Reaction mass of sodium aqua[5-{[2-hydroxy-4-(hydroxy-kO)-5-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl}phenyl]diazenyl}naphthalene-1-sulfonato(3-)]ferrate(1-) and sodium diaqua(chloro)[5-{[2-hydroxy-4-(hydroxy-kO)-5-{[2-(hydroxy-kO)-3,5-dinitrophenyl]diazenyl-kN2}phenyl]diazenyl}naphthalene-1-sulfonato(3-)]ferrate(1-)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Remarks:

Department of Toxicology, BASF AG

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Analogue Substance_01

IUPAC Name:

Analogue Substance_01

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr . Karl Thomae GmbH, Biberach a. d. Riss, FRG
- Age at study initiation: 42 d
- Weight at study initiation: mean 162 g (males), 134 g (females)
- Housing: singly in type DK III stainless steel wire cages, supplied by BECKER & CO., Castrop-Rauxel, FRG (floor area about 900 cm2)
- Diet (e.g. ad libitum): was ground Kliba rat/mouse "A" maintenance diet, GLP 343 meal, supplied by Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): tap water; ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 -70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Drinking water solutions were prepared twice a week by weighing the test substance and then disolving it in-drinking water using a magnetic stirrer. The stability of the drinking water solutions was confirmed over a period of 5 days. The concentration of the drinking water solutions was verified on the basis of one sample of each concentration, taken at the start of the study, in a double determination.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content of Säurebraun 6229 in the drinking water was determined by the UV-VIS method.

Duration of treatment / exposure:

28 days

Frequency of treatment:

continously by drinking water

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 5000, 10000 ppm
Basis:
nominal in water

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at each weighing, thus once a week


BODY WEIGHT: Yes
- Time schedule for examinations: All the animals were weighed once a week. The body weight was always determined on the same day of the week and at the same time of the day


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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


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 (if drinking water study): Yes
- Time schedule for examinations: determined twice a week


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: 9 days (female animals, 10000 ppm ) or 25 days after the beginning of administration (blood sampling 1)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all
- Parameters examined: hemoglobin, erythrocytes, hematocrit, mean hemoglobin content per erythrocyte, mean corpuscular volume, mean corpuscular hemoglobin concentration, platelets, leukocytes, differential blood count, prothrombin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 9 days (female animals, 10000 ppm ) or 25 days after the beginning of administration (blood sampling 1)
- Animals fasted: no
- How many animals: all
- Parameters examined: total bilirubin, creatinine, urea, sodium, potassium, total protein, glucose, inorganic phosphate, calcium, chloride, triglycerides, cholesterol albumin, glutamic-pyruvic transaminase, alkaline phosphatase, glutamic-oxalacetic transaminase


URINALYSIS: Yes
- Time schedule for collection of urine: 29 days after the beginning of treatment
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: pH, protein, glucose, ketones, bilirubin, blood, nitrite, urobilinogen, sediment


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes: liver, kidneys, spleen, adrenals, heart, testes, gastrointestinal tract and all gross lesions

Statistics:

- ANOVA followed by DUNNETT's test: body weights
- WILLIAMS test: absolute organ weights
- t- test: blood and plasma examinations
- chi-square test in corresponding two by two contingency tables: urinalysis

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
10000 ppm:
- 2/5 animals died 14 days after the beginning of the administration period.
- 1 female animal was sacrificed in a moribund state on day 18 after the beginning of the study; due to their poor state of health, remainin g females were sacrificed prematurely on day 9 after the beginning of the administration period
- a blackish brown colored feces from day 1 on; yellowish brown colored urine from day 10 on
5000 ppm:
- blackish brown colored feces in males and females from day 1 on
- addition showed yellowish brown colored urine from day 10 on
- one female showed a yellowish brown smear in the anogenital region, and this started with day 7 after the beginning of the administratio n period
500 ppm:
- blackish brown colored feces in males and females
- addition showed yellowish brown colored urine from day 10 on


BODY WEIGHT AND WEIGHT GAIN
10000 ppm:
- pronounced reduction in body weight in 3/5 females and reduction in body weight throughout the study in males
5000 ppm:
- reduction in body weight in the male and female animals throughout the study


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
10000 ppm:
- severe reduction in feed consumption in 3/5 females
- in males, reduction in feed consumption in the 1st week of the study followed by an increase to above the control values
5000 ppm:
- reduced feed consumption in the female animals in the 1st to 3rd week of the study and in the male animals in the first half of the study


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
10000 ppm:
- extreme reduction in water consumption in 4/5 females
- reduced water consumption in 3/5 males in the 1st week of the study followed by an increase in water consumption to above the control values
5000 ppm:
- reduced drinking water consumption in 3 of 5 male and female animals, approaching or exceeding the control values during the study


HAEMATOLOGY
10000 ppm:
- in females increase in the following values: prothrombin time, hemoglobin, erythrocyte count, hematocrit, mean corpuscular volume (MC V), polymorphonuclear neutrophils and atypical lymphocytes as well as polychromasia. Decrease in mean hemoglobin content per erythro cyte (MCH), mean corpuscular hemoglobin concentration (MCHC) and platelet count
- in males increase in the polymorphonuclear neutrophil count and increased incidence of polychromasia and anisocytosis
5000 ppm
- reduction in glucose and hemoglobin values in the male animals
- increase in polymorphonuclear neutrophils and monocytes in both sexes and increase in the cholesterol and leukocyte, lymphocyte and platelet counts in the male animals
- increased polychromasia in the male and female rats and increased anisocytosis in the male animals


CLINICAL CHEMISTRY
10000 ppm:
- In females increase in the following values: urea, sodium, inorganic phosphate, chloride, glutamic pyruvic transaminase, glutamic oxalac etic transaminase. Decrease in total protein and albumin
- In males increased urea levels
5000 ppm:
- reduction in total protein and albumin concentrations in both sexes


URINALYSIS
5000 ppm:
- increase in the inorganic phosphate concentration and urea levels and increased excretion of renal epithelial cells in the female animals

GROSS PATHOLOGY
10000 ppm:
- Necrotizing typhlitis and edemas in the cecum in males and females
5000 ppm.
- necrotizing typhlitis and edemas of the cecum in the male and female animals
- necrotizing colitis in the male rats

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

45 - 63 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

other: clinicochemical and hematological parameters

2

Dose descriptor:

NOAEL

Effect level:

47 - 73 mg/kg bw/day (actual dose received)

Sex:

female

Basis for effect level:

other: clinicochemical and hematological parameters

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Based on additional testing , the authors concluded that the increase in bilirubin detected in the urine is due to an interfering reaction of

the test substance with the test method used. Therefore, the increased bilirubin levels in the urine are not regarded as having any pathognostic relevance.

Substance intake (mg/kg bw/d):

Dose group	Sex	Days
		1 - 7	8 - 14	15 - 21	22 - 28
500 ppm	male	63	54	46	45
	female	73	57	53	47
5000 ppm	male	536	636	728	736
	female	489	635	732	665
10000 ppm	male	897	1984	1670	1465
	female	772	x	x	x

Body weights (g):

Dose group	Sex	Day
		0	7	14	21	28
control	male	161.0 ± 6.6	211.3 ± 12.6	249.6 ± 15.1	277.6 ± 14.0	296.8 ± 15.2
	female	133.5 ± 4.4	160.6 ± 7.1	182.7 ± 5.8	195.8 ± 10.6	209.4 ± 15.1
500 ppm	male	162.0 ± 6.6	212.3 ± 11.9	252.1 ± 18.0	283.7 ± 17.9	312.3 ± 19.9
	female	133.9 ± 4.8	162.6 ± 6.6	186.0 ± 10.8	203.4 ± 11.7	210.4 ± 12.9
5000 ppm	male	160.3 ± 5.3	169.7 ± 15.6*	184.3 ± 26.9**	212.7 ± 19.2**	235.8 ± 20.6**
	female	133.3 ± 3.6	130.5 ± 23.1	143.4 ± 34.9*	175.0 ± 13.2*	185.9 ± 12.1*
10000 ppm	male	161.0 ± 5.2	146.8 ± 39.2**	209.1 ± 15.3**	244.8 ± 9.9*	273.9 ± 5.7
	female	133.5 ± 4.0	107.3 ± 33.2**	x	x	x
Different from control: *p<0.05; **p<0.01 (Anova + Dunett's, two sided)