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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 31 January 1979 to 30 April 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

In this No GLP compliant study, animals were treated only 5 days per week instead of 7 required in the OECD guideline.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

In this No GLP compliant study, animals were treated only 5 days per week instead of 7 required in the OECD guideline.

GLP compliance:

no

Remarks:

NTP practices

Limit test:

no

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
- 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
Appropriate amounts of HC Red No.3 and corn oil were manually shaken for 1 minute, then mixes with a magnetic stirrer until the suspension was uniform.

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 Laboratories
- Females (if applicable) nulliparous and non-pregnant:Not specified
- Age at study initiation: 7-8 weeks
- Weight at study initiation:164±2g ( males) ; 121±2 (females)
- Fasting period before study: not specified
- Housing: 5 rats were housed in polycarbonate cages
- Diet (e.g. ad libitum): Wayne Lab Blox pellets (Allied Mills) ad libitum
- Water (e.g. ad libitum): ad libitum, provided by automatic watering system
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): : 30-50% relative humidity
- Air changes (per hr): 15 room air chages per hour
- Photoperiod (hrs dark / hrs light): 12h fluorescent light/12 h dark

IN-LIFE DATES: From: approximately 10 January 1979 To: 6 May 1979

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected for each chemical in order to provide more rigorous challenge than would be possible through dermal application.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Appropriate amounts of HC Red No.3 and corn oil were manually shaken for 1 minute, then mixes with a magnetic stirrer until the suspension was uniform.

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:

13 weeks

Frequency of treatment:

Once daily, 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

62 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 6

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals were used per sex per dose.

Control animals:

yes

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Not specified
- Rationale for animal assignment (if not random): Assigned to cage according to a table of random numbers and then to group according to an another table of random numbers

Examinations

Observations and examinations performed and frequency:

Animals were observed twice daily for viability and were weekly observed for clinical examination.

Sacrifice and pathology:

A necropsy and histopathology were performed on animals from vehicle control to 1000 mg/kg dose groups. Tissues were : gross lesions, skin, mandibular, lymph node, mammary gland, salivary gland, thigh muscle, sciatic nerve, femur including marrow, thymus, lungs and bronchi, trachea, heart, thyroid gland, parathyroids, esophagus, small intestine, colon, 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, pituitary gland.Kidneys and Thyroid glands of rats administered 250 or 500mg/kg also examined microscopically.

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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The urine of dosed animals was orange to purple throughout the studies.

Mortality:

no mortality observed

Description (incidence):

None of the rats died before the end of the studies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Final body mean weights relative to vehicle controls were 7% lower for mal rats that received 1000mg/kg and 5% lower for the male rat group that received 500 mg/kg. Final body mean body weights of dosed female rats were greater then those of the 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:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Granules of a brown to golden brown pigment were found in the cytoplasm of the thyroid gland follicular epithelial cells in 10/10 males and 10/10 females that received 1000 mg/kg and 10/10 males and 7/10 females that received 500 mg/kg but not in any of the rats that received 250 mg/kg. Similar pigment was found in the cytoplasm of convoluted tubular epithelial cells in the kidneys of all rats that received 1000 mg/kg, in 7/10 males and 10/10 females that received 500 mg/kg, and 6/10 males and 7/10 females that received 250 mg/kg. No other microscopic observations attributable to HC red No3 administration were noted.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table1 :Summary of the results

			MeanBodyWeighs(grams)
		Survival	Initial		Final		Change		Final weight relative to control (percent)
	Dose (mg/kg)		Mean	SD	Mean	SD	Mean	SD
Male	0	10/10	164	2	357	5	193	5
	62	10/10	159	4	352	4	193	4	98.8
	125	10/10	161	4	350	6	189	4	98
	250	10/10	160	3	355	6	196	5	99.6
	500	10/10	134	5	338	6	184	4	94.7
	1000	10/10	157	4	333	6	176	3	93.3
Female	0	10/10	121	2	196	2	75	2
	62	10/10	118	3	199	3	81	2	101.6
	125	10/10	119	2	202	2	83	3	102.9
	250	10/10	122	2	205	3	83	1	104.8
	500	10/10	123	2	202	4	79	2	102.9
	1000	10/10	117	3	198	3	81	2	101.0

 

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions, the test item HC Red No.3 induced low decrease in relative bodyweight for male rats of the high dose group and of the 500 mg/kg dose group. Intense pigmentation was observed in the thyroid and kidneys at the 1000 mg/kg/ay dose group. The selected high dose for carcinogenicity test was 500 mg/kg/day. The NOAEL was defined as 250 mg/kg bw/day based on the bodyweight decrease.

Executive summary:

The aim of the no GLP study was to investigate the potential toxicity of the test item HC Red No. 3 in rats which were exposed during 13 weeks. The study was performed in order to select appropriate doses for a 2 years carcinogenesis study.

F344/N rats were used in this study. Animals were exposed during 13 weeks, daily , 5 days per weeks. Clinical exmaination, bodyweights and morbidity observations were performed. At the termination of the study, necropsy and histopathology were performed on vehicule group and high dose group. Further analysis of thyroid and kidneys were performed.

None of the rats died before the end of the studies. The urine of dosed animals was orange to purple throughout the studies. Final body mean weights relative to vehicle controls were 7% lower for male rats that received 1000mg/kg and 5% lower for the male rat group that received 500 mg/kg. Final body mean body weights of dosed female rats were greater then those of the vehicle controls.

Granules of a brown to golden brown pigment were found in the cytoplasm of the thyroid gland follicular epithelial cells in 10/10 males and 10/10 females that received 1000 mg/kg and 10/10 males and 7/10 females that received 500 mg/kg but not in any of the rats that received 250 mg/kg. Similar pigment was found in the cytoplasm of convoluted tubular epithelial cells in the kidneys of all rats that received 1000 mg/kg, in 7/10 males and 10/10 females that received 500 mg/kg, and 6/10 males and 7/10 females that received 250 mg/kg. No other microscopic observations attributable to HC red No3 administration were noted.

Under the experimental conditions, the test item HC Red No.3 induced low decrease in relative bodyweight for male rats of the high dose group and of the 500 mg/kg dose group. Intense pigmentation was observed in the thyroid and kidneys at the 1000 mg/kg/ay dose group. The selected high dose for carcinogenicity test was 500 mg/kg/day. The NOAEL was defined as 250 mg/kg bw/day based on the bodyweight decrease. In this No GLP compliant study, animals were treated only 5 days per week instead of 7 required in the OECD guideline. Complete analysis were not performed (as haematology, clinical biochemistry, urinalysis). This study did not follow modern standard for a 90 days repeated toxicity study but was relevant for dose range finding for a chronic study.