A mixture of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium ((1-(4(or 5)-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-pentylphenylazo)-2-naphtholato))chromate(1-)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 1988 - July 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD testing guideline compliant study with well-characterized test material.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium ((1-(4(or 5)-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-pentylphenylazo)-2-naphtholato))chromate(1-)

EC Number:

403-720-7

EC Name:

A mixture of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium ((1-(4(or 5)-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-pentylphenylazo)-2-naphtholato))chromate(1-)

Cas Number:

117527-94-3

Molecular formula:

C21H21N304.C16H11N304.Cr unspecified.C12-C14 chain unspec.

IUPAC Name:

hexachromium(3+) hexakis(2-methyltridecan-2-aminium) tetrakis(1-[(1E)-2-(4-nitro-2-oxidophenyl)diazen-1-yl]naphthalen-2-olate) tetrakis(1-[(1E)-2-(5-nitro-2-oxidophenyl)diazen-1-yl]naphthalen-2-olate) tetrakis(1-[(1E)-2-[5-(2-methylbutan-2-yl)-3-nitro-2-oxidophenyl]diazen-1-yl]naphthalen-2-olate)

Details on test material:

- Lot/batch No.: 217342.89

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH Wiga, Sulzfeld, West Germany
- Age at study initiation: 6 weeks old
- Weight at study initiation: males: 188 - 258 g and females: 111 - 179 g
- Housing: polycarbonate cages
- Diet: standard pelleted laboratory diet, free acccess
- Water: Tap water, ad libitum
- Acclimation period: 14 days ( 7 or 9 days pre- and 7 or 5 days post randomisation)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 7.5
- Photoperiod (hrs dark / hrs light): 12 hours each/day

IN-LIFE DATES: 25. October - 21. December 1988

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1 % methyl cellulose

Details on oral exposure:

VEHICLE
- Concentration in vehicle: Homogenous suspension in 1% methyl cellulose at concentrations of 0.5 (w/w), 2.0% (w/w) and 10% (w/w).
- Amount of vehicle (if gavage): 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations prepared during weeks 1, 2,3 and 4 were analysed to check the accuracy of preparation. Concentrations of the test article in vehicle was determined on day 2, 9, 16 and 23. Actual concentrations of preparations were in agreement with the treatment levels as per protocol.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Once daily, approximately the same time each day, 7 days per week.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day (m/f)
Basis:
other: nominal

No. of animals per sex per dose:

Animals per Group: 10 males (5 toxicity, 5 recovery) and 10 females (5 toxicity, 5 recovery).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A 10 day range finding study was performed (with 3 rats/sex/group at dose levels of 100, 300 and 1000 mg/kg/day) to provide a basis for selection of dose levels for the 28 day study. In this dose range finding investigation, no abnormalities related to treatment were observed for clinical appearance, body weight, food consumption, liver weights or macroscopic appearance across the treatment.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily during treatment and recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily for 28 days

BODY WEIGHT: Yes
- Time schedule for examinations: weekly and the day immediately before termination of toxicity and recovery group (days 28 and 42) prior to overnight fasting.

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

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: On days 24 (toxicity and recovery groups) and 35 (recovery group).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination (puncture of the retro-orbital sinus) at week 4 (day 29; toxicity and recovery group) and at week 6 (day 43; recovery group).
- Anaesthetic used for blood collection: Yes (light ether anesthesia )
- Animals fasted: Yes (overnight before blood sampling, but water was provided)
- How many animals: all rats/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination (puncture of the retro-orbital sinus) at week 4 (day 29; toxicity and recovery group) and at week 6 (day 43; recovery group).
- Animals fasted: Yes (overnight before blood sampling, but water was provided)
- How many animals: all rats/sex/group

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight from animals deprived of food and water
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume/V0LUME/18H, Specific gravity, pH, Protein,Glucose, Ketones, haemoglobin, bilirubin, urobilirubin, appearance, colour, leukocytes, sediment (red and white blood cells)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsies: All animals were necropsied and descriptions of all macroscopic abnormalities recorded. All animals surviving were killed by injection of pentobarbital and subsequent exsanguination on day 29 (toxicity group) and day 43 (recovery group).

HISTOPATHOLOGY: Yes
Slides were prepared of adrenals, heart, kidneys, liver and spleen, collected at termination from all animals of the control and high dose group. In addition, all gross lesions of all dose groups were processed and tissue slides were prepared. Based on treatment related morphological changes, the adrenal glands were also examined from all rats of group 2 (50 mg/kg bw/d) and 3 (200 mg/kg bw/d).

Statistics:

The following statistical methods were used to analyse the body weight, food consumption, organ weights and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences. If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places.
Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS
The only clinical observation made during treatment or recovery period was purple appearance (grade 1 - minimal) over the whole body of all animals treated at 1000 mg/kg bw/d on days 12, 18, 21 and 28. In addition, two males receiving 1000 mg/kg bw/d showed blue appearance (grade 1 - minimal) over the whole body on day 7 of the recovery period. No clinical signs were noted in animals receiving 50 or 200 mg/kg bw/d.

MORTALITY
One female animal was found dead on day 3 of dosing as a result of a defective automatic drinking nozzle. Animal 8 was killed for humane reasons on day 6 of the recovery period due to an apparent extensive inflammation around the eye following blood sampling procedures. There were no deaths that could be related to treatment during the whole study period.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as control values during the whole study period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no statistically significant differences in appetite between treated and control animals before and after allowance for body weight. Fluctuations in food consumption by treated and control females were attributed to incidental high flood scatter by some females.

OPHTHALMOSCOPIC EXAMINATION
On day 24, opthalmoscopic examination of all animals of the toxicity and recovery groups did not show any abnormality that was considered to have arisen as a result of treatment. In addition, no ophthalmoscopic abnormalities were found in animals of the recovery groups on day 35.

HAEMATOLOGY
After 4 weeks of treatment (day 29):
Significantly low reticulocyte counts were noted in males receiving 200 or 1000 mg/kg bw/d and females reseiving 1000 mg/kg bw/d. Given that large unstained cells may also be erythrocytes in an early stage of development, the biological significance of the statistically sinificantly high value obtained in males receiving 1000 mg/kg bw/d should also be considered. The number of large unstained cells of males receiving 50 or 200 mg/kg bw/d and treated females and the number of reticulocytes of males receiving 50 mg/kg bw/d and females receiving 50 or 200 mg/kg bw/d remained in the same range as controls.
Statistically significantly high haemoglobin (Hb) values were noted in males receiving 200 or 1000 mg/kg bw/d and a significantly decreased mean cell volume (MCV) was noted in females receiving 1000 mg/kg bw/d. However both changes were within the biologically normal range for this age and strain rats and therefore not considered of toxicological significance.
Multiple changes of mature differential white blood cells in comparison with controls were noted in treated males, achieving a level of statistical significance for neutrophils in males receiving 50 mg/kg bw/d, eosinophils and monocytes in males receiving 1000 mg/kg bw/d, basophils in all treated males, and lymphocytes in males receiving 200 or 1000 mg/kg bw/d. Females were not thus affected and statistically significant differences were limited to monocytes only. However, all differences were within the range normally expected for rats of this age and strain. In the case of basophils in males receiving 1000 mg/kg bw/d, the statistically significantly increased values were, in the absence of a treatment related distribution and in the absence of a corrobarative effect in females and other haematological parameters not considered to be of toxicological significance. Although statistically significant decreases of thromboplastin time and partial thromboplastin time in comparison with controls were noted among males receiving 50 or 200 mg/kg bw/d, all decreases were considered to be within normal biological limits and not of toxicological significance.
Lymphocyte and large unstained cell values were excluded for two males at 1000 mg/kg bw/d following technical failure during analysis.
After 2 weeks recovery (week 6, day 43):
Although some incidental statistically significant differences of haematological parameters were noted in treated animals in comparison with controls (i.e. MCV in males receiving 200 mg/kg/day; RBC, MCH and PTT in female receiving 1000 mg/kg/day) all values were between normally expected limits and not considered to be toxicologically significant. The following animals had insufficient sample for the determination of methaemoglobin: 10, 16, 17, 18, 26, 27, 29, 30, 36, 50, 68, 69, 70 and 77.

CLINICAL CHEMISTRY
AFTER 4 WEEKS OF TREATMENT (DAY 29):
Total bilirubin levels in the blood serum of males and females receiving 1000 mg/kg bw/d were significantly increased compared to control levels. There was no such effect on bilirubin levels in the blood serum of animals receiving 50 or 200 mg/kg bw/d (although a level of statistical significance was observed in males and females receiving 200 mg/kg bw/d). Other biochemistry parameters of treated animals, achieving a level of statistical significance in their difference from controls included glucose, alkaline phosphatase (ALP), calcium and total protein (Protein T.) and were considered all consequences of out-of-range control values. The remaining statistically significant differences (i.e. inorganic phosphate, creatinine, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, potassium and chloride) were either within the range normally expected or (in the absence of any corroborative effects in the opposite sex, histopathological evidence or differences in organ weights) considered not to be of toxicological significance.
AFTER 2 WEEKS RECOVERY (WEEK 6, DAY 43):
There were no differences of toxicological significance noted among treated rats when compared to control rats.

URINALYSIS
DURING WEEK 4 OF TREATMENT (TOXICITY DAY 24, RECOVERY DAY 25):
Urine production over approximately 18 hours was noted as statistically significantly increased in males of the toxicity group receiving 1000 mg/kg bw/d
when compared to controls on day 24. On day 25, overnight urine production of males assigned to the recovery group and treated at 1000 mg/kg bw/d did not significantly differ from control animals. Therefore the increased urinary volume of day 24 was not considered to have arisen as an effect of treatment.
A possible difference in urine composition between treated and control rats was a slight to occasional increase (leucocyt-N test) or slight to moderate increase(WBC - microscope) of white blood cells in males only receiving 1000 mg/kg/day on days 24 and 25.
DURING WEEK 2 OF RECOVERY (DAY 39):
Urinary parameters of treated rats during week 2 of the recovery period did not differ from those of the controls.

ORGAN WEIGHTS
Comparing organ weights of treated animals with those of controls did not show any statistically significant difference of toxicological importance before or after adjustment for differences in body weight.

GROSS PATHOLOGY
Several rats from the high dose group showed bluish discolouration of the pancreas and testes. The remaining few macroscopic findings (i.e. pelvic dilation of the kidneys and reddened stomach) were unremarkable and amongst those commonly encountered in rats at these ages and in this strain.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination did not show any microscopic correlates to the macroscopically noted tissue discolouration. This was therefore concluded to be due to passive staining of the tissues by the test article (a dyestuff).
One high dose female had a slight degree of submucosal oedema in the stomach, which may have been due to a local irritant effect of the test article.
The only other treatment-related finding in this study was a severe degree of focal cortical hypertrophy in the adrenal glands of two high dose female rats. In each case, this was characterised by a large focus of pale staining and enlarged (but otherwise normal in appearance) zona fasiculata cells.
Other microscopic findings recorded were spontaneous in nature and are within the normal range of background alterations which may be encountered in this age and strain of rat.

Effect levels

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 2: selected haematolgy findings
	control	50 mg/kg bw	200 mg/kg bw	1000 mg/kg bw	control after recovery	1000 mg/kg bw after recovery
Reticulocytes, males	0.033	0.032	0.026*	0.017*	0.036	0.04
St. Dev	0.003	0.003	0.003	0.005	0.004	0.002
N	10	10	10	10	4	5
Reticulocytes, females	0.032	0.026	0.026	0.017*	0.035	0.03
St. Dev	0.007	0.005	0.007	0.006	0.003	0.004
N	10	10	10	9	5	4
Large unstained cells, males	0.037	0.039	0.057	0.073*	0.044	0.037
St. Dev	0.023	0.017	0.028	0.048	0.036	0.011
N	10	10	10	8	4	5
p < 0.05