100, 300 or 1000 mg/kg bw/day for 13-weeks. In male rats exposed to the high dose, there was a slight reduction in body weight, and a statistically significant increase in liver, brain and kidney weight relative to body weight. Absolute and relative liver weight was increased at 300 mg/kg body weight/day. In female rats, relative liver weight was increased at both 300 and 1000 mg/kg body weight/day and absolute liver weight was increased at 1000 mg/kg body weight/day. No gross or histopathological abnormalities were identified in liver, kidney or other organs.

At all dose levels, slight skin reactions were observed at a greater incidence in treated than the control animals and showed a dose response. Desquamation was present in the control, low-, mid- and high-dose groups at incidences of 33, 54, 83 and 96%, respectively. Erythema was not seen in control animals, but was present in treated rats in incidence of 38, 42 and 67%; 100, 300, 1000 mg/kg body weight/day, respectively. In the high dose rats, one had skin thickening and two had edema.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: At 300 mg/kg/d, increases in absolute and relative liver weight were observed in both sexes, but without histopathological findings reported for the liver (or other organs).
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOEL
Remarks:
local
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Increased rate of erythema was observed at all dose levels, but erythema Draize scores were not provided. Also, no histopathological changes in the skin were noted. Desquamation noted in all study animals, including controls.
Critical effects observed:
not specified

The 13-week study showed limited effects on body or organ weights or both in both sexes exposed to 300 or 1000 mg/kg body weight/day by dermal exposure. Histopathological findings were not present in any of the organs examined.

These results are consistent with the lack of toxicity observed in an oral toxicity study that was performed by Johnson et al. (1992). They administered DPO in the feed at dietary concentrations of 200, 1000 and 5000 ppm for 13 weeks. The top dose was equal to approximately 500-600 mg/kg body weight/day. The study by Johnson et al. (1992) found no compound-related effects on body weight, food consumption, hematology, serum chemistry, urinalysis, necropsy finding or histopathological examination of tissues and organs. There were some changes in body weight and associated changes in organ weights at the high dose level. These effects, however, were discounted because they were caused by a decrease in food consumption resulting from the decreased palatability of the diet at the 5000 ppm level.

In this study, male rats exposed to 1000 mg/kg body weight/day exhibited a statistically significant increase in relative brain weight, but absolute brain weight was

unaffected. A relative but not absolute brain weight is a result of body weight reduction and lacks biological significance (Feron et al., 1973; Oishi et al., 1979).

The increases in liver weights at the two higher doses in both sexes was not accompanied by any evidence of gross or histopathological findings of toxic effects, such as lesions or serum enzyme changes. Such increases in liver weight are commonly the result of microsomal enzyme induction and represent a physiological adaptation rather than an adverse effect (Glaister, 1986).

Increased relative kidney weight was observed only in the high-dose males, but not in any of the female groups. In addition, the increased relative kidney weight did not demonstrate a dose-related increase, and was not associated with evidence of renal dysfunction or histopathological lesions. Taking all these finding together, the kidney weight changes appear to be questionable and lack biological significance. There were adverse effects on the skin at all dose levels. Subchronic exposure produced limited effects.

Conclusions:
The systemic no-observed-efect level (NOEL) in this study is 100 mg/kg/day. Adaptive changes were observed at 1000 mg/kg/day. Organ weight changes were judged to lack biological significance and the no-observed adverse-effect level (NOAEL) was determined to be 1000 mg/kg/day by the authors.
Executive summary:

Diphenyl ether (DPE) was investigated in a dermal subchronic toxicity. The 13-week study was performed with groups of 12 Sprague-Dawley rats/sex/dose that received semi-occluded daily dermal applications of DPO for 6 h/day. All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the DEP vehicle at concentrations to administer 0, 100, 300 or 1000 mg/kg/day. At the high dose level, there was a slight reduction in body weight gain in males (13%) that was not reported to be statistically significant, increase in albumin (5-6%) and phosphate (10-15%) levels in both sexes, a reduction of cholesterol in females (14%), an increase in kidney (17%) and brain (8%) weights in males, and an increase in liver weight (18-19%) in both sexes. No histopathological lesions were seen in any organ examined, including skin. At 300 mg/kg body weight/day, the only notable findings were an increase in liver weight (10%) in both sexes and a slight increase in albumin (5%) in females. In addition, increased rate of skin irritation reactions at the site of application was observed in all DPO dose groups (Draize scores not given). The systemic no-observed-effect level (NOEL) in this study is 100 mg/kg/day. The systemic findings were judged to lack biological significance and the no-observed-adverse-effect level (NOAEL) was determined to be 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
acceptable quality

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The publication lists no information on guideline/s and GLP but the report contains sufficient data for interpretation of study results.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The 13-week subchronic toxicity study was performed with groups of 12 Sprague-Dawley rats/sex/dose. Rats received semi-occluded daily dermal applications of DPO for 6 h/day. All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the diethyl phthalate (DEP) vehicle at concentrations to administer 0, 100, 300 or 1000 mg DPO/kg body weight/day.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Sprague-Dawley CD rats were obtained from Charles River (UK) Ltd (Margate, Kent, UK).
Type of coverage:
semiocclusive
Vehicle:
other: diethyl phthalate
Details on exposure:
Four groups of 12 male and 12 female Sprague-Dawley rats were used. The hair on their backs was clipped before dosing and then twice weekly. Three groups were dosed daily with DPO via the dermal route of application using a semiocclusive dressing for a period of 6 h/day for 13 weeks. Doses of 2 ml/kg were applied to gauze squares ( ~ 4 cm x 4 cm) with an aluminium foil back. These patches were placed on the shaved skin on the animals' backs and held in place by a semi-occlusive dressing (50 mm Micropore, 3M) and tape (50 mm Blenderm, 3M). The gauze squares and dressing were removed after 6 h, and the dosed area was washed with DEP to remove the non-absorbed DPO. The rats were dosed at a constant volume of 2 ml/kg body weight and concentrations in DEP were calculated to achieve dose levels of 100, 300 or 1000 mg/kg body weight/day.
One control group (12 males/12 females) received DEP at a dose volume of 2 ml/kg.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day
Remarks:
Doses / Concentrations:
Solvent control, 100, 300 or 1000 mg/kg body weight/day
Basis:
other: nominal
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The 13-week subchronic toxicity study was performed with groups of 12 Sprague-Dawley rats/sex/dose. Rats received semi-occluded daily dermal applications of DPO for 6 h/day. All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the diethyl phthalate (DEP) vehicle at concentrations to administer 0, 100, 300 or 1000 mg/kg bw/day.

During the study, the animals were assessed for general clinical signs, skin irritation (erythema, eschar, edema and thickening), body weight, food consumption and water consumption. During week 13, samples from 10 males and 10 females from all groups were taken for urinalysis, hematology and clinical blood chemistry parameters.

After 13 weeks of dosing, animals were killed by CO2 narcosis, and necropsied. Selected organs were examined and weighed. Histopathologic
examinations were performed on major tissues from all animals in the control and high-dose groups; kidneys from all dose groups were examined histopathologically.

Hematology, clinical chemistry, organ weight and body weight data were statistically analyzed for homogeneity of variance by using the 'F-max' test. If the group variances appeared homogeneous a parametric ANOVA was used, and pair wise conparisons were made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, a non-parametric test such as Kruskal Wallis ANOVA was used. Organ weights were also analyzed conditional on body weight (i.e. covariance and relative analyses). Histology data were analyzed using Fisher's Exact Probability test.
Positive control:
No data
Observations and examinations performed and frequency:
During the study, the animals were assessed for general clinical signs, skin irritation (erythema, eschar, edema and thickening), body weight, food consumption and water consumption.
Sacrifice and pathology:
During week 13, samples from 10 males and 10 females from all groups were taken for urinalysis, hematology and clinical blood chemistry parameters. After 13 weeks of dosing, animals were killed by CO2 narcosis, and necropsied. Selected organs were examined and weighed. Histopathologic
examinations were performed on major tissues from all animals in the control and high-dose groups; kidneys from all dose groups were examined histopathologically.
Other examinations:
No data
Statistics:
Hematology, clinical chemistry, organ weight and body weight data were statistically analyzed for homogeneity of variance by using the 'F-max' test. If the group variances appeared homogeneous a parametric ANOVA was used, and pair wise conparisons were made via Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, a non-parametric test such as Kruskal Wallis ANOVA was used. Organ weights were also analyzed conditional on body weight (i.e. covariance and relative analyses). Histology data were analyzed using Fisher's Exact Probability test.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the DEP vehicle at concentrations to administer 0, 100, 300 or 1000 mg/kg bw/day for 13-weeks. In male rats exposed to the high dose, there was a slight reduction in body weight, and a statistically significant increase in liver, brain and kidney weight relative to body weight. Absolute and relative liver weight was increased at 300 mg/kg body weight/day. In female rats, relative liver weight was increased at both 300 and 1000 mg/kg body weight/day and absolute liver weight was increased at 1000 mg/kg body weight/day. No gross or histopathological abnormalities were identified in liver, kidney or other organs.

At all dose levels, slight skin reactions were observed at a greater incidence in treated than the control animals and showed a dose response. Desquamation was present in the control, low-, mid- and high-dose groups at incidences of 33, 54, 83 and 96%, respectively. Erythema was not seen in control animals, but was present in treated rats in incidence of 38, 42 and 67%; 100, 300, 1000 mg/kg body weight/day, respectively. In the high dose rats, one had skin thickening and two had edema.
Dose descriptor:
NOEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: At 300 mg/kg/d, increases in absolute and relative liver weight were observed in both sexes, but without histopathological findings reported for the liver (or other organs).
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOEL
Remarks:
local
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Increased rate of erythema was observed at all dose levels, but erythema Draize scores were not provided. Also, no histopathological changes in the skin were noted. Desquamation noted in all study animals, including controls.
Critical effects observed:
not specified

The 13-week study showed limited effects on body or organ weights or both in both sexes exposed to 300 or 1000 mg/kg body weight/day by dermal exposure. Histopathological findings were not present in any of the organs examined.

These results are consistent with the lack of toxicity observed in an oral toxicity study that was performed by Johnson et al. (1992). They administered DPO in the feed at dietary concentrations of 200, 1000 and 5000 ppm for 13 weeks. The top dose was equal to approximately 500-600 mg/kg body weight/day. The study by Johnson et al. (1992) found no compound-related effects on body weight, food consumption, hematology, serum chemistry, urinalysis, necropsy finding or histopathological examination of tissues and organs. There were some changes in body weight and associated changes in organ weights at the high dose level. These effects, however, were discounted because they were caused by a decrease in food consumption resulting from the decreased palatability of the diet at the 5000 ppm level.

In this study, male rats exposed to 1000 mg/kg body weight/day exhibited a statistically significant increase in relative brain weight, but absolute brain weight was

unaffected. A relative but not absolute brain weight is a result of body weight reduction and lacks biological significance (Feron et al., 1973; Oishi et al., 1979).

The increases in liver weights at the two higher doses in both sexes was not accompanied by any evidence of gross or histopathological findings of toxic effects, such as lesions or serum enzyme changes. Such increases in liver weight are commonly the result of microsomal enzyme induction and represent a physiological adaptation rather than an adverse effect (Glaister, 1986).

Increased relative kidney weight was observed only in the high-dose males, but not in any of the female groups. In addition, the increased relative kidney weight did not demonstrate a dose-related increase, and was not associated with evidence of renal dysfunction or histopathological lesions. Taking all these finding together, the kidney weight changes appear to be questionable and lack biological significance. There were adverse effects on the skin at all dose levels. Subchronic exposure produced limited effects.

Conclusions:
The systemic no-observed-efect level (NOEL) in this study is 100 mg/kg/day. Adaptive changes were observed at 1000 mg/kg/day. Organ weight changes were judged to lack biological significance and the no-observed adverse-effect level (NOAEL) was determined to be 1000 mg/kg/day by the authors.
Executive summary:

Diphenyl ether (DPE) was investigated in a dermal subchronic toxicity. The 13-week study was performed with groups of 12 Sprague-Dawley rats/sex/dose that received semi-occluded daily dermal applications of DPO for 6 h/day. All groups were dosed at a constant 2 ml/kg body weight volume of DPO in the DEP vehicle at concentrations to administer 0, 100, 300 or 1000 mg/kg/day. At the high dose level, there was a slight reduction in body weight gain in males (13%) that was not reported to be statistically significant, increase in albumin (5-6%) and phosphate (10-15%) levels in both sexes, a reduction of cholesterol in females (14%), an increase in kidney (17%) and brain (8%) weights in males, and an increase in liver weight (18-19%) in both sexes. No histopathological lesions were seen in any organ examined, including skin. At 300 mg/kg body weight/day, the only notable findings were an increase in liver weight (10%) in both sexes and a slight increase in albumin (5%) in females. In addition, increased rate of skin irritation reactions at the site of application was observed in all DPO dose groups (Draize scores not given). The systemic no-observed-effect level (NOEL) in this study is 100 mg/kg/day. The systemic findings were judged to lack biological significance and the no-observed-adverse-effect level (NOAEL) was determined to be 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2.5 mg/cm²
Study duration:
subchronic
Species:
rat
Quality of whole database:
acceptable quality

Additional information

Oral: In the GLP, guideline 1990 key study (ks) (Klimisch=1), dietary administration of Diphenyl Oxide (DPO) 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.

Dermal: In the 2003 ks (Klimisch=2, no GLP or guideline information), the systemic no-observed-effect level (NOEL) in this study is 100 mg/kg body weight/day. Systemic toxicity was manifested by decreased body weight at 1000 mg/kg body weight/day. Organ weight changes were judged to lack biological significance and the no-observed adverse-effect level (NOAEL) was determined to be 1000 mg/kg body weight/day.

Inhalation: In the only available inhalation study, rats, rabbits and dogs were exposed to DPO at mean exposure concentrations of 0, 4.9 and 10.0ppm vapour. Exposures were 7 hours per day, 5 days per week for a total of 20 exposures. An additional group of rats (10 per sex) were exposed to 0 or 20 ppm DPO for a total of 20 exposures. No untoward effects were observed in any of the animals exposed to 4.9ppm (139 mg/m3) vapour. However at 10 and 20 ppm, eye and nasal irritation were observed in rabbits (10 ppm) and rats (20 ppm). Aside from this irritation no other effects were discerned.

Considering the available data, DPO appears to have minimal systemic toxicity following repeated dosing via inhalation, oral and dermal routs. In fact, the highest dose tested appears to have been the limiting factor in deriving a NOAEL for systemic toxicity.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP, guideline study with K1 score; see discussion

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
see discussion

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
see discussion

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
see discussion

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
see discussion

Justification for classification or non-classification

Not classified - Based on a subchronic NOAEL of 301 mg/kg/day in rats following oral administration of DPO for 13 and a subchronic NOAEL of 1000 mg/kg/day in rats dermally exposed to DPO.