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Repeated dose toxicity: oral

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Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 July 1981 to 22 February 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Non-GLP study published by recognised Governmental body (US National Cancer Institute). Study and results are well documented with full statistical analysis included. This study is used in place of sub acute / sub chronic study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Unspecified.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Non-GLP study published by recognised Governmental body (US National Cancer Institute). Study and results are well documented with full statistical analysis included. This study is used in place of sub acute / sub chronic study required under section 7.5 of this data set.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
No specified guideline is stated within the report.

The basis of the study includes:
> a range finding study (to assess dose levels for 14 sub-acute range finder).
> a 14 day sub-acute range finder to assess dose levels for 90-day range finder.
> a 90-day range finder in order to specify dose levels for the 23 month study assessed.

The 23 month study was conducted in rodents. Rats (5 weeks old at study start) were fed diets containing appropriate levels of the test substance ad libitum 7 days/week for 103 weeks. Rats were housed 4/cage. All animals were observed twice daily for signs of toxicity. Mean body weights of animals by cage were recorded every 2 weeks for the first 13 weeks, and monthly thereafter. Clinical signs were recorded monthly. Moribund animals and animals that survived to the end of the study were killed and necropsied. Animals that were found dead were necropsied, unless precluded by autolysis or cannibalization. Examinations for grossly visible lesions were performed on major tissues or organs. Tissues were preserved, and 41 and 42 tissues were examined microscopically in rats and mice, respectively.
Analyses of the stability of 4,4’-oxydianiline in feed were performed by assaying dimethyl formamide extracts from samples of diet mixtures containing 100,000 ppm that had been stored at -20º, 5º, 25º, or 45ºC for 2 weeks. The concentrations of the test substance in the extracts were determined by vapor-phase chromatography. Selected batches of the formulated diets (200 and 800 ppm) administered during the study were analyzed for accuracy of dose level by spectrophotometric analysis.
GLP compliance:
no
Specific details on test material used for the study:
4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: NCI Frederick Cancer Research Center (Frederick, Maryland)
- Age at study initiation: Rats: 4 weeks at receipt, then observed for 10 days for the presence of parasites or other diseases, and then assigned to various groups so that the average initial body weight per group was approximately the same.
- Weight at study initiation: Rats: Males: 87g Females: 82g
- Fasting period before study: None
- Housing: solid-bottom polycarbonate cages (Lab Products, Inc., Garfield, New Jersey) containing Aspenbed^(aspen chips, American Excelsior, Summerville, Mass.). Rat cages were covered with a nonwoven fiber filter (Webrex). Rats were housed five per cage in the Subchronic studies and four per cage in the chronic studies. Rats were housed in separate rooms, and control animals were housed in the same room as the respective dosed animals. Cages, bottles, sipper tubes, and stoppers were changed twice per week. Feed hoppers were changed once per week. Stainless steel cage racks and the disposable filters were changed once every 2 weeks.
- Diet (e.g. ad libitum): Wayne Lab Blox animal meal (Allied Mills, Chicago, 111.); milled. Ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 -32 C
- Humidity (%): 5%-82%
- Air changes (per hr): 10 to 12 changes of room air per hour
- Photoperiod (hrs dark / hrs light): 12 hours : 12 hours.

IN-LIFE DATES: Not specified.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Test diets were prepared by first mixing the chemical with an aliquot of Wayne Lab Blox animal meal (Allied Mills, Chicago, 111.) using a mortar and pestle. This mixture was placed in a Patterson Kelly twin-shell blender with the remainder of the feed and mixed for 20 minutes. Test diets were sealed in labelled plastic bags.
- Storage temperature of food: 4 C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the stability of 4,4'-oxydianiline in feed were performed at Midwest Research Institute by assaying dimethyl formamide extracts from samples of diet mixtures containing 100,000 ppm that had been stored at -20 , 5°, 25°, or 45°C for 2 weeks. The concentrations of the test chemical in the extracts were determined by vapor-phase chromatography. 4,4'-Oxydianiline at 100,000 ppm was stable in feed for 2 weeks at 45°C.
Selected batches of the formulated diets administered during the chronic study were analyzed at EG&G Mason Research institute for accuracy of dose level. The test feeds were first extracted with 95% ethanol, and concentrations of the test chemical in the extracts were determined by spectrophotometric analysis at 247 nm. The mean concentration of 12 feed samples containing a theoretical level of 200 ppm was 200 +/-29 ppm, and the mean concentration of 14 samples measured in duplicate and containing a theorical level of 800 ppm was 780 +/-103 ppm.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
Ad libitum 7 days/week
Post exposure period:
None specified.
Dose / conc.:
200 ppm (nominal)
Remarks:
in the diet
Dose / conc.:
400 ppm (nominal)
Remarks:
in the diet
Dose / conc.:
500 ppm (nominal)
Remarks:
in the diet
No. of animals per sex per dose:
50 per species per sex per dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The dose selected for the carcinogenicity study were based on the resuts from the Range-Finding and 14-Day Repeated-Dose Studies and from the 90-day subchronic study.

> Range-Finding and 14-Day Repeated-Dose Studies
In the range-finding study conducted to determine the doses for the 14-day repeated-dose study, the test chemical was diluted in corn oil and administered by gavage to groups of two males and two females. The animals received the dose levels of 100 - 300 - 1,000 - 3,000 and 10,000 mg/kg bw. They were observed for 7 days and then killed and necropsied. To solubilize the 4,4'-oxydianiline, the 10,000 mg/kg dose was prepared in 10% DMSO (dimethyl sulfoxide) in corn oil.
All male rats receiving the three highest doses (1,000, 3,000, and 10,000 mg/kg bw) and all female rats receiving the two highest doses (3,000 and 10,000 mg/kg bw) died.
Intestinal hemorrhage was observed in rats at the two highest doses. Labored respiration was observed in the rats receiving the three highest doses of 1,000, 3,000, and 10,000 mg/kg bw.

In the fourteen-day repeated-dose studies conducted to determine the doses to be used in the 90-day subchronic studies, groups of five males and five females were fed diets containing the different concentrations of the test chemical. The animals received the dose levels of 300 - 1,000 - 3,000 - 10,000 and 30,000 ppm. They were observed daily, and after 14 days, all survivors were killed and necropsied.
All rats receiving 10,000 or 30,000 ppm and 4/5 male rats and 5/5 female rats receiving 3,000 ppm died. The LD50 calculated for male rats was 2,240 ppm and 1,730 ppm for females.
4,4'-Oxydianiline caused liver enlargement at all doses, jaundice at the two highest doses, hemorrhages of the digestive tract at the three highest doses, and hemorrhages of the renal medullae at the highest dose. Rats receiving doses greater than 1,000 ppm were emaciated because of decreased food consumption.

> Subchronic Studies
In 90-day subchronic feeding studies conducted to determine the concentrations of 4,4'-oxydianiline to be used in the chronic 2-year studies, groups of 10 males and 10 females were fed diets containing 0 - 3 - 10 - 30 - 100 or 300 ppm for 90 days. All animals were observed twice daily for mortality. Individual animal weights, food consumption, appear-ance, and behavior were recorded weekly. After 13 weeks, all the animals were killed and necropsied. Representative tissues were examined microscopically. Because no compound-related clinical signs, body weight changes, or pathologic changes were observed, a second subchronic study was carried out using diets containing 0 - 300 - 600 - 1,000 and 2,000 ppm.
A dose-related increase in mortality and decrease in weight gain were observed in both sexes of rats, and alopecia, labored respiration, and cyanosis were observed with the two highest doses (1,000 and 2,000 ppm). All rats receiving 600 ppm or more had diffuse parenchymatous goiter. In addition, pituitary hyperplasia, testicular degeneration, prostatic atrophy, seminal vesicular atrophy, and renal microlithiasis were detected in most of the rats receiving 600 ppm or more.
Because of the weight gain depression and the thyroid effects observed in the second subchronic study, doses selected for the chronic study in rats were 200, 400, and 500 ppm. The highest dose (500 ppm) was included to enhance the possiblity of detecting a thyroid response.

Chronic Study (carcinogenicity study):
On the basis of the results obtained in the Range-Finding and 14-Day Repeated-Dose Studies and in the 90-day subchronic study, the chronic study was conducted as follows:
Number of animals per group: 50.
Doses administered were 0 - 200 - 400 and 500 ppm.
Duration: 103 weeks.
Positive control:
None.
Observations and examinations performed and frequency:
All animals were observed twice daily for signs of toxicity. Mean body weights of animals by cage were recorded every 2 weeks for the first 13 weeks and monthly thereafter. Clinical signs were recorded monthly. Mori-bund animals and animals that survived to the end of the bioassay were killed using carbon dioxide and necropsied.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: Monthly

BODY WEIGHT: Yes
- Time schedule for examinations: every 2 weeks for the first 13 weeks and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Examinations for grossly visible lesions were performed on major tissues or organs. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Prepar-ations of the following were examined microscopically: tissue masses, abnormal lymph nodes, skin, mandibular lymph nodes, mammary gland, salivary gland, thigh muscle, sciatic nerve, bone marrow, costochondral junction (rib), thymus, larynx, trachea, lungs and bronchi, heart, thyroid, parathy-roid, esophagus, stomach, duodenum, jejunum, ileum, colon, mesenteric lymph nodes, liver, gallbladder (mice), pancreas, spleen, kidneys, adrenals, bladder, seminal vesicles/prostate/testes, ovaries/uterus, nasal cavity, brain, pituitary, eyes, and spinal cord. Special staining techniques were utilized as necessary.

Necropsies were performed on all animals found dead, unless precluded in whole or in part by autolysis or cannibalization. Thus, the number of ani-mals from which particular organs or tissues were examined microscopically varies and does not necessarily represent the number of animals that were placed on study in each group.
Other examinations:
None
Statistics:
Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958) and are presented in the form of graphs. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone’s (1975) extensions of Cox's methods for testing for a dose-related trend.
As a part of the tumour analyses, the one-tailed Fisher exact test (Cox, 1970) was used to compare the tumour incidence of a control group with that of a group of dosed animals at each dose level. When results for three dosed groups are com¬pared simultaneously with those for a control group, a correction to ensure an overall significance level of 0.05 may be made. The Bonferroni inequality (Miller, 1966) requires that the P value for any comparison be less than or equal to 0.017.
The Cochran-Armitage test for linear trend in proportions, with continuity correction (Armitage, 1971), was also used. When a linear trend is assumed, this test determines if the slope of the dose-response curve is different from zero at the one-tailed 0.05 level of significance. Unless otherwise noted, the direction of the significant trend is a positive relationship. This method also provides a two-tailed test of departure from linear trend.
The approximate 95% confidence interval for the relative risk of each dosed group compared with its control was calculated from the exact interval on the odds ratio (Gart, 1971).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Labored breathing was observed in all the female rats receiving the highest dose (500 ppm).
Mortality:
mortality observed, treatment-related
Description (incidence):
In male rats, results of the Tarone test for positive dose-related trend in mortality indicate no significant differences.
However, this test indicates that there was a significantly shortened survival in the high-dose female rats (P less than 0.001) when compared with any of the other groups (52% of the high dose group lived to 78 weeks compared with over 90% in the other groups). At the end of the study (weeks 105 to 106), the female survivors included 40/50 (80%) of the matched controls, 38/50 (76%) of
the low-dose group, 34/50 (68%) of the mid-dose group, and 13/50 (26%) of the high-dose group. Survival in the low- and mid-dose female rats and in the matched controls was comparable.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A dose-related depression in mean body weight gain was observed for all groups of dosed rats.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Nonneoplastic lesions were observed in males and in female rats.

In male rats, the main findings observed are:
> inflammation of lungs in 10% of animals in low and mid dose groups, in 16% of animals in high dose group (0% in the control group)
> lesion of the hematopoietic system observed in the spleen in 16% of animals in low dose group, 20% of animals in the mid dose group and 16 % in the high dose group (6% in the control group)
> cystic hepatic lesions in 8% of animals in the mid dose group and 6% in the high dose group (0% in the control group)
> mineralization in kidney in 2% of animals in the low dose group and in 22% of animals in the high dose group (0% in the control group)
> epithelial hyperplasia in kidney in 4%, 8% and 14% of animals in the low, mid and high dose groups respectiveley (2% in the control group)
> follicular cyst in thyroid in 24% and 6% of animals in the mid and high dose groups respectively (0% in the control group) and follicular-cell hyperplasia in 2%, 24% and 26% of animals in the low, mid and high dose groups respectively (0% in the control group); C-cell hyperplasia in 4 and 7% of animals in low and mid dose groups (2% in the control group)
> testis mineralization in 2%, 4% and 8% in the low, mid and high dose groups respectively (0% in the control group); testis atropy in 2%, 6% and 14% of animals in the low, mid and high dose groups respectively (4% in the control group); interstitial cell hyperplasia in testis in 6% of animals in the high dose group (0% in the control group)

In female rats, the main findings observed are:
> inflammation of lungs in 4%, 12% a,d 12% of animals in low, mid and high dose groups respectively (4% in the control group)
> hemosiderosis in the spleen in 10% of animals in the high dose group (0% in the control group or other treated groups)
> mineralization in kidney in 20%, 14% and 33% of animals in the low, mid and high dose group respectively (6% in the control group) and focal calcification in 29% of animals in the high dose group (0% in the control group)
> epithelial hyperplasia in kidney (pelvis) in 4%, 10% and 8% of animals in the low, mid and high dose groups respectiveley (0% in the control group)
> pituitary hyperplasia in 11% of animals in the high dose group (0% in the control group)
> follicular-cell hyperplasia in 2%, 13% and 44% of animals in the low, mid and high dose groups respectively (0% in the control group)
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below, "Details on results"
Other effects:
not specified
Details on results:
CLINICAL SIGNS
Labored breathing was observed in all the female rats receiving the highest dose (500 ppm).

MORTALITY
Over 90% of the male rats in each group lived to 78 weeks or more. Those surviving to the end of the study at 105 to 106 weeks included 25/50 (50%) of the matched controls, 34/50 (68%) of the low-dose, 35/50 (70%) of the mid-dose, and 30/50 (60%) of the high-dose group.
The high-dose female rats died earlier than did those in the other female groups, and only 52% of this group lived to 78 weeks, compared with over 90% in the other groups. At the end of the study (weeks 105 to 106), the survivors included 40/50 (80%) of the matched controls, 38/50 (76%) of the low-dose group, 34/50 (68%) of the mid-dose group, and 13/50 (26%) of the high-dose group.

BODY WEIGHT AND WEIGHT GAIN
A dose-related depression in mean body weight gain was observed for all groups of dosed rats. Labored breathing was observed in all the female rats receiving the highest dose (500 ppm). The incidence of exophthalmia was comparable in dosed and control groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Noted as reduced intake during the range finding and chronic studies; reflected by reduced weight gain.

HAEMATOLOGY
See below under "GROSS PATHOLOGY & HISTOPATHOLOGY: NON-NEOPLASTIC AND NEOPLASTIC".

ORGAN WEIGHTS
Not detailed specifically. Increased liver weights and thyroid were noted in

GROSS PATHOLOGY & HISTOPATHOLOGY: NON-NEOPLASTIC AND NEOPLASTIC
A variety of neoplasms were seen in control and dosed rats. None was associated with the test chemical except for those of the thyroid gland and liver.
The thyroid glands of most of the treated rats were grossly enlarged. Histologically, a cyst was considered to be follicular when it contained eosinophilic or pale colloid and was lined by cuboidal epithelial cells. A diffuse follicular enlargement or papillary ingrowths of the epithelium producing follicles of various sizes were characteristic features of follicular hyperplasia. The epithelial cells were either cuboidal or columnar.
Follicular neoplasms of the thyroid gland occurred in one control and in 107 treated rats. Follicular adenomas were encapsulated and they compressed the adjacent tissue. Both macro- and micro-follicular variants were observed. Colloid was conspicuous in macrofollicular tumors. The epithelial cells were cuboidal or columnar. Cytoplasm of the cells was homogeneous and the nuclei were hyperchromatic. The infiltration of tumor cells in the capsule and/or blood vessel was considered essential for the diagnosis of follicular carcinoma. The carcinomas involved one or both lobes and compressed the trachea in a few rats. Papillary arrangement of the cells was common in many tumors. Squamous metaplasia was noted in one tumor.
Areas of necrosis were common in the large tumors. Foci of mineralization, cholesterol clefts, and golden brown pigment were additional features. Stroma was hyalinized in 20 tumors, and stromal reaction (as evidenced by presence of fibroblasts) was found in a few tumors. Follicular carcinoma had metastasized to the lung in two female rats (one mid-dose; one high-dose).
Neoplastic nodules and carcinomas occurred in the livers of some treated rats. Multiple neoplastic nodules compressed the adjacent hepatic tissue. Cells in the nodules were larger than the normal hepatocytes. Cytoplasm of the cells was either acidophilic, basophilic, or vacuolated. Nuclei were vesicular and hyperchromatic. Hepatocellular carcinoma was well differentiated and involved a part or an entire lobe of the liver. Fibrous tissue septa separated the tumor parenchyma into nodules of various sizes. Acinar and trabecular forms were observed in these tumors. As in the neoplastic nodules, cytoplasm of the cells was acidophilic, basophilic, or vacuolated.
Nuclei were hyperchromatic, and the nucleoli were prominent. Both normal and abnormal mitotic figures were present. Multinucleate cells were found in a few tumors. Hemorrhage and necrosis occurred in the large tumors.
In some of the hepatocellular neoplasms in rats of the mid-dose and high-dose groups, the tumor cells appeared to have undergone cystic degeneration. Such areas contained a few blood cells and/or a lacy material which stained light blue.
Except for focal mineralization of the kidney and transitional cell hyperplasia of the renal pelvis in a few treated rats, there were no other chemical-related nonneoplastic lesions.
The results of the histopathologic examination indicated that, under conditions of this study, 4,4'-oxydianiline was carcinogenic to F344 rats, causing both increased incidences of follicular-cell neoplasms of the thyroid gland and liver neoplasms.

OTHER FINDINGS
Statistical analysis was undertaken on the primary tumors that occurred in at least two animals of one group and at an incidence of at least 5% in one or more than one group.

The Cochran-Arraitage test indicates significant dose-related trends (P less than 0.001) in the incidence of animals with follicular-cell adenomas or carcinomas in the thyroid of both sexes. The incidences in the mid and the high-dose groups of either sex are significantly higher (P less than
0.001) than in the control group. The historical incidence in the bioassay program is 25/2,230 (1.1%) in male F344 rats and 12/2,194 (less than 1%) in females as compared with the incidences of the control group (2% in male F344 rats and 4% in female F344 rats) which were observed in this study.
In male rats, the Cochran-Armitage test indicates a significant positive trend (P less than 0.001) in the incidence of animals with hepatocellular carcinomas or neoplastic nodules and a departure from linear trend due to sharp increases in the two higher dosed groups. The incidences in all the dosed groups were significantly higher (P less than 0.001) than the incidence in the control group. The historical incidence in the bioassay program, accumulated to date, in male F344 rats with these types of tumors from all laboratories, is 26/2,230 (1.2%). This incidence is comparable with the 1/50 (2%) observed in this control group. In female rats, a significant positive trend (P less than 0.001) in the incidence of animals with neoplastic nodules or hepatocellular carcinomas was observed. The incidences in the high-dose group and mid-dose group were significantly higher (P less than 0.001) than the incidence in the controls. The historical incidence in the bioassay program, accumulated to date, in female F344 rats with these types of tumors is 25/2,194 (less than 1%).

In male rats, the incidence of leukemias occurs with a negative trend (P less than 0.001) with significantly lower incidence (P less than 0.001) in each of the dosed groups than in the control group. For male F344 rats, the historical incidence accumulated from all laboratories is lower (235/2,230 or 10%) than the incidence in the control group of male rats (23/50 or 46%) observed in this study. In females, the Cochran-Armitage test indicates a significant negative trend (P=0.019) as a result of lower incidence (P=0.028) in the high-dose group than in the control group. This may be a consequence of the early mortality observed in the high-dose group females.
A negative trend (P less than 0.001) and a significantly lower incidence (P less than 0.030) of fibroadenomas in the mammary gland of the dosed groups were observed in female rats. The control group incidence of 16/50 (32%) is almost double the historical incidence of 378/2,194 (17%) for this type of lesion.
The incidences of female rats with endometrial stromal polyps or sarcomas of the uterus and tumors of the pituitary are lower in the high dose group than in the control group. These results may have been affected by the shortened survival in the high-dose group.

The statistical analyses indicate that the occurrence of liver and thyroid tumors in both sexes of rats is related to the administration of 4,4'-oxydianiline.
Relevance of carcinogenic effects / potential:
Under the conditions of this bioassay, 4,4'-Oxydianiline was carcinogenic for male and female F344 rats, inducing hepatocellular carcinomas or neoplastic nodules and follicular-cell adenomas or carcinomas of the thyroid.
Key result
Dose descriptor:
dose level:
Effect level:
200 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
histopathology: non-neoplastic
histopathology: neoplastic
Remarks on result:
other: Effect type: carcinogenicity
Key result
Dose descriptor:
dose level:
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
histopathology: non-neoplastic
histopathology: neoplastic
Remarks on result:
other: Effect type: carcinogenicity
Key result
Dose descriptor:
dose level:
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
histopathology: non-neoplastic
histopathology: neoplastic
Remarks on result:
other: Effect type: carcinogenicity
Key result
Critical effects observed:
yes
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

The following table contains the incidences of the above mentioned tumors in male and female rats.

 

Dose (ppm)

0

200

400

500

Hepatocellular carcinoma:

 

 

 

 

Males

0/50

4/50

23/50

22/50

Females

0/50

0/49

4/50

6/50

 

Hepatocellular neoplastic nodule:

Males

1/50

9/50

18/50

17/50

Females

3/50

0/49

20/50

11/50

 

Hepatocellular carcinoma or neoplastic nodule:

Males

1/50

13/50

41/50

39/50

Females

3/50

0/49

24/50

17/50

 

Thyroid follicular cell adenoma:

Males

1/46

1/47

8/46

13/50

Females

0/49

2/48

17/48

16/50

 

Thyroid follicular cell carcinoma:

Males

0/46

5/47

9/46

15/50

Females

0/49

2/48

12/48

7/50

 

Thyroid follicular cell adenoma or carcinoma:

Males

1/46

6/47

17/46

28/50

Females

0/49

4/48

29/48

23/50

 

The following table contains incidences of the non-neoplastic lesions:

 

Kidney mineralization:

Males

0/50

1/50

0/50

11/50

Females

3/49

10/50

7/50

16/49

 

Epithelial hyperplasia of renal pelvis:

Males

1/50

2/50

4/50

7/50

Females

0/49

2/50

5/50

4/49

  

Conclusions:
Under the conditions of this study, the test substance was carcinogenic to F344 rats, causing both increased incidences of follicular-cell neoplasms of the thyroid gland and liver neoplasms in rats.
Executive summary:

A study for assessment of possible carcinogenicity was conducted on the test item by feeding diets containing 200, 400, or 500 ppm of the test chemical to groups of 50 male or female F344 rats for 104 weeks.

Matched controls consisted of 50 untreated rats. All surviving animals were killed at 104 to 105 weeks.

A dose-related decrement in mean body weight gain was observed for all groups of dosed rats. Survival was significantly shortened in the high-dose female rats.

In male and female rats, hepatocellular carcinomas or neoplastic nodules occurred at incidences that were dose-related, and the incidences in all dosed groups (except low-dose females) were higher than those in the controls.

The occurrence of follicular-cell adenomas or carcinomas of the thyroid was dose-related. Among groups of male and female rats,.the incidences in the mid- and high-dose groups of either sex were significantly higher than those of the corresponding controls.

Under the conditions of this bioassay, 4,4'-oxydianiline was carcinogenic for male and female F344 rats, inducing hepatocellular carcinomas or neoplastic nodules and follicular-cell adenomas or carcinomas of the thyroid.

Reason / purpose:
reference to same study
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Unspecified.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Non-GLP study published by recognised Governmental body (US National Cancer Institute). Study and results are well documented with full statistical analysis included. This study is used in place of sub acute / sub chronic study required under section 7.5 of this data set.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
No specified guideline is stated within the report.

The basis of the study includes:
> a range finding study (to assess dose levels for 14 sub-acute range finder).
> a 14 day sub-acute range finder to assess dose levels for 90-day range finder.
> a 90-day range finder in order to specify dose levels for the 23 month study assessed.

The 23 month study was conducted in rodents. Mice (6 weeks old at study start) were fed diets containing appropriate levels of the test substance ad libitum 7 days/week for 103 weeks. Mice were housed 5/cage during the study. All animals were observed twice daily for signs of toxicity. Mean body weights of animals by cage were recorded every 2 weeks for the first 13 weeks, and monthly thereafter. Clinical signs were recorded monthly. Moribund animals and animals that survived to the end of the study were killed and necropsied. Animals that were found dead were necropsied, unless precluded by autolysis or cannibalization. Examinations for grossly visible lesions were performed on major tissues or organs. Tissues were preserved, and 41 and 42 tissues were examined microscopically in rats and mice, respectively.
Analyses of the stability of 4,4’-oxydianiline in feed were performed by assaying dimethyl formamide extracts from samples of diet mixtures containing 100,000 ppm that had been stored at -20º, 5º, 25º, or 45ºC for 2 weeks. The concentrations of the test substance in the extracts were determined by vapor-phase chromatography. Selected batches of the formulated diets (200 and 800 ppm) administered during the study were analyzed for accuracy of dose level by spectrophotometric analysis.
GLP compliance:
no
Specific details on test material used for the study:
4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: NCI Frederick Cancer Research Center (Frederick, Maryland)
- Age at study initiation: Mice: 6 weeks
- Weight at study initiation: Mice: Males: 17.3g Females: 15.2g
- Fasting period before study: None
- Housing: solid-bottom polycarbonate cages (Lab Products, Inc., Garfield, New Jersey) containing Aspenbed^(aspen chips, American Excelsior, Summerville, Mass.). Mouse cages were covered with spun-bonded Filtek^ filter bonnets (Lab Products). Mice were housed five per cage. Mice were housed in separate rooms, and control animals were housed in the same room as the respective dosed animals. Cages, bottles, sipper tubes, and stoppers were changed twice per week. Feed hoppers were changed once per week. Stainless steel cage racks and the disposable filters were changed once every 2 weeks.
- Diet (e.g. ad libitum): Wayne Lab Blox animal meal (Allied Mills, Chicago, 111.); milled. Ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 -32 C
- Humidity (%): 5%-82%
- Air changes (per hr): 10 to 12 changes of room air per hour
- Photoperiod (hrs dark / hrs light): 12 hours : 12 hours.

IN-LIFE DATES: Not specified.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Test diets were prepared by first mixing the chemical with an aliquot of Wayne Lab Blox animal meal (Allied Mills, Chicago, 111.) using a mortar and pestle. This mixture was placed in a Patterson Kelly twin-shell blender with the remainder of the feed and mixed for 20 minutes. Test diets were sealed in labelled plastic bags.
- Storage temperature of food: 4 C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the stability of 4,4'-oxydianiline in feed were performed at Midwest Research Institute by assaying dimethyl formamide extracts from samples of diet mixtures containing 100,000 ppm that had been stored at -20 , 5°, 25°, or 45°C for 2 weeks. The concentrations of the test chemical in the extracts were determined by vapor-phase chromatography. 4,4'-Oxydianiline at 100,000 ppm was stable in feed for 2 weeks at 45°C.
Selected batches of the formulated diets administered during the chronic study were analyzed at EG&G Mason Research institute for accuracy of dose level. The test feeds were first extracted with 95% ethanol, and concentrations of the test chemical in the extracts were determined by spectrophotometric analysis at 247 nm. The mean concentration of 12 feed samples containing a theoretical level of 200 ppm was 200 +/-29 ppm, and the mean concentration of 14 samples measured in duplicate and containing a theorical level of 800 ppm was 780 +/-103 ppm.
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
Ad libitum 7 days/week
Post exposure period:
No specified.
Dose / conc.:
150 ppm (nominal)
Remarks:
in the diet
Dose / conc.:
300 ppm (nominal)
Remarks:
in the diet
Dose / conc.:
800 ppm (nominal)
Remarks:
in the diet
No. of animals per sex per dose:
50 per species per sex per dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The dose selected for the carcinogenicity study were based on the resuts from the Range-Finding and 14-Day Repeated-Dose Studies and from the 90-day subchronic study.

In the range-finding study conducted to determine the doses for the 14-day repeated-dose study, the test chemical was diluted in corn oil and administered by gavage to groups of two males and two females. Doses administered wre 100 - 300 - 1,000 - 3,000 and 10,000 mg/kg bw. The animals were observed for 7 days and then killed and necropsied. To solubilize the 4,4'-oxydianiline, the 10,000 mg/kg bw dose was prepared in 10% DMSO (dimethyl sulfoxide) in corn oil.
No mortality occurred among the male mice, but both female mice receiving the 3,000 mg/kg dose died. Labored respiration was observed in the mice receiving the three highest doses that subsequently died after receiving the 3,000 mg/kg dose. Enlarged lymph nodes were observed in the mice at all doses.
In the fourteen-day repeated-dose studies conducted to determine the doses to be used in the 90-day subchronic studies, groups of five males and five females were fed diets containing the test chemical. Doses administered wre 300 - 1,000 - 3,000 - 10,000 and 30,000 ppm. The animals were observed daily, and after 14 days, all survivors were killed and necropsied.
All the animals receiving the 10,000- or 30,000-ppm dose died. Three of five male mice and 1/5 female mice receiving 3,000 ppm 4,4'-oxydianiline died. The LD50 calculated for male mice was 2,820 ppm and 4,470 ppm for females.
4,4'-Oxydianiline caused liver enlargement at all doses, jaundice at the two highest doses, hemorrhages of the digestive tract at the three highest doses, and hemorrhages of the renal medullae at the highest dose. Lymphatic enlargement was observed in all dosed mice but in only 2/9 male controls and 1/8 female controls.

Subchronic Studies
In 90-day subchronic feeding studies conducted to determine the concen-trations of 4,4'-oxydianiline to be used in the chronic 2-year studies, groups of 10 males and 10 females were fed diets containing 0, 3, 10, 30, 100, or 300 ppm for 90 days. All animals were observed twice daily for mortality. Individual animal weights, food consumption, appear-ance, and behavior were recorded weekly. After 13 weeks, all the animals were killed and necropsied. Representative tissues were examined microscop-ically as described in the section on chronic studies. Because no compound-related clinical signs, body weight changes, or pathologic changes were observed, a second subchronic study was carried out using diets containing 0, 300, 600, 1,000, and 2,000 ppm. The doses administered in the second study, survival, and mean body weights of the dosed and control groups are shown in Tables 3 and 4 below.
None of the mice died, but a dose-associated decrease in weight gain was observed in male mice at all doses and in female mice at doses of 600 ppm and higher. Mice receiving the two highest doses were lethargic toward the end of the study. Most mice receiving 600 ppm or more had thyroid hyper-trophy and hyperplasia. Hyperplastic goiter was observed in mice receiving the highest dose (2,000 ppm). Pituitary hypertrophy and hyperplasia were associated with thyroid changes in some female mice receiving 1,000 ppm and in nearly all mice receiving 2,000 ppm. Testicular degeneration was found in most male mice receiving 1,000 or 2,000 ppm.
Because of the weight gain depressions and thyroid effects observed in the second subchronic study, doses selected for the chronic study were 150, 300, and 800 ppm. The highest dose (800 ppm) was included to enhance the possibility of detecting a thyroid response.

Chronic Study (carcinogenicity study):
On the basis of the results obtained in the Range-Finding and 14-Day Repeated-Dose Studies and in the 90-day subchronic study, the chronic study was conducted as follows:
Number of animals per group: 50.
Doses administered were 0 - 150 - 300 and 800 ppm.
Duration: 103 weeks.
Positive control:
None.
Observations and examinations performed and frequency:
All animals were observed twice daily for signs of toxicity. Mean body weights of animals by cage were recorded every 2 weeks for the first 13 weeks and monthly thereafter. Clinical signs were recorded monthly. Mori-bund animals and animals that survived to the end of the bioassay were killed using carbon dioxide and necropsied.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: Monthly

BODY WEIGHT: Yes
- Time schedule for examinations: every 2 weeks for the first 13 weeks and monthly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No data

CLINICAL CHEMISTRY: No data

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Examinations for grossly visible lesions were performed on major tissues or organs. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Prepar-ations of the following were examined microscopically: tissue masses, abnormal lymph nodes, skin, mandibular lymph nodes, mammary gland, salivary gland, thigh muscle, sciatic nerve, bone marrow, costochondral junction (rib), thymus, larynx, trachea, lungs and bronchi, heart, thyroid, parathy-roid, esophagus, stomach, duodenum, jejunum, ileum, colon, mesenteric lymph nodes, liver, gallbladder (mice), pancreas, spleen, kidneys, adrenals, bladder, seminal vesicles/prostate/testes, ovaries/uterus, nasal cavity, brain, pituitary, eyes, and spinal cord. Special staining techniques were utilized as necessary.

Necropsies were performed on all animals found dead, unless precluded in whole or in part by autolysis or cannibalization. Thus, the number of ani-mals from which particular organs or tissues were examined microscopically varies and does not necessarily represent the number of animals that were placed on study in each group.
Other examinations:
None
Statistics:
Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958) and are presented in the form of graphs. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone’s (1975) extensions of Cox's methods for testing for a dose-related trend.
As a part of the tumour analyses, the one-tailed Fisher exact test (Cox, 1970) was used to compare the tumour incidence of a control group with that of a group of dosed animals at each dose level. When results for three dosed groups are com¬pared simultaneously with those for a control group, a correction to ensure an overall significance level of 0.05 may be made. The Bonferroni inequality (Miller, 1966) requires that the P value for any comparison be less than or equal to 0.017.
The Cochran-Armitage test for linear trend in proportions, with continuity correction (Armitage, 1971), was also used. When a linear trend is assumed, this test determines if the slope of the dose-response curve is different from zero at the one-tailed 0.05 level of significance. Unless otherwise noted, the direction of the significant trend is a positive relationship. This method also provides a two-tailed test of departure from linear trend.
The approximate 95% confidence interval for the relative risk of each dosed group compared with its control was calculated from the exact interval on the odds ratio (Gart, 1971).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A compound-related increase in the number of mice with discharging, cloudy, or swollen eyes was also observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The result of the Tarone test for positive dose-related trend in mortality is not significant in either sex; the survival of the low and mid-dose female groups was significantly less than that of the matched control group (P=0.028 and P=0.036, respectively).
In male mice, 35/50 (70%) of the matched-control group, 39/50 (78%) of the low-dose group, 33/49 (67%) of the mid-dose, and 34/50 (68%) of the high-dose group were alive at the end of the bioassay at 105 to 106 weeks.
In females, 42/50 (82%) of the control group, 33/50 (66%) of the low-dose and mid-dose groups, and 42/50 (84%) of the high-dose group lived to the end of the bioassay.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A dose-related depression in mean body weight gain was observed for all groups.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Non-neoplastic lesions were observed in males and in female mice.

In male mice, the main findings observed are:
> lesion of the hematopoietic system observed in the spleen in 14%, 19% and 24% of animals in the low, mid and high dose groups respectively (8% in the control group)
> thyroid follicular cell hyperplasia in 53% of animals in the high dose group (0% in the control group and in other treated groups)
> inflammation in the harderian gland in 10%, 8% and 12% of the low, mid and high dose groups respectively (0% in the control group)

In female rats, the main finidngs observed are:
> inflammation of lungs in 2%, 2% and 8% of animals in low, mid and high dose groups respectively (2% in the control group)
> lesion of hematopoiesis observed in the spleen in 19%, 10% and 24% of animals in the low, mid and high dose group respectively (10% in the control group)
> nephropathy in 6% and 10% of animals in the mid and high dose group respectively (2% in the control group)
> thyroid follicular cell hyperplasia in 52% of animals in the high dose group (0% in the control group and in other treated groups)
> lesion of hydrometra in uterus in 17%, 32% and 31% of animals in low, mid and high dose groups respectively (15% in the control group)
> cyst in ovary in 10, 12 and 12 % of animals in the low, mid and high dose group respectively (2% in the control group)
> inflammation in the harderian gland in 4% and 8% of the animals in the low and mid dose groups respectively (0% in the control group)
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A variety of neoplasms were seen in control and treated mice.
Neoplasms or lesions associated with 4,4'-oxydianiline administration were hepatocellular neoplasms, adenomas of the harderian gland, and proliferative lesions of the thyroid gland.
see below, "Details on results".
Other effects:
not specified
Details on results:
CLINICAL SIGNS
A compound-related increase in the number of mice with discharging, cloudy, or swollen eyes was also observed.

MORTALITY
The result of the Tarone test for positive dose-related trend in mortality is not significant in either sex; the survival of the low and mid-dose female groups was significantly less than that of the matched control group (P=0.028 and P=0.036, respectively).
In male mice, 35/50 (70%) of the matched-control group, 39/50 (78%) of the low-dose group, 33/49 (67%) of the mid-dose, and 34/50 (68%) of the high-dose group were alive at the end of the bioassay at 105 to 106 weeks.
In females, 42/50 (82%) of the control group, 33/50 (66%) of the low-dose and mid-dose groups, and 42/50 (84%) of the high-dose group lived to the end of the bioassay. Sufficient numbers of animals in all groups were at risk for the development of late-appearing tumors

BODY WEIGHT AND WEIGHT GAIN
A dose-related depression in mean body weight gain was observed for all groups of dosed mice and a compound-related increase in the number of mice with discharging, cloudy, or swollen eyes was also observed.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Noted as reduced intake during the range finding and chronic studies in both species; reflected by reduced weight gain.

ORGAN WEIGHTS
Not detailed specifically. Increased liver weights and thyroid were noted in

GROSS PATHOLOGY & HISTOPATHOLOGY: NON-NEOPLASTIC and NEOPLASTIC
A variety of neoplasms were seen in control and treated mice. Neoplasms or lesions associated with 4,4'-oxydianiline administration were hepatocellular neoplasms, adenomas of the harderian gland, and proliferative lesions of the thyroid gland.
Hepatocellular adenomas compressed the adjacent tissue. Cells in the adenomas were large and usually acidophilic. Nuclei were hyperchromatic. Mitotic figures were not numerous. Hepatocellular carcinomas involved a part or an entire lobe of the liver. The lobular architecture was not maintained.
Cell plates were two or more cells thick. A pleomorphism in the size of cells was seen, and cytoplasmic inclusions were present in some cells. The nuclei had coarse chromatin, and nucleoli were predominant. An occasional multinucleated cell was noticed. Both normal and abnormal mitotic figures were sometimes numerous.
Markedly distended sinusoids and cavernous vascular spaces were present in a few tumors. The cells lining such vascular channels were fusiform and occasionally spherical. Cytoplasm of these cells was inconspicuous, and the nuclei were hyperchromatic. Islands of neoplastic hepatocytes were encircled by fusiform cells, and these tumors were diagnosed as hemangiomas or hemangiosarcomas.

Areas of necrosis and hemorrhage were common in the large tumors. The hepatocellular carcinomas had metastasized to the lung in 12 male mice (control, 2; low-dose, 4; mid-dose, 5; high-dose, 1) and in none of the female mice. Toxic, nonneoplastic hepatic lesions were not seen in dosed mice.

Adenomas of the harderian gland were found in 1/50 control males, 17/50 low-dose males, 13/49 mid-dose males, 17/50 high-dose males, 2/50 control females, 15/50 low-dose females, 14/50 mid-dose females, and 12/50 high-dose females. The harderian gland was histologically evaluated only when it was enlarged. Adenomas of the harderian gland involved either a part or an entire gland and were characterized either as a papillary ingrowth of the epithelium into the lumen of the distended acini or as a solid sheet of cells. These cells were columnar, and they contrasted with the cuboidal cells in the normal gland. Cytoplasm of the cells had fine vacuoles, and nuclei were of uniform size and hyperchromatic. Mitotic figures were numerous. Porphyrin pigment was not found in any of these tumors. Clusters of inflammatory cells were scattered around the gland.

In high-dose mice, follicular-cell hyperplasia of the thyroid gland occurred in 26/49 males and 25/48 females. Adenomas were found in 2/47 middose males, 2/49 high-dose males, and 7/48 high-dose females. A diffuse enlargement of the follicles or irregular papillary ingrowth of the epithelium was considered to be follicular hyperplasia. The epithelial cells were cuboidal, and the nuclei were hyperchromatic. The adenoma compressed the adjacent tissue. Follicular arrangement of the cells was maintained and cells were columnar or cuboidal. Cytoplasm of the cells was basophilic or eosinophilic, and nuclei were hyperchromatic.

Under the conditions of this bioassay, 4,4'-oxydianiline was found to be carcinogenic to B6C3F1 mice, causing an increased incidence of neoplasms of liver, harderian glandand thyroid gland.

STATISTICAL ANALYSIS:
In male mice, the Fisher exact test shows that the combined incidence of animals with hepatocellular adenomas or carcinomas is significantly higher in the low-dose group than in the controls (P=0.015). For the bioassay program, the historical incidence of male B6C3F1 mice with these tumors is 651/2,843 (23%), which is lower than the mid- and high-dose group incidences of 34/49 (69%) and 36/50 (72%), respectively.
In female mice, a significant positive linear trend is observed (P less than 0.001) in relation to increasing dose in the incidence of animals with hepatocellular adenomas or carcinomas. The incidence in the high-dose group is also significantly higher (P less than 0.001) than that of the controls.
In male mice, a significant positive linear trend (P=0.004) was observed in the incidence of animals with adenomas, NOS (not otherwise specified), in the harderian glands. The incidences in all dosed groups were significantly higher (P less than 0.001) than the incidence in the control group. A departure from the linear trend is indicated due to a higher incidence (34%) in the low-dose group compared with the mid-dose group (27%).
In female mice, the Fisher exact test shows that the incidence of adenomas, NOS, in the harderian gland is significantly higher (P less than 0.005 in all dosed groups) than that in the control group. A departure from linear trend was observed due to the higher incidence in the low-dose group than in the other dosed groups. The historical incidence among female B6C3F1 mice for this kind of tumor is 9/2,917 (0.3%). This figure is lower than the 2/50 (4%) reported in the controls in this study. In female mice, a positive linear trend (P less than 0.001) is indicated in the incidence of animals with follicular-cell adenomas in the thyroid.
The incidence in the high-dose group is significantly higher (P=0.007) than in the controls. The incidence observed in the control group of this study (0%) is not significantly different from the historical incidence of 32/2,917 (1%) in the bioassay program's accumulated data.
In male mice, there is a positive linear trend (P=0.001) in the incidence of adenomas, NOS, in the pituitary. The incidence in the high-dose group is higher than that in the controls, but the P=0.023 observed is above the P=0.017 level required for significance when the Bonferroni inequality criterion is used to assess the comparison of three dosed groups with a single control group.
In male mice, the Cochran-Armitage test indicates a significant dose related trend (P=0.011) in the incidence of hemangiomas of the circulatory system. The incidences in the mid- and high-dose groups are also in the control group; but the significance levels observed in the groups do not meet the significance level required (P less than 0.017 he Bonferroni inequality criterion is applied.
In male mice, a negative trend is indicated (P=0.015) for animals with alveolar/bronchiolar adenomas or carcinomas in the lung; negative trend with significantly lower incidence in the high-dose group also observed in the incidence of malignant lymphomas in the hematopoisystem of both sexes.

The statistical analyses indicate that the occurrences of tumors in harderian gland of male and female mice are related to the administration 4,4'-oxydianiline. There is also an association with liver tumors and with thyroid tumors in females.
Relevance of carcinogenic effects / potential:
4,4'-Oxydianiline was carcinogenic for male and female B6C3F1 mice, inducing adenomas, NOS, in the harderian glands and hepatocellular adenomas or carcinomas. 4,4'-Oxydianiline also induced follicular-cell adenomas of the thyroid in female mice.
Key result
Dose descriptor:
dose level:
Effect level:
150 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
histopathology: neoplastic
Remarks on result:
other: Effect type: carcinogenicity
Key result
Dose descriptor:
dose level:
Effect level:
300 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
histopathology: neoplastic
Remarks on result:
other: Effect type: carcinogenicity
Key result
Dose descriptor:
dose level:
Effect level:
800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
histopathology: neoplastic
Remarks on result:
other: Effect type: carcinogenicity
Key result
Critical effects observed:
yes
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
System:
eye
Organ:
other: harderian gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

 The following table contains the incidences of the above mentioned tumors in male and female mice.

 

Dose (ppm)

0

150

300

800

 

 

 

 

 

Harderian Gland adenomas

Males

1/50

17/50

13/49

17/50

Females

2/50

15/50

14/50

12/50

 

 

 

 

 

Hepatocellular adenomas

Males

11/50

13/50

11/49

10/50

Females

4/50

6/49

9/48

14/50

 

 

 

 

 

Hepatocellular carcinomas

Males

18/50

27/50

23/49

26/50

Females

4/50

7/49

6/48

15/50

 

 

 

 

 

Hepatocellular adenomas or carcinomas

Males

29/50

40/50

34/49

36/50

Females

8/50

13/49

15/48

29/50

 

 

 

 

 

Thyroid follicular cell adenomas

Females

0/46

0/43

0/42

7/48

 

 

 

 

 

Pituitary adenomas:

Males

1/37

0/44

0/34

7/35

 

 

 

 

 

Circulatory System hemangiomas:

Males

0/50

0/50

5/49

5/50

 

 

 

 

 

 

Conclusions:
Under the conditions of this study, the test substance was carcinogenic to B6C3F1 mice, inducing adenomas, NOS, in the harderian glands and hepatocellular adenomas or carcinomas. 4,4'-Oxydianiline also induced follicular-cell adenomas of the thyroid in female mice.
Executive summary:

A study for assessment of a possible carcinogenicity was conducted by feeding diets containing 150, 300, or 800 ppm to groups of 50 male or female B6C3F1 mice for 104 weeks. Matched controls consisted of 50 untreated mice of each sex. All surviving animals were killed at 104 to 105 weeks.

A dose-related decrement in mean body weight gain was observed for all groups of dosed mice.

Survival was significantly shortened in the low- and mid-dose female mice.

In male and female mice, adenomas in the harderian glands occurred in all dosed groups at incidences that were significantly higher than the incidence in the matched controls.

In low-dose male mice and in high-dose female mice, hepatocellular adenomas or carcinomas occurred at incidences significantly higher than those in the matched controls.

In female mice, follicular-cell adenomas in the thyroid occurred with a positive linear trend, and in a direct comparison the incidence in the high-dose group was also significantly higher than that in the controls.

Tumors occurring among male mice at increased incidences which could not be statistically related to the chemical were adenomas in the pituitary and hemangiomas of the circulatory system.

Under the conditions of this bioassay, 4,4'-oxydianiline was carcinogenic for male and female B6C3F1 mice, inducing adenomas in the harderian glands, hepatocellular adenomas or carcinomas in both sexes, and follicular-cell adenomas in the thyroid of females.

Reason / purpose:
reference to same study
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 July 1981 to 22 February 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Non-GLP study published by recognised Governmental body (US National Cancer Institute). Study and results are well documented with full statistical analysis included. This study is used in place of sub acute / sub chronic study.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A 90-day Subchronic feeding studies were conducted to determine the concentrations of 4,4'-oxydianiline to be used in the chronic 2-year studies. Groups of 10 males and 10 females mice were fed diets containing 0, 3, 10, 30, 100, or 300 ppm for 90 days. All animals were observed twice daily for mortality. Individual animal weights, food consumption, appearance, and behavior were recorded weekly. After 13 weeks, all the animals were killed and necropsied. Representative tissues were examined microscopically as described in the section on chronic studies.
Because no compound-related clinical signs, body weight changes, or pathologic changes were observed, a second Subchronic study was carried out using diets containing 0, 300, 600, 1,000, and 2,000 ppm.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: NCI Frederick Cancer Research Center (Frederick, Maryland)
- Age at study initiation: 5-week old
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: solid-bottom polycarbonate cages (Lab Products, Inc., Garfield, New Jersey) containing Aspenbed^(aspen chips, American Excelsior, Summerville, Mass.) / housed five per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 32 °C
- Humidity (%): 5 - 82 %
- Air changes (per hr): Incoming air was filtered by 2-inch fiberglass Tri-dek 125-40 filters with 10 to 12 changes of room air per hour
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was provided on a 12-hour per day cycle
Route of administration:
oral: feed
Details on route of administration:
gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analysis of formulated diets were not conducted in this study. However they were conducted in the further chronic study (2-year study) on 200 and 800 ppm formulations.
Duplicate samples of 2 g each were extracted with 50 ml of 95% ethanol in 100-ml ground glass stoppered graduated cylinders by repeated inversions of the cylinders during a 15-minute period. The feed particles were allowed to settle overnight, and the absorbance of the supernatants was measured at 247 nm in a Beckman DU Spectrophotometer after appropriate dilutions with 95% ethanol. The absorbance readings were adjusted with a "blank" extract from a 2-g feed sample from the same bag as the sample and were worked up in the same manner. Concentrations were determined by direct comparisons with standard solutions of the test compound. Recovery was determined by working up controlled feed mixtures simultaneously with the samples. The controls were prepared by spiking blank feed samples in duplicate. "Corrected" concentrations were adjusted for average recovery loss.

Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
300 ppm
Dose / conc.:
600 ppm
Dose / conc.:
1 000 ppm
Dose / conc.:
2 000 ppm
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
All animals were observed twice daily for mortality. Individual animal weights, food consumption, appearance, and behavior were recorded weekly. After 13 weeks, all the animals were killed and necropsied. Representative tissues were examined microscopically.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mice receiving the two highest doses were lethargic toward the end of the study.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A dose-associated decrease in weight gain was observed in male mice at all doses and in female mice at doses of 600 ppm and higher.
Description (incidence and severity):
Most mice receiving 600 ppm or more had thyroid hypertrophy and hyperplasia. Hyperplastic goiter was observed in mice receiving the highest dose (2,000 ppm). Pituitary hypertrophy and hyperplasia were associated with thyroid changes in some female mice receiving 1,000 ppm and in nearly all mice receiving 2,000 ppm. Testicular degeneration was found in most male mice receiving 1,000 or 2,000 ppm.
Key result
Dose descriptor:
dose level: in feeding diet
Effect level:
600 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
dose level: in feeding diet
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Key result
Dose descriptor:
dose level: in feeding diet
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
dose level: in feeding diet
Effect level:
1 000 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 ppm
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
endocrine system
Organ:
pituitary gland
Treatment related:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 ppm
System:
male reproductive system
Organ:
testes
Treatment related:
yes

Analytical results of 200 and 800 ppm formulated diets from the chronic study (2 -year study):

 Theorical concentration (ppm)  Number of samples  Sample analytical mean (ppm)  Coefficient of variation (%)  Acceptable range (ppm)
 200 12  200  14.5  160 -240
 800  14  780  13.2  650 -1050
Conclusions:
This study showed effects on thyroid at dietary concentrations of 4,4'-oxydianiline of 600 ppm and above, on testes and pituitary gland at dietary concentrations of 4,4'-oxydianiline of 1000 ppm and above.
Executive summary:

A 90-day Subchronic feeding studies were conducted to determine the concentrations of 4,4'-oxydianiline to be used in the chronic 2-year studies. Groups of 10 males and 10 females mice were fed diets containing 0, 300, 600, 1,000, and 2,000 ppm for 90 days.

This study showed effects on thyroid at dietary concentrations of 4,4'-oxydianiline of 600 ppm and above, on testes and pituitary gland at dietary concentrations of 4,4'-oxydianiline of 1000 ppm and above.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
A 90-day Subchronic feeding studies were conducted to determine the concentrations of 4,4'-oxydianiline to be used in the chronic 2-year studies. Groups of 10 males and 10 females rats were fed diets containing 0, 3, 10, 30, 100, or 300 ppm for 90 days. All animals were observed twice daily for mortality. Individual animal weights, food consumption, appearance, and behavior were recorded weekly. After 13 weeks, all the animals were killed and necropsied. Representative tissues were examined microscopically as described in the section on chronic studies.
Because no compound-related clinical signs, body weight changes, or pathologic changes were observed, a second Subchronic study was carried out using diets containing 0, 300, 600, 1,000, and 2,000 ppm.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): 4,4'-Oxydianiline
- Substance type: Organic
- Physical state: Solid
- Supplier: E. I. DuPont de Nemours and Company (Wilmington, Del.)
- Lot/batch No.: In two batches. Lot No. 387 was used in the first and second Subchronic studies and Lot No. 82/02 was used in the chronic studies
- Analytical purity: Amine titration of Lot Nos. 387 and 82/02 indicated purities of 99.9%+0.6% and 98.9%+0.2%, respectively
- Impurities (identity and concentrations): Results from vapor-phase chromatography of Lot No. 387 indicated an impurity constituting 0.29% of the major peak, whereas Lot No. 82/02 contained two impurities, each with an area of 0.1% of the major peak. The nuclear magnetic resonance spectrum of Lot No. 82/02 indicated a trace impurity at 3.28 to 3.42 ppm. The impurities were not identified.
- Structural formula attached as image file (if other than submission substance): Included within the report.
- Stability under test conditions: Analyses of the stability of 4,4'-oxydianiline in feed were performed at Midwest Research Institute by assaying dimethyl formamide extracts from samples of diet mixtures containing 100,000 ppm that had been stored at -20 deg C, 5°, 25°, or 45 C for 2 weeks. The concentrations of the test chemical in the extracts were determined by vapor-phase chromatography 4,4'-Oxydianiline at 100,000 ppm was stable in feed for 2 weeks at 45 C.
- Storage condition of test material: Stored at 4 C in the original container
- Molecular formula (if other than submission substance): Not specified.
- Molecular weight (if other than submission substance): Not specified.
- Smiles notation (if other than submission substance): Not specified.
- InChl (if other than submission substance): Not specified.
- Composition of test material, percentage of components: Not specified.
- Isomers composition: Not applicable.
- Purity test date: Not specified.
- Expiration date of the lot/batch: Not specified.
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable.
Specific details on test material used for the study:
4,4'-oxydianiline
4,4'-diaminodiphenyl ether
CAS 101-80-4

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female

Administration / exposure

Route of administration:
oral: feed
Doses / concentrationsopen allclose all
Dose / conc.:
300 ppm
Dose / conc.:
600 ppm
Dose / conc.:
1 000 ppm
Dose / conc.:
2 000 ppm
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Alopecia, labored respiration, and cyanosis were observed with the two highest doses (1,000 and 2,000 ppm).
Mortality:
mortality observed, treatment-related
Description (incidence):
A dose-related increase in mortality was observed in both sexes of rats with the two highest doses (1,000 and 2,000 ppm).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A dose-related decrease in weight gain was observed in both sexes of rats with the two highest doses (1,000 and 2,000 ppm).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
All rats receiving 600 ppm or more had diffuse parenchymatous goiter. In addition, pituitary hyperplasia, testicular degeneration, prostatic atrophy, seminal vesicular atrophy, and renal microlithiasis were detected in most of the rats receiving 600 ppm or more.

Effect levels

Key result
Dose descriptor:
dose level: in feeding diet
Effect level:
600 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 ppm
System:
endocrine system
Organ:
thyroid gland
Treatment related:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
600 ppm
System:
male reproductive system
Organ:
seminal vesicle
testes
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
This study showed effects on thyroid, testes/seminal vesicles and kidneys at dietary concentrations of 4,4'-oxydianiline of 600 ppm and above, equivalent to approximately 30 mg/kg /day using standard food consumption rates
Executive summary:

A 90-day Subchronic feeding studies were conducted to determine the concentrations of 4,4'-oxydianiline to be used in the chronic 2-year studies. Groups of 10 males and 10 females rats were fed diets containing 0, 300, 600, 1,000, and 2,000 ppm for 90 days.

This study showed effects on thyroid, testes/seminal vesicles and kidneys at dietary concentrations of 4,4'-oxydianiline of 600 ppm and above, equivalent to approximately 30 mg/kg /day using standard food consumption rates.