4,4'-oxydianiline

Carcinogenicity

Description of key information

Studies on rats and mice to assess the potential carcinogenic effect of 4,4'-oxydianiline were conducted for the Carcinogenesis Testing Program, National Cancer Institute (NCI)/National Toxicology Program (NTP).

These studies were conducted by feeding diets the test chemical to groups of 50 male or female F344 rats and groups of 50 male or female B6C3F1 mice for 104 weeks. Matched controls consisted of 50 untreated rats and 50 untreated mice of each sex. All surviving animals were killed at 104 to 105 weeks.

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.

4,4'-Oxydianiline was also 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.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

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 or test system 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:

body weight and weight gain

clinical signs

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:

body weight and weight gain

clinical signs

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:

body weight and weight gain

clinical signs

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.

Reference 2

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

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 or test system 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:

body weight and weight gain

clinical signs

histopathology: neoplastic

histopathology: non-neoplastic

mortality

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:

body weight and weight gain

clinical signs

histopathology: neoplastic

histopathology: non-neoplastic

mortality

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:

body weight and weight gain

clinical signs

histopathology: neoplastic

histopathology: non-neoplastic

mortality

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

T25

0.31 mg/kg bw/day

Study duration:

chronic

Species:

rat

System:

hepatobiliary

Organ:

liver

thyroid gland

Justification for classification or non-classification

The above results triggered classification in agreement with the current classification under the CLP Regulation (EC No 1272/2008) as follows:

 

Carc. 1B (Hazard statement: H350: May cause cancer)

Additional information

The test substance was carcinogenic to F344 rats and B6C3F1 mice, causing both increased incidences of follicular-cell neoplasms of the thyroid gland and liver neoplasms in rats, as well as an increased incidence of neoplasms of the liver, harderian gland, and thyroid gland in mice.

The data available within the Carcinogenicity studies available on the registered substance provided some challenges with regards to derivation of a suitable endpoint for use in hazard assessment, as the studies available are feeding studies that do not provide a definitive dose-response endpoint.  The main issue was that it was difficult to calculate a daily dose level per kg bodyweight during the exposure period, as actual dose levels were not available to do this.  It was therefore decided to use an approach with an average calculation for the food consumption.  In order to so, the use of the guidance detailed in the“Guidelines For Setting Specific Concentration Limits For Carcinogens In Annex I Of Directive 67/548/EEC - Inclusion Of Potency Considerations - Commission Working Group On The Classification And Labelling Of Dangerous Substances”; as the reference was the most viable approach.  This document contains some default values for dose calculation (Table 1, page 10) on dietary studies. The document also specified the use of T25 values, so due consideration was given to this mode of assessment.

 

Further review of guidance documentation indicated that the approach to use T25 values was suitable for assessment, as this is detailed in the following documents:

 

ECHA Guidance Document,CHAPTER R.8 - DOSE [CONCENTRATION]-RESPONSE REGARDING HUMAN HEALTH, R.8.5.2.1 The ‘Linearised’ approach which states that:

 

a) Select the relevant dose-descriptor(s), i.e. T25 and BMD(L)10

 

The T25 should be used as the default dose-descriptor in relation to linear extrapolation. The linear approach is used when there is an absence of sufficient information on modes of action or when mode of action information indicates that the dose-response curve at low dose is or is expected to be linear. The BMD10 i.e. the Benchmark-dose representing a 10% response should be used in certain cases in addition to the T25 when data are adequate for modelling purposes. Thus, based on the available data a decision is made on what dose descriptor to use.

 

In addition, APPENDIX R. 8-6 Animal dose descriptors for non-threshold carcinogenic responses states that:

 

Use of dose descriptors

Within the EU context the T25 dose-descriptor is in use for inclusion of potency considerations in setting specific concentration limits for carcinogens in Annex I of Directive 67/548/EEC (EC, 1998). Additionally, a T25 has been used by some Member States for risk characterisation within the framework of existing substances (EC Regulation 1488/94; EC, 1994) and for risk characterisation of chemicals in general. Moreover, the Scientific Committee on Consumer Products (previously: Scientific Committee on Cosmetics and Non-Food Products) uses the T25 method for safety evaluation in relation to non-threshold carcinogens SCCNFP/00690/03.

 

The BMD10 has regulatory use as its lowest confidence value (i.e. BMDL10) for the assessment of risks posed by food ingredients by EFSA (EFSA, 2005). A comparable dose descriptor, ED10, has regulatory use as its lowest confidence value (i.e. LED10) in cancer risk assessment practice by US EPA (US EPA, 2005).

 

Therefore, the conclusion drawn was that for an EU registration, it was appropriate to use a T25 rather than a BMDL10 value for the purposes of the calculation for this endpoint.

Evaluation of the T25 values from Carcinogenicity studies in the rat, mouse has therefore been undertaken. The results of these evaluation provides the following results:

 

NTP 80 14 (1980) Bioassay of 4 4 oxydianiline for possible carcinogenicity - Results

 

The T25 dose descriptor in the Rat is therefore calculated to be 4.90 mg/kg/day based on occurrence of hepatocellular carcinoma or neoplastic nodules.

 

The T25 dose descriptor in the Mouse is therefore calculated to be 12.4 x 10E-03 mg/kg/day based on occurrence of Harderian Gland adenomas.

 

An evaluation of the potential potency of the carcinogenic effects was then taken, using the reference source: Guidelines For Setting Specific Concentration Limits For Carcinogens In Annex I Of Directive 67/548/EEC - Inclusion Of Potency Considerations - Commission Working Group On The Classification And Labelling Of Dangerous Substances.

This document details that T25 values can be used to place substances classified as a carcinogen into arbitrarily selected ranges that define potency. As such, it is possible to identify carcinogens of high and low potency. For the purpose of assigning specific concentration limits, it is proposed that:

 

Carcinogens of high potency: T25 value < 1 mg/kg bodyweight/day

 

Carcinogens of medium potency: 1 mg/kg bodyweight/day < T25 value < 100 mg/kg bodyweight/day

 

Carcinogens of low potency: T25 value > 100 mg/kg bodyweight/day.

 

Based on this guidance, it is deemed appropriate, on a worst case basis, to assign the lowest T25 value for the purposes of calculation of the DMEL for the substance as a potential Carcinogen of medium potency; this being the T25 dose descriptor in the Mouse at 12.4 x 10E-03 mg/kg bodyweight/day. 

However, as this value is calculated from harderian gland adenomas, a further evaluation of the appropriate value was undertaken. Whilst harderian gland’s are known to exist within rodents, the existence within humans is still debatable. As such, it was concluded that calculation of DMEL using a T25 value derived from a biological endpoint that may not exist inhumans was not appropriate. As such, other endpoints were evaluated. As hepatocellular adenoma and carcinoma occurred in both species evaluated within the NTP studies, there is strong indication that liver is the target organ for tumours and hepatocellular adenoma and carcinoma are endpoints considered relevant for humans.

 

As such, it was therefore considered appropriate to utilise the T25 lowest value for these endpoints, which was determined to be 4.90 mg/kg/day based on occurrence of hepatocellular carcinoma or neoplastic nodules.

 

It is this value that has been used to derive the DMELs presented within this evaluation report.

 

Derivation of T25 value is determined by the following calculations, utilising the reference source Guidelines For Setting Specific Concentration Limits For Carcinogens In Annex I Of Directive 67/548/EEC - Inclusion Of Potency Considerations - Commission Working Group On The Classification And Labelling Of Dangerous Substances

Provisional evaluation of potency classification

 

T25 values can be used to place substances classified as a carcinogen into arbitrarily selected ranges that define potency. As such, it is possible to identify carcinogens of high and low potency. For the purpose of assigning specific concentration limits, it is proposed that:

 

Carcinogens of high potency: T25 value < 1 mg/kg bodyweight/day

 

Carcinogens of medium potency: 1 mg/kg bodyweight/day < T25 value < 100 mg/kg bodyweight/day

 

Carcinogens of low potency: T25 value > 100 mg/kg bodyweight/day.

 

Tabulated Data – taken from NTP 80 14 (1980) Bioassay of 4 4 oxydianiline for possible carcinogenicity

The following table contains the incidences of the tumors in male and female rats dosed over 103 weeks. 50 per species per sex per dose. 

Dose (ppm)	0	200	400	500
Hepatocellular carcinoma:
Males	0/50 (0%)	4/50(8%)	23/50(46%)	22/50(44%)
Females	0/50(0%)	0/49(0%)	4/50(8%)	6/50(12%)
Hepatocellular neoplastic nodule:
Males	1/50(2%)	9/50(18%)	18/50(36%)	17/50(34%)
Females	3/50(6%)	0/49(0%)	20/50(40%)	11/50(22%)
Hepatocellular carcinoma or neoplastic nodule:
Males	1/50(2%)	13/50(26%)	41/50 (82%)	39/50(78%)
Females	3/50(6%)	0/49(0%)	24/50(48%)	17/50(34%)
Thyroid follicular cell adenoma:
Males	1/46(2%)	1/47(2%)	8/46(17%)	13/50(26%)
Females	0/49(0%)	2/48(4%)	17/48(35%)	16/50(32%)
Thyroid follicular cell carcinoma:
Males	0/46(0%)	5/47(11%)	9/46 (20%)	15/50(30%)
Females	0/49(0%)	2/48(4%)	12/48(25%)	7/50(14%)
Thyroid follicular cell adenoma or carcinoma:
Males	1/46(2%)	6/47(13%)	17/46(37%)	28/50(56%)
Females	0/49(0%)	4/48(8%)	29/48(60%)	23/50(46%)

The highest incidence at the lowest dose occured in Rats males, 400 ppm with occurrence of hepatocellular carcinoma or neoplastic nodules.

 

Control: 1/50 ( 2%)

400 ppm diet: 41/50 (82%)

net %: 80%

 

Daily dose per kg bodyweight during the exposure period:

 

As no information on specific food consumption is available, nor is bodyweight specified, it is deemed appropriate to utilise standard values as detailed in Table 1 of the above mentioned reference source. Table 1: Default values for dose calculation, experimental period, weights, and intake by diet, water, and inhalationdetails that a male adult rat will consume a default amount of 20 g diet per day, and weighs approximately 500 g (0.5 kg), with a default lifespan of 2 years (104 weeks)

 

Dose level (in diet): 400 ppm = 400 mg/kg = 0.4 mg/g diet.

 

Therefore dose level consumed = 20 g per day x 0.4 mg/g = 8 mg / day consumption.

 

Male rat bodyweight (default) = 0.5 kg.

 

Consumption = 8 mg / day x 1/0.5 kg

 

Exposure Level of 16 mg/kg bodyweight/day exposure.

 

Dose at this incidence of Hepatocellular carcinoma or neoplastic nodule when administration started after 5 weeks and exposure is for 103 weeks.

 

= (Study length / lifespan)²x Dose Exposure Level (proposed by Peto et al, 1984)

 

= (103/104)² x 16 mg/kg bodyweight/day

 

= 15.69 mg/kg bodyweight/day.

 

T25 after 24 months: 

 

= (25% / % effected animals after 24 months) x Incident dose

 

 = (25/80) x 15.69

 

= 4.90 mg/kg/day

The T25 dose descriptor in the Rat is therefore 4.90 mg/kg/day

Based on the above reference document, the test material would be considered as a:

 

Carcinogens of medium potency: 1 mg/kg bodyweight/day < T25 value < 100 mg/kg bodyweight /day

 

Reliability

The above study have been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because although one is a published literature based data study, the reference states a recognised scientific method and detailed observations. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

Carcinogenicity: via oral route (target organ): other: all gross lesions and masses