2-(4-amino-2-nitroanilino)ethanol

Carcinogenicity

Administrative data

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 11 March 1978 To 27 November 1981

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

This study was performed in rats which were treated 5 days per week instead of 7 days per week required in the guideline. Chosen doses were low and not apprpriate to induce low threshold effect as mentionned in the guideline.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1986

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

F344/N rats, groups of 50 males and 50 females (7-8 weeks old), were exposed to HC Red n° 3 by oral gavage 5 days per week for 105 weeks. The rats received 0 (control), 250 (low dose), and 500 mg/kg bw (high dose). The animals were observed twice daily.At the termination of the study, gross necropsy and hispathological analysis were performed.

GLP compliance:

no

Remarks:

NTP practices

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-amino-2-nitroanilino)ethanol

EC Number:

220-701-7

EC Name:

2-(4-amino-2-nitroanilino)ethanol

Cas Number:

2871-01-4

Molecular formula:

C8H11N3O3

IUPAC Name:

2-(4-amino-2-nitroanilino)ethanol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 5890377 (used date : 27.11.79) ; C080480 (used date : 24.10.80)
- Expiration date of the lot/batch: not specified
- Purity test date:not specifed

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at 22°C
- Stability under test conditions: Stable in corn oil
- Solubility and stability of the test substance in the solvent/vehicle: Stable in vehicle for 7 days at room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The dose preparation method consisted of suspending the HC Red NO.3 in corn oil on a weight/volume basis with a magnetic stirrer. The dose mixtures were prepared weekly and stored at 22°C until used for dosing.

Test animals

Species:

rat

Strain:

Fischer 344

Remarks:

F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory
- Females (if applicable) nulliparous and non-pregnant:Not specified
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 286g (males) ; 189 g(females)
- Fasting period before study: not specified
- Housing: 5 rats were housed in polycarbonate cages
- Diet (e.g. ad libitum): NIH07 Open Formula ad libitum
- Water (e.g. ad libitum): ad libitum, provided by automatic watering system
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 30-70% except 12/79 : 40-60%
- Air changes (per hr): 15 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12h fluorescent light/12 h dark

IN-LIFE DATES: From: approximately 7 November 1979 To: 11 December 1981

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose preparation method consisted of suspending the HC Red NO.3 in corn oil on a weight/volume basis with a magnetic stirrer. The dose mixtures were prepared weekly and stored at 22°C until used for dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): 5mL /kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Doses mixture of HC Red in corn oil were analyzed periodically by spectrophotometric quantitation. It was estimated that doses were formulated within specifications 86% of the time during the 2 years study

Duration of treatment / exposure:

105 weeks

Frequency of treatment:

Once a day, five days per week

Post exposure period:

24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

50 animales per sex per dose were used

Control animals:

yes

yes, concurrent vehicle

Details on study design:

- Toxicokinetic data not specified
- Dose selection rationale: Selected doses were based on a previous 13 weeks repeated toxicity study in which rats were dosed with up 1000 mg/kg/day.
- Rationale for animal assignment (if not random): assignaed to cages according to one table of random numbers and then to groups according to another table of random numbers
- Rationale for selecting satellite groups: not performed

Examinations

Observations and examinations performed and frequency:

All animals were observed twice daily, and clinical signs were recorded once per week. Body weights by cage were recorded once per week for the first 12 weeks of the studies and once month thereafter. Mean bodyweights were calculated for each group. Moribund animals were killed, as were animals that survived to the end of the studies.

Sacrifice and pathology:

A necropsy was performed on all animals, including those found dead unless they were excessively autolyzed or cannibalized. Thus, the number of animals from which particular organs or tissues were examined microscopically varies and is not necessarily equal to the number of animals that were placed on study in each group. 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. Tissues examined microscopically were : gross lesions, skin, mandibular, lymph node, mammary gland, salivary gland, thigh muscle, sciatic nerve, femur including marrow, thymus, costochondral junction, larynx, lungs and bronchi, trachea, heart, thyroid gland, parathyroids, esophagus, stomach, duodenum, jejunum, eyes, tissue masses with regional lymph node, ileum, colon, cecum, rectum, liver, mesenteric lymph node, inguinal lymph node, pancreas, spleen, kidneys, adrenal glands, urinary bladder, testis/epididymus/seminal vesicles/prostate or ovaries/uterus/fallopian tube/vagina, nasal cavity, brain, preputial gland, pituitary gland, spinal cord, external and middle ear.

Statistics:

Survival analysis of survival was estimated by the product limit procedure of Kaplan and Meier (1958). Statistical analyses for a possible dose-related effect on survival for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone's (1975) life table test for dose related trend. All reported P value for the survival analysis were two sided. The incidence of neoplastic or nonneoplastic lesions was given as the ratio of number of animals in which that site was examined. Tumor incidence was evaluated by three methods. The two that adjust for interconcurrent mortality emplyed the classical method for combining contingency tables developed by Mantel and Haznszel (1959). Test of significance included pairwise comparisons of high dose and low dose groups with vehicle controls and tests for overall dose-response trends. For studies in which compound administration has little effect on survival , the results of the three alternatives analysis will generally be similar. When differing results were obtained by the three methods, the final interpretation if the data will depend on the extent to which the tumor under consideration is regarded as being the cause of death. All reported P value for tumor analysis were one-sided

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No compound related clinical signs were observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No significant differences in survival were observed between any groups of either sex.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Throughout most of the study, there was little or no difference in the mean body weights of dosed rats either sex as compared with those vehicle controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Pigmentation of various tissues was a common observation. The pigment was not identified but was presumed to be a derivative of HC Red n° 3. Very minimal nephropathy was found in dosed female rats, but its relationship to HC Red n° 3 was considered equivocal.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There was an increase in the incidence of mammary gland fibroadenomas or cystadenomas in low dose female rats. The incidence of this lesion in high dose female rats was not increased (vehicle control, 14/50, 28%; low dose, 25/50, 50%; high dose, 11/50, 22%). Largely because of the lack of a dose response, the increased incidence in the low dose females was not considered to be due to HC Red n° 3. No increased incidences of neoplasms were seen in male rats.
Transitional cell papillomas of the urinary bladder were detected in one high dose male rat, two low dose female rats, and one high dose female rat; none was observed in the vehicle controls. These uncommon neoplasms were found in animals that survived to the termination of the study and were not accompanied by other proliferative lesions.

Other effects:

not examined

Relevance of carcinogenic effects / potential:

There was no evidence of carcinogenicity for male and female F344/N rats given 250 or 500 mg/kg/day.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

500

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

gross pathology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

Applicant's summary and conclusion

Conclusions:

It was concluded that under the conditions of this 2-year gavage study of HC Red n° 3, there was no evidence of carcinogenicity for male or female F344/N rats given 250 or 500 mg/kg bw/day. Both sexes of the rats may have been able to tolerate higher doses of HC Red n° 3. The rats were treated only 5 days per weekd instead of 7 days per week required in the OECD 451 method. Therefore, the sensitivity of this study for detecting carcinogenesis may have been limited.

Executive summary:

The purpose of this no GLP compliant study was to evaluate to potential carcinogenicity of the test item HC Red No. 3 when administered orally to F344/N rats during a Two-years carcinogenicity study.

F344/N rats, groups of 50 males and 50 females (7-8 weeks old), were exposed to HC Red n° 3 by oral gavage 5 days per week for 105 weeks. The rats received 0 (control), 250 (low dose), and 500 mg/kg bw (high dose). The animals were observed twice daily.At the termination of the study, gross necropsy and hispathological analysis were performed. The animals were observed twice daily.

No significant differences in survival were observed between any groups of either sex. Neither did the treatment affect body weight or body weight gains. No compound-related clinical signs were observed. Pigmentation of various tissues was a common observation. The pigment was not identified but was presumed to be a derivative of HC Red n° 3. Very minimal nephropathy was found in dosed female rats, but its relationship to HC Red n° 3 was considered equivocal.

There was an increase in the incidence of mammary gland fibroadenomas or cystadenomas in low dose female rats. The incidence of this lesion in high dose female rats was not increased (vehicle control, 14/50, 28%; low dose, 25/50, 50%; high dose, 11/50, 22%). Largely because of the lack of a dose response, the increased incidence in the low dose females was not considered to be due to HC Red n° 3. No increased incidences of

neoplasms were seen in male rats. Transitional cell papillomas of the urinary bladder were detected in one high dose male rat, two low dose female rats, and one high dose female rat; none was observed in the vehicle

controls. These uncommon neoplasms were found in animals that survived to the termination of the study and were not accompanied by other proliferative lesions.

It was concluded that under the conditions of this 2-year gavage study of HC Red n° 3, there was no evidence of carcinogenicity for male or female F344/N rats given 250 or 500 mg/kg bw/day. Both sexes of the rats may have been able to tolerate higher doses of HC Red n° 3. The rats were treated only 5 days per weekd instead of 7 days per week required in the OECD 451 method. Therefore, the sensitivity of this study for detecting carcinogenesis may have been limited.