(p-ammoniophenyl)bis(2-hydroxyethyl)ammonium sulphate

Carcinogenicity

Description of key information

N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate did not induce tumours after oral administration in a long-term rat study. The substance has also been studied alone and after mixing with hydrogen peroxide by topical application to mice and rats. However, as known carcinogens have been tested by the same experimental procedures without any tumour formation, it is not possible to draw any conclusions from these experiments with regard to potential carcinogenic effects.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, followed method comparable to guideline with deviation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

No information on clinical chemistry or urinalysis provided

GLP compliance:

no

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc. (Atsugi, Japan)
- Age at study initiation: 6 wk
- Weight at study initiation: Not reported
- Fasting period before study: No
- Housing: In groups of five, in transparent polypropylene cage on hardwood chip bedding
- Diet: Certified powdered CE-2 diet (CLEA Japan Co., Ltd, Tokyo Japan); ad libitum
- Water: ad libitum
- Acclimation period: 1 wk, body weight and health condition were monitored during acclimation.

ENVIRONMENTAL CONDITIONS
- Temperature: 22±2°C
- Humidity: 55±10%
- Air changes: More than 15 air changes per h with all fresh system.
- Photoperiod: 12 h light/12 h dark cycle per d

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION: Test substance was incorporated in to the diet at required levels. Corn oil (Nihon Shokuhin Kako Co., Ltd. Tokyo Japan) was added to all diets at a concentration of 2% before mixing in order to prevent contamination with test compound as dust.
- Rate of preparation of diet: Not reported
- Mixing appropriate amounts with: Certified powdered CE-2 diet (CLEA Japan Co., Ltd, Tokyo Japan)
- Storage temperature of food: Not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of stability and homogeneity of test substance in prepared diet confirmed that test substance was stable at room temperature and the homogeneity was found to be satisfactory. Content analysis was performed at monthly intervals by Japan Food Research, Tokyo and each dietary levels analyzed was within the acceptable range.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

continuously in diet, ad libitum

Post exposure period:

no

Dose / conc.:

300 ppm

Remarks:

nominal in diet (daily average intake for males 12 mg/kg/day and for females 14 mg/kg/day)

Dose / conc.:

1 000 ppm

Remarks:

nominal in diet (daily average intake for males 40 mg/kg/day and for females 48 mg/kg/day)

Dose / conc.:

3 000 ppm

Remarks:

nominal in diet (daily average intake for males 121 mg/kg/day and for females 146 mg/kg/day)

No. of animals per sex per dose:

50 animals/sex/group; A group of other 40 animals served as biological monitors

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose selection was based on 13 wk range finding preliminary study, details are provided in "Any other information on materials and methods incl. tables" section.
- Rationale for animal assignment: Animals were randomized to different treatment and control group using a computerized stratified body weight technique; so that the weight distribution within each group was similar and initial mean body weights were approximately equal.
- Rationale for selecting satellite groups: No satellite group were included in the study

Positive control:

no

Observations and examinations performed and frequency:

MORTALITY/MORBIDITY: Yes
- Time schedule: Daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations included: Signs of abnormalities

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for the first 14 wk and every other week thereafter

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was measured over a 2-day period before each weighing
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE : No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At wk 104
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight)
- How many animals: 12 animals/sex/group
- Parameters examined: Erythrocyte and leucocyte counts, haemoglobin concentrations, haematocrit values and platelet counts

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE: At week 104, all surviving rats were deprived of food, but not water, overnight and then killed under ether anaesthesia

GROSS PATHOLOGY: Yes, gross observations were made at autopsy and recorded.
- Samples of heart, spleen, adrenals, thyroids, liver, kidneys, gonads, brain, aorta, lymph nodes, thymus, pituitary, adrenals, trachea, lung, tongue, salivary glands, oesophagus, stomach, small intestine, large intestine, pancreas, kidney, urinary bladder, testis, prostate, seminal vesicle, epididymis, ovary, uterus, mammary gland, vagina, spinal cord, peripheral nerve, eye, Harderian gland, skin, skeletal muscle, bone and bone marrow, and any other tissues of abnormal appearance were fixed in 10% buffered formalin.

HISTOPATHOLOGY: Yes; A full histopathological examination was performed on haematoxylin-eosin stained tissue sections of organs from the control and high-dose rats, as well as from animals with unscheduled deaths or in a moribund condition during the course of the study. Histopathological examination for terminal sacrifice rats of the low and mid dose groups were restricted to the lungs, liver, kidneys, any gross lesions and the thyroids.

Other examinations:

- Absolute and relative organ weight: At terminal sacrifice, heart, spleen, adrenals, thyroids (after fixation), liver, kidneys, gonads and brain from each rat were weighed and organ to body weight ratios determined.
- Blood smears stained with Wright-Giemsa stain were also prepared from all animals, either killed in a moribund condition or sacrificed at termination, to allow diagnosis of haematopoietic neoplasms.

Statistics:

For body weight, haematology, blood biochemistry and organ weight data, the significance of intergroup data were assessed using two tailed student's t-test. Insufficient homogeneity of variance was corrected with respect to degrees of freedom.
- For incidences of non-neoplastic and neoplastic lesions, the significance of differences observed between the control and treated groups were evaluated by Fisher's exact probability test.
- The level of significance was taken at P<0.05 or 0.01.
- Differences in the survival period between the control and treated groups were analyzed by generalized Wilcoxon and Cox-Mantel tests.

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):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No clinical signs related to treatment were apparent in any of the rats during the 104 wk of the experiment. At the end of the study, the survival rates of control, low mid and high dose rats were 86, 80, 84 and 84%, respectively, for males and 78, 80, 80 and 80%, respectively, for females. There were no significant differences in mortality between controls and treated animals during the course of the study.

BODY WEIGHT AND WEIGHT GAIN: Statistically significant lower body weights, or a tendency for lower body weights, were observed in both sexes of high dose group from wk 2 to termination. No body weight retardation was found in mid and low dose group animals.

FOOD CONSUMPTION: Food consumption in controls and treated animals showed no clear difference.

TEST SUBSTANCE INTAKE: For the three treated groups, the intakes of test substance were calculated from the nominal dietary levels. The average test substance intakes for males and females were 12 and 14 mg/kg body weight/day, respectively, in the 300 ppm group; 40 and 48 mg/kg body weight/day, respectively, in the 1000 ppm group; and 121 and 146 mg/kg body weight/day, respectively, in the 3000 ppm group.

HAEMATOLOGY: No treatment-related adverse effects were apparent in the haematology results

ORGAN WEIGHTS: Slightly increased values for thyroid weight and the thyroid to body weight ratio in the high dose group male, compared with those of the control male, were significant. Thyroid weight, but not the thyroid to body weight ratio, in the mid dose male was also significantly higher than the respective control values. However, the findings were not supported by histopathological observations.
- Based on histopathological observations and evidences from previous studies, differences in thyroid weight and thyroid to body weight ratio were not considered to be treatment related toxic effects.

GROSS PATHOLOGY: No treatment related macroscopic changes were found in treated animals at autopsy or on detailed examination after fixation.

HISTOPATHOLOGY: NON-NEOPLASTIC: A wide range of non-neoplastic lesions were evident. But no treatment-related changes were found.

HISTOPATHOLOGY: NEOPLASTIC: A conspicuous decrease in the incidence of fibromas in the skin/subcutis was found in high dose group males. No significant differences from controls were observed in the incidences of other types of tumour. There were no differences in the development of any tumour category between control and treated groups for either sex.
- Histopathology of thyroid: Follicular cell adenomas were found in one low dose group male and a male and a female from mid dose group. Follicular cell carcinomas were found in one low dose group male and two females of the mid dose group. However, such tumours were not found in either sex of the high dose group. C-cell adenomas and carcinomas randomly occurred in controls and treated animals. None of these lesions was considered to be related to treatment.

Relevance of carcinogenic effects / potential:

N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate was not carcinogenic to Fischer 344/DuCrj rats of either sex in chronic feeding study.

Dose descriptor:

NOAEL

Effect level:

>= 3 000 other: ppm in diet

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Overall effects

Remarks on result:

other: Effect type: carcinogenicity

Dose descriptor:

NOAEL

Effect level:

1 000 other: ppm in diet

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Body weight

Remarks on result:

other: Effect type: toxicity

Dose descriptor:

NOAEL

Effect level:

>= 121 mg/kg bw/day

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: Overall effects

Remarks on result:

other: Effect type: carcinogenicity

Dose descriptor:

NOAEL

Effect level:

>= 146 mg/kg bw/day

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: Overall effects

Remarks on result:

other: Effect type: carcinogenicity

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: Body weight

Remarks on result:

other: Effect type: toxicity

Dose descriptor:

NOAEL

Effect level:

48 mg/kg bw/day

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: Body weight

Remarks on result:

other: Effect type: toxicity

Conclusions:

N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate was not carcinogenic to Fischer 344/DuCrj rats of either sex in chronic feeding study.

Executive summary:

The toxicology and carcinogenic potential of N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate (4APE) was determined by following a method equivalent or similar to OECD guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies).

This study was designed to assess the chronic toxicity and carcinogenicity of test substance when administered to Fischer 344/DuCrj rats (50/sex/group) at dose levels of 0, 300, 1000 and 3000 ppm in the diet (equivalent to 12, 40, and 121 mg/kg bw/day (males); 14, 48 and 146 mg/kg bw/day (females)).

A total of 400 CD-1 Fischer 344/DuCrj rats (200/sex) of 6 wk age (source: Charles River Japan Inc) were housed in groups of 5 in transparent polypropylene cages on hardwood chip bedding and maintained under standard laboratory conditions (temperature: 22±2°C, humidity: 55±10%, more than 15 air changes per h, 12 h light/12 h dark cycle per d). The animals were acclimated for 1 wk and fed on Purina Certified powdered CE-2 diet; ad libitum.

Dose selection was based on a 13 wk range finding preliminary study. Prior to treatment, animals were randomized to different treatment and control groups using a computerized stratified body weight technique; so that the weight distribution within each group was similar and initial mean body weights were approximately equal.

During treatment animals were observed daily for viability and signs of abnormalities. Body weight and food consumption were recorded at regular intervals during the treatment period. At wk 104, all surviving rats were deprived of food, but not water, overnight and then sacrificed under ether anaesthesia. Blood samples were collected from 12 animals/sex/group for evaluation of haematological parameters. Gross observations were made at autopsy and recorded. Blood smears stained with Wright-Giemsa stain were also prepared from all animals, either killed in a moribund condition or sacrificed at termination, to allow diagnosis of haematopoietic neoplasms. At terminal sacrifice, heart, spleen, adrenals, thyroids (after fixation), liver, kidneys, gonads and brain from each rat were weighed and organ to body weight ratios determined.

A full histopathological examination was performed on haematoxylin-eosin stained tissue sections of organs from the control and high-dose rats, as well as from animals with unscheduled deaths or in a moribund condition during the course of the study. Histopathological examination for terminal sacrifice rats of the low and mid dose groups were restricted to the lungs, liver, kidneys, any gross lesions and the thyroids.

No clinical signs related to treatment were apparent in any of the rats during the 104 wk of the experiment. There were no significant differences in mortality between controls and treated animals during the course of the study.

Statistically significant lower body weights, or a tendency for lower body weights, were observed in both sexes of the high dose group from wk 2 to termination. No treatment related effects were observed in food consumption, organ weight and gross pathology findings.

A wide range of non-neoplastic lesions were evident. But no treatment-related changes were found. There were no differences in the development of any tumour category between control and treated groups for either sex. None of neoplastic lesions was considered to be treatment related.

In conclusion, administration of N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate (4APE) to Fischer 344/DuCrj rats over a period of 104 wk at dose levels of 0, 300, 1000 and 3000 ppm in the diet (equivalent to 0, 12, 40, and 121 mg/kg bw/day (males); 0, 14, 48 and 146 mg/kg bw/day (females)) was determined to have no carcinogenic potential.

 

Based on decrease in body weight at highest dose level tested (3000 ppm in diet), the NOAEL for chronic toxicity was determined to be 1000 ppm in diet (equivalent to 40 mg/kg bw/day (males) and 48 mg/kg bw/day (females)).. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

40 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

NOAEL of 1000 ppm in diet (equivalent to 40 mg/kg bw/day (males) and 48 mg/kg bw/day (females)).

Justification for classification or non-classification

N,N-bis(2-hydroxyethyl)-p-phenylenediamine sulfate did not induce tumours after oral administration in a long-term rat study. The substance has also been studied alone and after mixing with hydrogen peroxide by topical application to mice and rats. However, as known carcinogens have been tested by the same experimental procedures without any tumour formation, it is not possible to draw any conclusions from these experiments with regard to potential carcinogenic effects.

Additional information

There was no evidence of carcinogenicity in a one hundred and four week study in F344/DuCrj rats fed a diet containing 300, 1000 or 3000 ppm N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate (3000 ppm equivalent to a dose of 121 mg/kg body weight/d in males and 146 mg/kg body weight/d in females). In addition, a medium term bioassay in F344/DuCrj male rats fed a diet containing 110, 330 or 1000 ppm N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate, indicated N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate  was neither hepatocarcinogenic nor a promoter of hepatic neoplasms at up to 1000 ppm (equivalent to 76 mg/kg body weight/d). The negative carcinogenicity data is consistent with the lack of evidence of in vivo genotoxicity of N,N-Bis(2-hydroxyethyl)-p-phenylenediamine sulfate.