2-[[4-[bis(2-hydroxyethyl)amino]-o-tolyl]azo]-5-nitrobenzonitrile

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from NTP report

Data source

Reference

Reference Type:

other: NTP report

Title:

Toxicology And Carcinogenesis Studies Of HC Yellow 4 (Cas No. 59820-43-8) In F344/N Rats And B6C3F1 Mice (Feed Studies)

Author:

U.S. Department Of Health And Human Services

Year:

1992

Bibliographic source:

National Toxicology Program No. 419, NIH No. 92-3150, PB931 23883, 1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

14 days repeated dose oral toxicity study was performed to determine the toxic nature of HC Yellow 4 using rats

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-(2-hydroxyethylamino)-5-nitrophenoxy]ethanol

Cas Number:

59820-43-8

Molecular formula:

2-[2-(2-hydroxyethylamino)-5-nitrophenoxy]ethanol

IUPAC Name:

2-[2-(2-hydroxyethylamino)-5-nitrophenoxy]ethanol

Details on test material:

- Name of test material: HC Yellow 4
- Molecular formula: C10H14N2O5
- Molecular weight: 242.2 g/mol
- Substance type: Organic
- Physical state: Fluffy yellow powder

Specific details on test material used for the study:

- Name of test material: HC Yellow 4
- IUPAC name: 2-[2-(2-hydroxyethylamino)-5-nitrophenoxy]ethanol
- Molecular formula: C10H14N2O5
- Molecular weight: 242.2 g/mol
- Substance type: Organic
- Physical state: Fluffy yellow powder
- Purity: > 93%

Test animals

Species:

rat

Strain:

Fischer 344

Remarks:

F344/N

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Kingston, NY)
- Age at study initiation: 7 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: Animals were housed in polycarbonate cages with Aspen Bed, heat-treated hardwood chips and Non-woven polyester filters
- Diet (e.g. ad libitum): NIH-Q7 Rat Ration, Open formula, mash ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 13-15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22˚C
- Humidity (%): 69%
- Air changes (per hr): 12-15/hr
- Photoperiod (hrs dark / hrs light): Fluorescent light 12 hrs/day

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The dosed feed route of administration was selected to ensure systemic exposure

Vehicle:

other: Feed

Remarks:

NIH-07 Rat Ration, Open formula, mash

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with feed at dose levels of 0, 5,000, 10,000, 20,000, 40,000, or 80,000 ppm (0, 500, 1000, 2000, 4000 or 8000 mg/Kg bw/day)

DIET PREPARATION
- Rate of preparation of diet (frequency): The dose formulations were stored for no longer than 2 weeks
- Mixing appropriate amounts with (Type of food): NIH-Q7 Rat Ration, Open formula, mash
- Storage temperature of food: The dose formulations were stored in the dark at 0˚ ± 5˚ C for no longer than 2 weeks.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Feed
- Concentration in vehicle: 0, 5,000, 10,000, 20,000, 40,000, or 80,000 ppm (0, 500, 1000, 2000, 4000 or 8000 mg/Kg bw/day)
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Studies were conducted by the analytical chemistry laboratory to determine the homogeneity and stability of 10,000 ppm HC Yellow 4 in feed. Homogeneity was confirmed using an ultraviolet spectroscopic method for sample analysis; stability of the dose formulations for at least 14 days when stored in the dark at temperatures up to 25° C was confirmed using a high-performance liquid chromatographic method.

All dose formulations analyzed for the 14-day study were within 10% of the target concentrations

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

0, 5,000, 10,000, 20,000, 40,000, or 80,000 ppm (0, 500, 1000, 2000, 4000 or 8000 mg/Kg bw/day)

No. of animals per sex per dose:

Total: 30 males and 30 females
0 mg/Kg bw: 5 males and 5 females
500 mg/Kg bw: 5 males and 5 females
1000 mg/Kg bw: 5 males and 5 females
2000 mg/Kg bw: 5 males and 5 females
4000 mg/Kg bw: 5 males and 5 females
8000 mg/Kg bw: 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Animals assigned to groups by weight, so that cage weights were approximately
equal (± 2 g)
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included. Signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On the day of necrospy

BODY WEIGHT: Yes
- Time schedule for examinations: At the start of experiment, on days 7 and 14, and at necropsy

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: No data
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: No data
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, Complete necropsies were performed on all animals. The brain, heart, right kidney, liver, lung, right testis, and thymus of survivors were weighed at necropsy

HISTOPATHOLOGY: Yes, Histopathology was performed on selected tissues from all rats in the 0, 20,000, 40,000, and
80,000 ppm (0, 2000, 4000 or 8000 mg/Kg/day) dose groups. Tissues examined included: Bone and marrow, Peyer's patch, spleen, and thymus. Tissues examined only for the 80,000 ppm dose group included: Brain, clitoral gland, and kidney. Tissues examined only for the 0, 20,000, and 40,000 ppm dose groups included: Mediastinal lymph node and testis.

Other examinations:

No data

Statistics:

No data

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical findings were attributed to HC Yellow 4 administration

Mortality:

no mortality observed

Description (incidence):

No mortality was noted during the study period

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The final mean body weights and mean body weight changes of males that received doses of 20,000 ppm (2000 mg/Kg bw/day) and above and females that received doses of 10,000 ppm (1000 mg/Kg bw/day) and above were significantly lower than those of the controls

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption by males that received doses of 20,000 ppm (2000 mg/kg bw/day) or greater and females that received doses of 10,000 ppm (1000 mg/Kg bw/day) or greater was lower than that of the controls during the first week.

During the second week, feed consumption by males in the 40,000 ppm (4000 mg/Kg bw/day) dose group was lower than controls; feed consumption by other male and female dose groups was similar to or higher than that of the controls. Because rats that received 40,000 ppm (4000 mg/Kg bw/day) did not gain weight, and the final mean body weights of rats that received 80,000 ppm (8000 mg/kg bw/day) were decreased approximately 30%, it was concluded that the feed consumption values were high and may have included feed scattered by animals searching for unadulterated feed.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Significant changes in absolute and relative organ weights were observed but were considered to be secondary to decreases in body weights

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

No data

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No significant alterations were noted at the mentioned dose level

2

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No significant alterations were noted at the mentioned dose level

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Survival, Mean Body Weights, and Feed Consumption of Rats in the 14-Day Feed Studies of HC Yellow 4

Dose	Survival	Mean body weight			Final Weight Relative to Controls (%)	Feed consumption (g/animals/day)
		Initial	Final	Change		Week 1	Week 2
Males
0	5/5	107±6	178±7	71±3		15.3	16.7
5000	5/5	108±5	173±6	66±3	97	15.6	16.7
10000	5/5	107±5	170±5	63±2	95	14.9	16.9
20000	5/5	107±4	148±6**	10±5**	83	10.7	14.4
40000	5/5	108±3	107±4**	-1±2**	60	5.9	11.6
80000	5/5	108±4	75±3**	-32±3**	42	8.7	16.3
Females
0	5/5	101±3	145±2	44±1		16.0	11.6
5000	5/5	101±3	138±5	37±2	95	15.0	10.4
10000	5/5	101±3	131±1**	29±3**	90	12.6	10.1
20000	5/5	101±2	130±3**	29±4**	90	7.2	11.7
40000	5/5	101±2	102±3**	0±3**	70	10.6	15.4
80000	5/5	102±3	72±3**	-29±4**	50	9.4	12.1

** Significantly different (p≤0.01) from the control group by Williams' or Dunnett's test

Applicant's summary and conclusion

Conclusions:

The No observed Adverse effect level (NOAEL) for male rats is considered to be 1000 mg/kg bw and female rats the NOAEL is considered to be 500 mg/Kg bw.

Executive summary:

14 days repeated dose oral toxicity study was performed to determine the toxic nature of HC Yellow 4 using rats. The test chemical was mixed with feed and dosed to F344/N 5 male and 5 female rats at dose levels of 0, 500, 1000, 2000, 4000 or 8000 mg/Kg bw/day for 14 days. During the study period, the animals were observed for signs of toxicity, death, changes in body weight and feed consumption, and they were subjected to gross and histo-pathology. No deaths were observed and no signs of toxicity attributable to the test chemical administration were noted. The final mean body weights and mean body weight changes of males that received doses of 20,000 ppm (2000 mg/Kg bw/day) and above and females that received doses of10,000 ppm (1000 mg/Kg bw/day) and above were significantly lower than those of the controls. Feed consumption by males that received doses of 20,000 ppm (2000 mg/kg bw/day) or greater and females that received doses of 10,000 ppm (1000 mg/Kg bw/day) or greater was lower than that of the controls during the first week. During the second week, feed consumption by males in the 40,000 ppm (4000 mg/Kg bw/day) dose group was lower than controls; feed consumption by other male and female dose groups was similar to or higher than that of the controls. Because rats that received 40,000 ppm (4000 mg/Kg bw/day) did not gain weight, and the final mean body weights of rats that received 80,000 ppm (8000 mg/kg bw/day) were decreased approximately 30%, it was concluded that the feed consumption values were high and may have included feed scattered by animals searching for unadulterated feed. Significant changes in absolute and relative organ weights were observed but were considered to be secondary to decreases in body weights. Based on these observations made, No observed Adverse effect level (NOAEL) for male rats is considered to be 1000 mg/kg bw and female rats the NOAEL is considered to be 500 mg/Kg bw.