Diphenylamine

Carcinogenicity

Description of key information

2 year oral feed carcinogenicity study of Diphenylamine in B6D2F1 Mice
There was some evidence for carcinogenicity of diphenylamine in male mice based on the increased incidences of vascular tumors in spleen and in all organs included spleen and liver. There was no evidence for carcinogenicity of diphenylamine in female mice.
In the present two-year feeding study, the effects on blood and hematopoietic system were observed for the lowest dose of 250 ppm in both males and females. The lowest observed adverse effect level (LOAEL) of diphenylamine in the diet was 250 ppm (male: 29kg body weight per day, female: 36 body weight per day).

2 year oral feed carcinogenicity study of Diphenylamine in F344 Rats
There was some evidence for carcinogenicity of diphenylamine in male and female rats, based on the increase incidence of vascular tumors in spleen and increase incidence of vascular tumors in all organs including in spleen and subcutus in males. 
Using blood and hematopoietic system and liver as endpoint markers, the no-observed-adverse-effect-level (NOAEL) of diphenylamine in the diet was 250 ppm (12 mg/kg body weight per day) for males. The NOAEL for females could not be estimated, the lowest observed-adverse-effect-level (LOAEL) of diphenylamine in the diet was 250 ppm (15 mg/kg body weight per day) for 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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Authoritative secondary source reporting data comparable to guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)

Deviations:

yes

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

GLP compliance:

yes (incl. QA statement)

Remarks:

self certified

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks

Route of administration:

oral: unspecified

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

12 or 24 months

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 200, 750, 3750 and 7500 ppm
Basis:
nominal in diet
(males)

Remarks:

Doses / Concentrations:
0, 150, 500, 2500 and 5000 ppm
Basis:
nominal in diet
(females)

No. of animals per sex per dose:

60 rats/sex/dose

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing, and weekly 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: Yes
- Time schedule for examinations: once prior to first administration, in week 53, and prior to terminal necropsy
- Dose groups that were examined: animals designed to interim sacrifice, and then all suviving animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at pre-test, week 26, 53 & 78 and at terminal necropsy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at pre-test, week 26, 53 & 78 and at terminal necropsy

URINALYSIS: Yes
- Time schedule for collection of urine: at pre-test, week 26, 53 & 78, and prior to terminal sacrifice

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

effects on bilirubin, alkaline phosphatase and aspartate aminotransferase

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased spleen and liver weights

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

splenic congestion, increased haemosiderosis and haematopoiesis

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No apparent decrease in death rate associated with treatment

BODY WEIGHT AND WEIGHT GAIN
2500/3750 ppm and 5000/7500 ppm: signicantly reduced group mean body weight values

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Significant variations in all treated groups, especially in 2500/3750 ppm and 5000/7500 ppm

OPHTHALMOSCOPIC EXAMINATION
No treatment or dose related effects

HAEMATOLOGY
150 ppm female: decrease of red blood cell count (week 26), increase of mean corpuscular volume (week 26), increase of mean corpusuclar haemoglobin (week 26)
500 ppm female: decrease of red blood cell count (week 26 & 53), increase of mean corpuscular volume (week 26), increase of mean corpusuclar haemoglobin (week 26)
2500-5000 ppm female: decrease of red blood cell count, decrease of haemoglobin, hematocrit,increase of mean corpuscular volum, increase of mean corpusuclar haemoglobin
750 ppm male: decrease of red blood cell count (week 26 & 102), increase of mean corpuscular volume (week 102), increase of mean corpusuclar haemoglobin concentrations (week 102)
3750 ppm male: decrease of red blood cell count (week 26, 53 & 102), decrease of hemoglobin (week 26 & 102), increase of mean corpuscular volume (week 26, 53 & 102), increase of mean corpuscular haemoglobin (week 26, 53 & 102), increase of mean corpusuclar haemoglobin concentrations (week 53 & 102)
7500 ppm male: decrease of red blood cell count (week 26, 53 & 102), decrease of hemoglobin (week 26 & 102), increase of mean corpuscular volume (week 26, 53 & 102), increase of mean corpuscular haemoglobin (week 26, 53 & 102), increase of mean corpusuclar haemoglobin concentrations (week 102), decreased platelet count (week 102)

CLINICAL CHEMISTRY
Majority of observed effects: 2500/3750 ppm and 5000/7500 ppm

URINALYSIS
Significant effects at all dose levels at 26 wks, but no treatment related effect

GROSS PATHOLOGY
Kidney lesions in all groups
750-7500 ppm males, 500-5000 ppm females: increased frequency of dark and/or enlarged spleens

HISTOPATHOLOGY: NON-NEOPLASTIC
Significant positive trend for non-neoplastic lesiosn related to red blood cell destruction
500/750-5000/7500 ppm: kidney and liver pigmentation
2500/3750 and 5000/7500 ppm: spleen congestion with haemosiderosis, erythroid hyperplasia of the bone marrow, haematopoiesis of the liver and spleen increased

Relevance of carcinogenic effects / potential:

All neoplasms were considered spontaneous or incidental

Dose descriptor:

NOEL

Effect level:

> 150 - < 200 ppm

Based on:

not specified

Sex:

male/female

Basis for effect level:

other: gross pathology; histopathology

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

7.5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: haematology; gross pathology; histopathology of the spleen, kidney and liver

Remarks on result:

other: Effect type: toxicity (migrated information)

Conclusions:

There was no evidence of carcinogenicity in this study

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Authoritative secondary source reporting data comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

yes

GLP compliance:

yes (incl. QA statement)

Remarks:

self certified

Species:

mouse

Strain:

CD-1

Sex:

male/female

Route of administration:

oral: unspecified

Vehicle:

corn oil

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

18 months

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 525, 2625 and 5250 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

60 mice/sex/dose

Control animals:

yes

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: during pre-test period, at initiation, and then weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes

OTHER: Survival rate

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

increased mortality

Mortality:

mortality observed, treatment-related

Description (incidence):

increased mortality

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

significant reduction

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

reduced red blood cell count, increased reticulocyte count

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

general increase

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

congestion and haemosiderosis of the spleen

Histopathological findings: neoplastic:

no effects observed

Relevance of carcinogenic effects / potential:

There were no indications of increased tumour incidence in any organ or tissue at terminal necropsy
There were no significant increases in neoplastic changes in any organ or tissue among the treated groups of either sex

Dose descriptor:

NOEL

Effect level:

< 73.2 mg/kg bw/day

Based on:

dissolved

Sex:

male

Basis for effect level:

other: haematology; histopathology

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOEL

Effect level:

< 90.5 mg/kg bw/day

Based on:

dissolved

Sex:

female

Basis for effect level:

other: haematology; histopathology

Remarks on result:

other: Effect type: toxicity (migrated information)

Dose descriptor:

NOAEL

Effect level:

73 mg/kg bw/day

Based on:

dissolved

Sex:

male/female

Basis for effect level:

other: mortality; body weight; haematology; gross pathology; histopathology

Remarks on result:

other: Effect type: toxicity (migrated information)

Conclusions:

Diphenylamine did not demonstrate a carcinogenic potential when fed to mice at levels up to 5250 ppm in the diet

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication which meets basic scientific principles

Qualifier:

no guideline followed

Principles of method if other than guideline:

Female Sprague-Dawley rats were orally administered a single maximum tolerated dose (MTD) of diphenylamine and observed during 6 months for neoplastic effects especially in the mammary tissues

GLP compliance:

not specified

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague-Dawley, Inc., Madison, Wisconsin, USA
- Age at study initiation: 50-55 days old
- Housing: stainless steel cages, 16x16x5 inches, no more than 5 rats/cage
- Diet : Purina laboratory chow ad libitum

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Analytical verification of doses or concentrations:

not specified

Frequency of treatment:

Single administration

Post exposure period:

6 months

Remarks:

Doses / Concentrations:
300 mg/rat
Basis:
nominal conc.

No. of animals per sex per dose:

20 rats tested with diphenylamine
89 control rats

Control animals:

yes, concurrent vehicle

Positive control:

7, 12-Dimethylbenzanthracene

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

2/20 dead

Mortality:

mortality observed, treatment-related

Description (incidence):

2/20 dead

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

similar in experimental and control groups

Gross pathological findings:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Conclusions:

Diphenylamine gave no indications of neoplastic effects in the major organs (mammary tissues, intestinal tract, pituitary) and the mortality before end of the experiment was 2/20

Reference 4

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2009-2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

Not Specified

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report

Species:

rat

Strain:

Fischer 344/DuCrj

Details on species / strain selection:

Historical control data available for the species and strain selected for the study

Sex:

male/female

Details on test animals or test system and environmental conditions:

No further details specified in the study report.

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

No details on specific exposure are specified in the study report. Both male and female animals were fed doses in diet as specified below.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The identity of the diphenylamine used in these experiments was confirmed by both infrared spectrometry and mass spectrometry. The chemical was analyzed by high performance liquid chromatography before and after use to affirm its stability. The concentrations of diphenylamine in the diet were determined by the high performance liquid chromatography at the time of preparation and on the 8th day after the preparation while stored at room temperature or stored in the refrigerator.

Duration of treatment / exposure:

2 years (104 weeks)

Frequency of treatment:

Daily

Post exposure period:

No post exposure period specified int he study report

Dose / conc.:

0 ppm

Dose / conc.:

250 ppm

Dose / conc.:

1 000 ppm

Dose / conc.:

4 000 ppm

No. of animals per sex per dose:

50 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

Groups of test animals were administered diphenylamine in their food for 2 years (104 weeks). Each group consisted of either 50 male or 50 female rats. The dietary concentration of diphenylamine were 0, 250, 1000 or 4000 ppm (w/w). Both sexes were administered each concentration of diphenylamine. The highest dose level was chosen so as not to exceed the maximum tolerated dose (MTD), based on both growth rate and toxicity in a previous 13-week toxicity study.

Positive control:

Not required for the study

Observations and examinations performed and frequency:

Animals were observed daily for clinical signs and mortality. Body weight, water consumption and food consumption were measured once a week for the first 14 weeks and every 4 weeks thereafter. Animals found dead, in a moribund state, or surviving to the end of the 2-year administration period.
Urinalysis was performed near the end of the administration period. Hematology and blood chemistry analysis were performed at the terminal necropsy: surviving animals were fasted overnight and bled under deep ether anethesia.

Sacrifice and pathology:

Organs and tissues were removed, weighed and examined for macroscopic lesions at necropsy. The organs and tissues were then fixed and embedded in paraffin. Five µm thick tissue sections were prepared and stained with hematoxylin and eosin and examined microscopically.

Statistics:

Incidences of neoplastic lesions were statistically analyzed by Fisher's exact test. Any positive dose-response trends of diphenylamine induction of neoplastic lesions were analyzed by Peto's test. Incidences of non-neoplastic lesions and urinalysis were analyzed by the Chi-square test. Changes in body weight, water consumption, food consumption, hematological and blood biochemical parameters, and organ weights were analyzed by Dunnett's test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Brown urine was observed in the 4000 ppm-fed males and females. Soiled fur around the genitalia was observed in the all treated females and 4000ppm-fed males.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No significant differences in survival rate was found between any diphenylamine-fed group of either sex and their respective controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Growth rates of the males fed 4000 ppm and all females fed diphenylamine were suppressed throughout most of the 2 year administration period, and growth rates of males fed 1000 ppm were suppressed in the earlier administration period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was decreased in the males fed 4000 ppm throughout of the 2 year administration period, and food consumption of males fed 1000 ppm were suppressed in the earlier administration period. Food consumption was decreased in females fed 1000 and 4000 ppm until the 78th week of the 2 year administration period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In blood and hematopietic system, methemoglobin concentration was increased in males fed 1000 ppm and above, and all female groups fed diphenylamine. Anemia caused by increase of methemoglobin concentration was observed in males fed 4000 ppm, and females fed 1000 ppm and above. Various anemia-related changes in hematology were observed in diphenylamine-fed groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Various anemia-related changes in biochemistry were observed in diphenylamine-fed groups.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Brown urine was observed in the 4000 ppm-fed males and females.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the spleen. Increased organ weights were observed.
In the liver, increased organ weights were observed in males and females fed 1000 ppm and above.
In the kidney, increased organ weights were observed in males fed 1000 ppm and above.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the spleen, engorgement of erythrocyte, increased extramedullary hematopoiesis (male only), deposit of hemosiderin (males only), capsular hyperplasia, angiectasis and focal fibrosis (female only) were observed.
In the liver, hepatocellular hypertrophy were observed in males and females fed 4000 ppm, and increased activity of deviation enzymes were observed in fed groups.
In the kidney, increased severities of chronic nephropathy was observed in males fed 4000 ppm, deposit of brown pigment in proximal tubule were observed in males and females fed 4000 ppm, mineralization in pelvis were observed in males fed 4000 ppm and females fed 1000 ppm and above. Plasma urea nitrogen was increased in males and females fed 4000 ppm.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The incidence of hemangioma and combined incidence of hemangioma and/or hemangiosarcoma (Peto test and Cochran-Armitage test) in spleen were increased in males. The incidence of hemangiosarcoma in all organs including subcutis and spleen increased in male (Peto test and Cochran-Armitage test), and the combined incidence of hemangioma and hemangiosarcoma were increased in male fed 4000 ppm (Fisher’[s exact test). In females, the incidence of adenocarcinoma and the combined incidence of adenoma and/or adenocarcinoma in uterus were increased (Peto test and Cochran-Armitage test).

Relevance of carcinogenic effects / potential:

The incidence of hemangioma and combined incidence of hemangioma and/or hemangiosarcoma (Peto test and Cochran-Armitage test) in spleen were increased in males. The incidence of hemangiosarcoma in all organs including subcutis and spleen increased in males (Peto test and Cochran-Armitage test), and the combined incidence of hemangioma and hemangiosarcoma were increased in males fed 4000 ppm (Fisher’s exact test). In females, the incidence of adenocarcinoma and the combined incidence of adenoma and/or adenocarcinoma in uterus were increased (Peto test and Cochran-Armitage test).

Key result

Dose descriptor:

NOAEL

Effect level:

250 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Using blood and hematopoietic system and liver as endpoint marker

Remarks on result:

other: 12 mg/kg body weight per day

Key result

Dose descriptor:

LOAEL

Effect level:

250 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Using blood and hematopoietic system and liver as endpoint markers

Remarks on result:

other: 15 mg/kg body weight per day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

250 ppm

System:

other: blood and hematopoietic system

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Incidences of selected neoplastic lesions of male rats in the 2-year feed carcinogenicity study of diphenylamine

Dose (ppm)		0	250	1000	4000	Peto test	Cochran-Armitage test
Number of examined animals		50	50	50	50
Benin tumor
Skin appendage	Keratoacanthoma	3	3	5	2
subcutis	Fibroma	2	11**	3	2
	Hemangioma	0	0	0	1
Pancreas	Islet cell adenoma	6	3	2	0*		↑
Spleen	Hemangioma	0	1	0	0		↑
Pituitary	Adenoma	17	19	11	8*
thyroid	C-cell adenoma	11	10	12	13
Adrenal	Pheochromocytoma	4	8	4	4
Testis	Interstitial cell tumor	37	40	46*	46*	↑	↑
Malignant tumor
Subcutis	Fibrosarcoma	0	2	0	1
	Hemangiosarcoma	0	0	0	1
Spleen	Hemangiosarcoma	0	0	0	3	↑↑	↑↑
	Mononuclear cell leukemia	5	3	2	1
Spleen	Hemangioma + Hemangiosarcoma	0	1	0	3	↑	↑
Subcutis	Fibroma + Fibrosarcoma	2	13**	3	3
All organs ^a)	Hemangioma	0	1	0	1
	Hemangiosarcoma	0	0	0	4	↑↑	↑↑
	Hemangioma + Hemangiosarcoma^b)	0	1	0	5*

 

Significant difference

*: p ≤ 0.05 (fisher test)

↑: p ≤ 0.05 increase (Peto, Cochran-Armitage test)

↓ p ≤ 0.05 decrease (Cochran-Armitage test)

**: p ≤ 0.01 (fisher test)

↑↑: p ≤ 0.01 increase (Peto, Cochran-Armitage test)

↓↓ p ≤ 0.01 decrease (Cochran-Armitage test)

a: all organs were consisted of subcutis and spleen

b: Combined analysis of Hemangioma + Hemangiosarcoma in all organs of Peto test and Cochran-Armitage test was not applied.

 

Incidences of selected neoplastic lesions of female rats in the 2-year feed carcinogenicity study of diphenylamine

Dose (ppm)		0	250	1000	4000	Peto test	Cochran-Armitage test
Number of examined animals		50	50	50	50
Benin tumor
Pituitary	Adenoma	11	13	12	16
thyroid	C-cell adenoma	7	9	7	5
uterus	Endometrial stromal polyp	5	2	6	7
	Adenoma	0	1	0	0
Mammary gland	Fibroadenoma	8	11	7	2*		↓
Malignant tumor
Spleen	Hemangiosarcoma	0	0	0	1
	Mononuclear cell leukemia	3	2	0	5
Pituitary	adenocarcinoma	2	1	4	0	↑
Uterus	Adenocarcinoma	1	0	0	4	↑↑	↑↑
uterus	Adeoma + adenocarcinoma	1	1	0	4	↑	↑

 

Significant difference

*: p ≤ 0.05 (fisher test)

↑: p ≤ 0.05 increase (Peto, Cochran-Armitage test)

↓ p ≤ 0.05 decrease (Cochran-Armitage test)

**: p ≤ 0.01 (fisher test)

↑↑: p ≤ 0.01 increase (Peto, Cochran-Armitage test)

↓↓ p ≤ 0.01 decrease (Cochran-Armitage test)

 

 

 

Conclusions:

There was some evidence for carcinogenicity of diphenylamine in male and female rats, based on the increase incidence of vascular tumors in spleen and increase incidence of vascular tumors in all organs including in spleen and subcutus in males.

Using blood and hematopoietic system and liver as endpoint markers, the no-observed-adverse-effect-level (NOAEL) of diphenylamine in the diet was 250 ppm (12 mg/kg body weight per day) for males. The NOAEL for females could not be estimated, the lowest observed-adverse-effect-level (LOAEL) of diphenylamine in the diet was 250 ppm (15 mg/kg body weight per day) for females.

Executive summary:

The tests were conducted and supported by the Ministry of health, Labour and Welfare of Japan. The study was conducted in accordance with the OECD Good Laboratory Practice and with reference to the OCED Guideline for Testing of Chemicals 451 “Carcinogenicity Studies”.

 

The carcinogenicity and chronic toxicity of diphenylamine were examined in F344/DuCrlClj rats. Groups pf test animals were administered diphenylamine in their food for 2 years (104 weeks). Each group consisted of either 50 male or 50 female rats. The dietary concentration of diphenylamine were 0, 250, 1000 or 4000 ppm (w/w). Both sexes were administered each concentration of diphenylamine.

 

The animals were observed daily for clinical signs and mortality. Body weight, water consumption and food consumption were measured once a week for the first 14 weeks and every 4 weeks thereafter. Animals found dead, in a moribund state, or surviving to the end of the 2-year administration period underwent complete necropsy.

 

No significant difference in survival rate was found between any diphenylamine-fed group of either sex and their respective controls. Brown urine was observed in the 4000 ppm-fed males and females. Growth rates of the males fed 4000 ppm and all females fed diphenylamine were suppressed throughout most of the 2-yesr administration period, and growth rates of the male fed 1000 ppm were suppressed in the earlier administration. Food consumption was decreased in the males fed 4000 ppm throughout the 2-year administration period, and food consumption of males fed 1000 ppm were suppressed in the earlier administration period. Food consumption was decreased in the females fed 1000 and 4000 ppm until the 78^th week of the 2-year administration.

 

The incidences of hemangioma and combined incidence of hemangioma and/or hemangiosarcoma in spleen were increased in males. The incidence of hemangiosarcoma in all organs including subcutis and spleen increased in male and the combined incidence of hemangioma and hemangiosarcoma was increased in males fed 4000 ppm. In females, the incidence of adenocarcinoma and the combined incidence of adenoma and/or adenocarcinoma in uterus were increased.

 

Anemia caused by the increase of methemoglobin concentration was observed in males fed 4000 ppm, and females fed 1000 ppm and above. Various anemia-related changes in hematology and biochemistry were observed in diphenylamine-fed groups.

 

In the spleen, increased organ weights, engorgement of erythrocyte, increased extramedullary hematopoiesis (male only), deposit of hemosiderin (males only), capsular hyperplasia, angiectasis and focal fibrosis (female only) were observed.

In the liver, increased organ weights were observed in males and females fed 1000 ppm and above, hepatocellular hypertrophy were observed in males and females fed 4000 ppm, and increased activity of deviation enzymes were observed in fed groups.

In the kidney, increased organ weights were observed in males fed 1000 ppm and above. Increased severities of chronic nephropathy was observed in males fed 4000 ppm, deposit of brown pigment in proximal tubule were observed in males and females fed 4000 ppm, mineralization in pelvis were observed in males fed 4000 ppm and females fed 1000 ppm and above. Plasma urea nitrogen was increased in males and females fed 4000 ppm.

 

Using blood and hematopoietic system and live as endpoint markers, the no-observed-adverse-effect-level (NOAEL) of diphenylamine in the diet was 250 ppm (12 mg/kg body weight per day) for males.

The NOAEL for female could not estimate, the lowest observed-adverse-effect-level (LOAEL) of diphenylamine in the diet was 250 ppm (15 mg/kg body weight per day for females.

 

There was some evidence for carcinogenicity of diphenylamine in male and females rats, based on increase in the incidence of vascular tumors in spleen and increase in the incidence of vascular tumors in all organs including in spleen and subcutus in males.  

Reference 5

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2009-2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

Not specified

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

No further details specified in the study report.

Species:

mouse

Strain:

other: B6D2F1

Details on species / strain selection:

Historical control data available for the species and strain selected for the study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

No further details specified in the study report.

Route of administration:

oral: feed

Vehicle:

not specified

Details on exposure:

Details on exposure not specified in the study report. Both male and female animals were fed doses in diet as specified below.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The identify of diphenylamine used in these experiments was confirmed by both infrared spectrometry and mass spectrometry. The chemical was analyzed by high performance liquid chromatography before and after use to affirm its stability. The concentrations of diphenylamine in the diet were determined by high performance liquid chromatography at the time of preparation and on the 8th day after preparation while stored at room temperature or stored in the refrigerator.

Duration of treatment / exposure:

2 years (104 weeks)

Frequency of treatment:

Daily

Post exposure period:

Post exposure period not require for the study

Dose / conc.:

0 ppm

Dose / conc.:

250 ppm

Dose / conc.:

1 000 ppm

Dose / conc.:

4 000 ppm

No. of animals per sex per dose:

50 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

Groups of test animals were administered diphenylamine in their food for 2 years (104 weeks). Each group consisted of either 50 male or 50 female rats. The dietary concentration of diphenylamine were 0, 250, 1000 or 4000 ppm (w/w). Both sexes were administered each concentration of diphenylamine. The highest dose level was chosen so as not to exceed the maximum tolerated dose (MTD), based on both growth rate and toxicity in a previous 13-week toxicity study.

Observations and examinations performed and frequency:

Animals were observed daily for clinical signs and mortality. Body weight, water consumption and food consumption were measured once a week for the first 14 weeks and every 4 weeks thereafter.
Animals found dead, in a moribund state, or surviving to the end of the 2-year administration period.
Urinalysis was performed near the end of the administration period.
Haematology and blood chemistry analysis were performed at the terminal necropsy: surviving animals were fasted overnight and bled under deep ether anaesthesia.

Sacrifice and pathology:

Organs and tissues were removed, weighed and examined for macroscopic lesions at necropsy. The organs and tissues were then fixed and embedded in paraffin. Five μm thick tissue sections were prepared and stained with hematoxylin and eosin and examined microscopically.

Statistics:

Incidences of neoplastic lesions were statistically analyzed by Fisher's exact test. Any positive dose response trends of diphenylamine induction of neoplastic lesions were analyzed by Peto's test. Incidences of non-neoplastic lesions and urinalysis were analyzed by the Chi-square test. Changes in body weight, water consumption, food consumption, hematological and blood biochemical parameters, and organ weights were analyzed by Dunnett's test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Brown urine was observed in the 4000 ppm-fed males and females.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

The markedly decreased survival rate of the 4000 ppm-fed male group was attributed to the increased number of deaths due to urinary retention. Survival rates of the females fed 4000 ppm increased more than the female control at the end of the administration period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights were suppressed in males fed 4000 diphenylamine after the 19th week of administration period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption in the all administered group were similar to the respective controls. Due to the markedly decreased survival rate caused by urinary retention and the marked body weight suppression, the high dose level of 4000 ppm for males was considered to exceed the MTD.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In blood hematopoietic system, methemoglobin concentration was increased in all groups of males and females fed diphenylamine. Anemia was caused by the increase of methemoglobin concentration was observed in all groups of males and females fed diphenylamine. Various anemia-related changes in hematology were observed in diphenylamine-fed groups. In the bone marrow, increased hematopoiesis was observed. Plasma urea nitrogen was increased in females fed 4000 ppm.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Various anemia-related changes in biochemistry were observed in diphenylamine-fed groups. In the bone marrow, increased hematopoiesis was observed. Plasma urea nitrogen was increased in females fed 4000 ppm.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Brown urine was observed in the 4000 ppm-fed males and females.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the spleen, increased organ weights were observed.
Kidney weights were increased in females fed 1000 ppm and above.

Description (incidence and severity):

In the bone marrow, increased hematopiesis was observed.
In the spleen, increased extramedullary hematopiesis, deposit of hemosiderin and engorgement of erythrocyte were observed.
In the liver, hepatocellular hypertrophy were increased in males and females fed 4000 ppm.
In the urinary system, urinary retention was observed in males fed 4000 ppm. Plasma urea nitrogen was increased in females fed 4000 ppm.
In the kidney pyelonephritis was observed in males fed 4000 ppm.
In the urinary bladder, dilation was observed in males fed 4000 ppm and hyaline droplet degeneration was observed in both males and females fed 4000 ppm. The inflammation in the urethra was observed in males fed 4000 ppm.
In lung, uremic pneumonitis was increased in males fed 4000 ppm and degeneration of blood vessels was observed in both males and females fed 4000 ppm.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The combined incidence of hemangioma and/or hemangiosarcoma in spleen were increased in males fed 1000 ppm. The incidence of hemangioma in all organs including subcutis, bone marrow, spleen, liver and heart was increased in males and the incidence of hemangioma and the combined incidence of hemangioma and/or hemangiosarcoma was increased in males fed 1000 ppm. The incidence of histiocytic sarcoma in uterus was increased in female mice fed 1000 ppm diphenylamine. But the incidence was within the range of maximum incidence of the JBRC historical control data, so the incidence of histiocytic sarcoma in uterus can not be attributed to the diphenylamine administration. No significant diphenylamine related increased in incidence of neoplastic lesions was found in females.

Relevance of carcinogenic effects / potential:

The combined incidence of hemangioma and/or hemangiosarcoma in spleen was increased in males fed 1000 ppm (Fisher’s exact test). The incidence of hemangioma in all organs including subcutis, bone marrow, spleen, liver and heart was increased in males (Peto test), and the incidence of hemangioma and the combined incidence of hemangioma and/or hemangiosarcoma was increased in males fed 1000 ppm (Fishers’s exact test). The incidence of histiocytic sarcoma in uterus was increased in female mice fed 1000 ppm diphenylamine. But the incidence was within range of maximum incidence of the JBRC to be attributed to the diphenylamine administration. No significant diphenylamine related incidence of neoplastic lesions was found in females.

Key result

Dose descriptor:

LOAEL

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: effects on blood and hematopoietic system

Remarks on result:

other: male: 29kg body weight per day, female: 36 body weight per day

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

250 ppm

System:

other: blood and hematopoietic system

Organ:

other: liver, kidney, urinary system

Treatment related:

yes

Dose response relationship:

not specified

Incidences of selected neoplastic lesions of male mice in the 2-year feed carcinogenicity study of diphenylamine

Dose (ppm)		0	250	1000	4000	Peto test	Cochran-Armitage test
Number of examined animals		50	50	50	50
Benin tumor
Subcutis	Hemangioma	0	0	1	0
Lung	Bronchiolar-alveolar adenoma	5	4	7	4
Bone marrow	Hemangioma	0	0	0	1
Spleen	Hemangioma	1	0	6	2
Liver	Hemangioma	2	2	5	3	↑
	Hepatocellular adenoma	9	14	10	2*		↓↓
Harderian gland	adenoma	4^a)	2	1	1
Malignant tumor
Lung	Bronchiolar-alveolar carcinoma	5	6	8	1
Lymph node	Malignant lymphoma	6	4	3	2
Spleen	Hemangiosarcoma	0	0	3	1
Heart	Hemangiosarcoma	0	1	0	0
Liver	Histiocytic sarcoma	5	1	1	1
	Hepatocellular carcinoma	7	15*	5	2		↓↓
	Hemangiosarcoma	0	1	2	1
Epididymis	Histiocytic sarcoma	1	1	3	1
Spleen	Hemangioma + Hemangiosarcoma	1	0	9**	3
liver	Hemangioma + Hemangiosarcoma	2	3	7	4	↑
All organs^(b)	Hemangioma	3	2	10*	6	↑
	Hemangiosarcoma	0	1	4	1
	Hemangioma + hemangiosarcoma ^(c)	3	3	14**	6

 

Significant difference

*: p ≤ 0.05 (fisher test)

↑: p ≤ 0.05 increase (Peto, Cochran-Armitage test)

↓ p ≤ 0.05 decrease (Cochran-Armitage test)

**: p ≤ 0.01 (fisher test)

↑↑: p ≤ 0.01 increase (Peto, Cochran-Armitage test)

↓↓ p ≤ 0.01 decrease (Cochran-Armitage test)

a: number of animals examined is 49

b: All organs were consisted of spleen, liver, subcutis, bone marrow and heart.

c: Combined analysis of hemangioma + hemangiosarcoma in all organs of Peto test and Cochran-Armitage test was not applied.

 

Incidences of selected neoplastic lesions of female mice in the 2-year feed carcinogenicity study of diphenylamine

Dose (ppm)		0	250	1000	4000	Peto test	Cochran-Armitage test
Number of examined animals		50	50	50	50
Benin tumor
Lung	Bronchiolar-alveolar adenoma	1	3	1	2
Liver	Hepatocellular adenoma	4	4	3	0		↓
Pituitary	Adenoma	2	0	5	4
Harderian gland	Adenoma	0	3	1	2
Malignant tumor
Lymph node	Malignant lymphoma	18	20	17	15
Spleen	Malignant lymphoma	0	3	1	0
Liver	Histiocytic sarcoma	4	0	1	1
	Hemangiosarcoma	1	1	2	3
Uterus	Histiocytic sarcoma	8	7	17*	12

 

Significant difference

*: p ≤ 0.05 (fisher test)

↑: p ≤ 0.05 increase (Peto, Cochran-Armitage test)

↓ p ≤ 0.05 decrease (Cochran-Armitage test)

**: p ≤ 0.01 (fisher test)

↑↑: p ≤ 0.01 increase (Peto, Cochran-Armitage test)

↓↓ p ≤ 0.01 decrease (Cochran-Armitage test)

Conclusions:

There was some evidence for carcinogenicity of diphenylamine in male mice based on the increased incidences of vascular tumors in spleen and in all organs included spleen and liver. There was no evidence for carcinogenicity of diphenylamine in female mice.

In the present two-year feeding study, the effects on blood and hematopoietic system were observed for the lowest dose of 250 ppm in both males and females. The lowest observed adverse effect level (LOAEL) of diphenylamine in the diet was 250 ppm (male: 29kg body weight per day, female: 36 body weight per day).

Executive summary:

The tests were contracted and supported by the Ministry of health, Labour and Welfare of Japan. The study was conducted in accordance with the OECD Good Laboratory Practice and with reference to the OCED Guideline for Testing of Chemicals 451 “Carcinogenicity Studies”.

 

The carcinogenicity and chronic toxicity of diphenylamine were examined in B6D2F1/Crlj mice. Groups of test animals were administered diphenylamine in their food for 2 years (104 weeks). Each group consisted of either 50 male or 50 female mice. The dietary concentration of diphenylamine were 0, 250, 1000 or 4000 ppm (w/w). Both sexes were administered each concentration of diphenylamine.

 

The animals were observed daily for clinical signs and mortality. Body weight, water consumption and food consumption were measured once a week for the first 14 weeks and every 4 weeks thereafter. Animals found dead, in a moribund state, or surviving to the end of the 2-year administration period underwent complete necropsy.

 

The markedly decreased survival rate of the 4000 ppm-fed male group was attributed to the increased number of deaths due to urinary retention. Survival rates of the females fed 4000 ppm increased more than the female control at the end of the administration period. Brown urine was observed in the 4000 ppm-fed males and females. Body weights were suppressed in males fed 4000 ppm diphenylamine throughout most of the 2-yesr administration period and in females fed 4000 ppm diphenylamine after 18^th week of the administration period. Food consumption in all the administered group were similar to the respective controls. Due to the markedly decreased survival rate caused by urinary retention and the marked body weights suppression, the high dose level of 4000 ppm for males was considered to exceed the MTD.

 

The combined incidence of hemangioma and/or hemangiosarcoma in spleen were increased in males fed 1000 ppm. The incidence of hemangioma in all organs including subcutis, bone marrow, spleen, liver and heart was increased in males and the incidence of hemangioma and the combined incidence of hemangioma and/or hemangiosarcoma was increased in males fed 1000 ppm. The incidence of histiocytic sarcoma in uterus was increased in female mice fed 1000 ppm diphenylamine. But the incidence was within the range of maximum incidence of the JBRC historical control data, so the incidence of histiocytic sarcoma in uterus can not be judged to eb attributed to the diphenylamine administration. No significant diphenylamine related increased in incidence of neoplastic lesions was found in females.

 

Anemia caused by the increase of methemoglobin concentration was observed in all groups of males and females fed diphenylamine. Various anemia-related changes in hematology and biochemistry were observed in diphenylamine-fed groups.

 

In the bone marrow, increased hematopiesis was observed.

In the spleen, increased organ weights, increased extramedullary hematopiesis, deposit of hemosiderin and engorgement of erythrocyte were observed.

In the liver, hepatocellular hypertrophy were increased in males and females fed 4000 ppm.

In the urinary system, urinary retention was observed in males fed 4000 ppm. Plasma urea nitrogen was increased in females fed 4000 ppm.

Kidney weights were increased in females fed 1000 ppm and above, pyelonephritis was observed in males fed 4000 ppm.

In the urinary bladder, dilation was observed in males fed 4000 ppm and hyaline droplet degeneration was observed in both males and females fed 4000 ppm. The inflammation in the urethra was observed in males fed 4000 ppm.

In lung, uremic pneumonitis was increased in males fed 4000 ppm and degeneration of blood vessels was observed in both males and females fed 4000 ppm.  

 

In the two-year feeding study, the effects on blood and hematopoietic system were observed for the lowest of 250 ppm in both males and females. The lowest observed-adverse-effect-level (LOAEL) of diphenylamine in the diet was 250 ppm (male: 29 mg/kg body weight per day. Female: 36 mg/kg body weight per day).

 

There was some evidence for carcinogenicity of diphenylamine in male mice, based on the increase incidences of vascular tumors in spleen and in all organs including spleen and liver. There was no evidence for carcinogenicity of diphenylamine in female mice.  

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

250 mg/kg bw/day

Study duration:

chronic

Species:

other: Rat/Mouse

Quality of whole database:

K2

System:

other: blood and hematopoietic system

Organ:

other: liver, kidney, urinary system

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information from two 2 year oral feed carcinogenicity studies in rats and mice, diphenylamine has shown potental for carcinogenicity. Therefore the substance is classified as a category 2 carcinogen in accordance with Regulation 1272/2008 (CLP)