Amines, C12-14-tert-alkyl, compds. with 1-[2-[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]diazenyl]-2-naphthalenol 1-[2-[2-hydroxy-4(or 5)-nitrophenyl]diazenyl]-2-naphthalenol chromium complexes

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:

04 Apr 2014 - 07 Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: U.S. EPA Health Effects Test Guidelines. OPPTS 870.3050; Jul 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass 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-4-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-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-(1,1-dimethylpropyl)phenylazo)-2-naphtholato))chromate(1-)

EC Number:

938-781-3

Cas Number:

117527-94-3

Molecular formula:

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

IUPAC Name:

reaction mass 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-4-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-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-(1,1-dimethylpropyl)phenylazo)-2-naphtholato))chromate(1-)

Details on test material:

- Physical state: solid/black
- Storage condition of test material: room temperature, avoid humidity

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42± 1 days
- Weight at study initiation: males: 149.5 - 167.1g; females: 123.3 - 140.9g
- Fasting period before study: no
- Housing: 5 animals per cage in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm^2).
- Diet: ground Kliba maintenance diet mouse/rat "GLP", ad libitum
- Water: supplied ad libitum from water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Apr 2014 To: 14 May 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: drinking water containing 0.5% Carboxymethylcellulose and Cremophor

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water containing 0.5% Carboxymethylcellulose and Cremophor was filled up to the desired volume, subsequently mixed with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.4; 2.0 and 10.0g/100ml
- Amount of vehicle (if gavage): 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The studies were carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test article in the drinking water containing 1% Carboxymethylcellulose and Cremophor at room temperature over a period of 4 days and at refrigerator over 7 days was proven before the start of the administration. Homogeneity analyses were performed in the highest and lowest concentration. Additionally, concentration control was performed in all concentrations at the beginning of the administration period.
The various analyses confirmed:
• the stability of the test-substance preparations for a period of 4 days at room temperature and 7 days at refrigerator conditions
• the homogeneous distribution of the test substance in the vehicle,
• the correctness of the prepared concentrations except the low dose of 40 mg/kg bw/d as only about 80% of the prepared concentrations were found.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
1000, 200 and 40 (corrected to 32 mg/kg bw after concentration analysis) mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: as requested by the sponsor
- Rationale for animal assignment: Random distribution according to weight among the individual test groups

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily before the administration as well as 2 hours and within 5 hours after the administration. A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: performed in all animals prior to the administration period and thereafter at weekly intervals. Parameters examined: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period and on day 0 (start of the administration period) and thereafter at weekly intervals.

FOOD CONSUMPTION
Food consumption was determined weekly over a period of 4 days and calculated as mean food consumption in grams per animal and day.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the administration period, in the morning
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RET), Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period, in the morning
- Animals fasted: Yes
- How many animals: all
- Parameters examined: "Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

URINALYSIS: Yes
- Time schedule for collection of urine: Day 24 of administration
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, Protein (PRO), Glucose (GLU), Ketones (KET), Urobilinogen (UBG), Bilirubin (BIL), Blood, Specific gravity (SP.GR.), Sediment, Color, turbidity (COL, TURB), Volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested: Home cage observations / Open field observations / Sensory motor tests/ reflexes / Motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix

HISTOPATHOLOGY: Yes
Organs were fixed followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below.

Other examinations:

To further classify the increase in eosinophilic droplets in the kidneys of male animals in test group 3 (1000 mg/kg bw/d), the kidneys of animal Nos. 1 and 16 were immunohistochemically stained against alpha 2u globuline. The immunorelevant organs and tissues were evaluated according to the following
parameters:
Thymus:
• Increased/decreased grade of cortico-medullar ratio (related only to area)
• Increase of starry sky cells
• Changes of cellular density in the cortex
• Changes of cellular density in the medulla
Spleen:
• Changes of the cellularity of PALS, lymphoid follicles, marginal zone, red pulp
• Altered cellular composition of follicles
• Altered number of germinal centers
Lymph nodes (mesenteric and axillary lymph nodes):
• Changes in the cellularity of follicles, interfollicular area, paracortical area, medulla
• Altered cellular composition of paracortex
• Altered number of germinal centers
• Hyperplasia of high endothelial venules
Peyer's patches (of the jejunum):
• Changes of the cellularity of follicles (including mantle zone and germinal centers)
• Changes of the cellularity of interfollicular area
Bone marrow:
• Changes of the cellularity
• Changes of the myeloid/erythroid ratio

Statistics:

• Body weight, body weight change: DUNNETT's test (two-sided)
• Feces, rearing, grip, strength forelimbs, grip strength, hindlimbs, footsplay, test, motor activity: KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided) if p-value was equal or less than 0.05
• Blood parameters: For parameters with bidirectional changes: KRUSKAL-WALLIS test and WILCOXON test (two-sided) if p-value was equal or less than 0.05; For parameters with unidirectional changes: WILCOXON-test (one-sided)
• Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity: WILCOXON-test (one-sided)
• Urine pH, volume, specific gravity, color and turbidity: KRUSKAL-WALLIS test and WILCOXON test (two-sided) if p-value was equal or less than 0.05
• Weight parameters: KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided) if p-value was equal or less than 0.05

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died prematurely in the present study. Black discolored feces were observed in all animals of test group 3 (1000 mg/kg bw/d) from study day 1 onwards and in all animals of test group 2 (200 mg/kg bw/d) from study day 9 until the end of the administration period. Black discolored skin was observed in all animals of test group 3 (1000 mg/kg bw/d) from study day 3 onwards and in all animals of test grou 2 (200 mg/kg bw/d) from study day 13 until the end of the administration period. Black discolored eyes were observed in all animals of test group 3 (1000 mg/kg bw/d) from study day 8 onwards and in all animals of test group 2 (200 mg/kg bw/d) from study day 15 until the end of the administration period. These findings were assessed as being related to treatment. Feces discoloration was not assessed to be adverse. The assessment of the discoloration of skin was not possible a an impairment of the functional integrity of this organ could not clearly be investigated in this study. The discoloration of eyes was assumed to be adverse. No signs were observed for male and female animals in test group 1 (40 mg/kg bw/d).

BODY WEIGHT AND WEIGHT GAIN
With regard to the mean body weights, no significant changes to the control values were observed in male and female animals of test groups 1, 2 and 3 (40, 200 and 1000 mg/kg bw/d). The same was true for mean body weight change values except the body weight change values examined in male animals of test group 3 (1000 mg/kg bw/d) which were statistically significantly lower on study days 7 (-20%) and 14 (-14%). However, as the difference to the control animals did not last over the entire study period, the changes were assessed as being incidental and not related to treatment.

FOOD CONSUMPTION
No test substance-related, adverse findings were observed. All recorded values were within the biological range typical for this strain of rats.

WATER CONSUMPTION
No test substance-related changes with regard to water consumption were observed.

HAEMATOLOGY
At the end of the administration period in females of test group 3 (1000 mg/kg bw/d), platelet counts were decreased. Although total white blood cell counts were not statistically increased, an increase of absolute monocyte cell counts above the historical control range was found in females of test group 3 (1000 mg/kg bw/d). In addition, absolute large unstained cell (LUCA) counts were increased in these individuals, but the mean was within the historical control range. Therefore, this last alteration was regarded as incidental and not treatment-related. Relative neutrophil cell counts were decreased and relative lymphocyte counts were increased in males of test group 1 (40 mg/kg bw/d), but the changes were not dosedependent. Relative reticulocyte cell counts were higher in rats of both sexes of test group 3 (1000 mg/kg bw/d) and additionally in males of test group 2 (200 mg/kg bw/d). Mean corpuscular volume (MCV was decreased in males of test group 3 (1000 mg/kg bw/d). The mentioned values were within historical control ranges and therefore the changes were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
At the end of the administration period in rats of both sexes of test group 3 (1000 mg/kg bw/d), creatinine, cholesterol and globulin levels were decreased and total bilirubin levels were increased. Already in test group 2 (200 mg/kg bw/d) in males creatinine values were decreased and in females total bilirubin levels were increased. In females of test group 3 (1000 mg/kg bw/d) total protein values were decreased and in males of the same test group triglyceride levels were decreased.

URINALYSIS
In rats of both sexes of test groups 2 and 3 (200 and 1000 mg/kg bw/d), conjugated bilirubin excretion via the urine was increased. Additionally in males of test group 3 (1000 mg/kg bw/d), urine volume was increased and the specific gravity of the urine was decreased. However, these alterations per se were regarded as treatment-related, but not adverse.

NEUROBEHAVIOUR
During Functional observational battery, deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental. Regarding the overall motor activity as well as single intervals, no test substance-related deviations were noted for male and female animals of test groups 1-3 (40, 200 and 1000 mg/kg bw/d).

ORGAN WEIGHTS
The weight changes of absolute and relative organ weights (see table below) of adrenal glands of test group 3 females (1000 mg/kg bw/d) and spleen in female animals of test groups 2 and 3 (200 and 1000 mg/kg bw/d) were regarded to be treatment-related as they were slightly above the historical control data (for test group 3). Nevertheless, no histopathologic correlates were observed. Therefore, the change was regarded to be not adverse. The increase of relative liver weight in male animals of test group 3 (1000 mg/kg bw/d) was still within historical control data and no histopathologic correlate was observed. It was regarded to be not related to treatment and not adverse.
The decrease of absolute heart weight in male animals of test group 3 (1000 mg/kg bw/d) was not regarded to be treatment-related as no histopathologic correlate was observed and the relative organ weight was not statistical significantly altered. In females of test group 3 (1000 mg/kg bw/d), the relative liver weight was significantly increased and outside historical control data. Therefore, a treatment-related effect could not be excluded but was not regarded to be adverse as no histopathologic finding was observed. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

GROSS PATHOLOGY
All animals of test groups 2 and 3 (200 and 1000 mg/kg bw/d) revealed a grey to dark grey discoloration of the carcass and all organs. In some animals of test group 1 (40 mg/kg bw/d), only the content of the digestive tract revealed a grey discoloration. These findings were regarded to be treatment-related. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were observed in the lungs of male and females with incidences and grading according to the table below.
In all animals of test group 3 (1000 mg/kg bw/d) there was a (multi)focal to extensive inflammation within the lungs. Mainly areas at the brochio-alveolar transition were affected. Inflammatory foci were mainly composed of macrophages revealing a foamy cytoplasm and a lower number of neutrophils. This finding was regarded to be treatment-related. Female animal No. 26 of test group 1 (40 mg/kg bw/d) revealed a severe inflammation with single
multinucleated giant cells in the lungs. This was evidence for a foreign body reaction most likely caused by a gavage error. Therefore, this finding was application-related but not related to treatment with the test substance.
Male animals of test group 3 (1000 mg/kg bw/d) revealed a slightly higher incidence of eosinophilic droplets in the tubules of the kidneys when compared to the other test groups and control animals. An immunohistochemical staining against alpha 2µ globulin was positive in animal No. 16 of test group 3 (1000 mg/kg bw/d) and to lesser extent in control animal No. 1. Peri-/vasculitis was observed in a single control female as well as in four male animals of test group 3 (1000 mg/kg bw/d). The finding is commonly observed in the rat liver, especially in male animals (Ruben et al., 2000). All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Signs of systemic toxicity were determined at the next higher dose level of 200 mg/kg bw/d and above.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Absolute organ weights

When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (40)	2 (200)	3 (1000)	1 (40)	2 (200)	3 (1000)
Adrenal glands				106%	104%	131%*
Heart	98%	97%	84%*
Spleen				99%	114%*	130%*

*p ≤ 0.05; **p ≤ 0.01

Relative organ weights

When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly changed (statistically significant changes printed in bold):

	Male animals			Female animals
Test group	1	2	3	1	2	3
(mg/kg bw/d)	(40)	(200)	(1000)	(40)	(200)	(1000)
Adrenal glands				105%	107%	137%**
Liver	105%	105%	111%*	101%	105%	117%**
Spleen				100%	118%*	136%**

*p ≤ 0.05; **p ≤ 0.01

Gross Lesions

	Male animals				Female animals
Test group	0	1	2	3	0	1	2	3
(mg/kg bw/d)	0	(40)	(200)	(1000)	0	(40)	(200)	(1000)
No. of animals	5	5	5	5	5	5	5	5
General observation Discoloration	0	0	5	5	0	0	5	5
Cecum Discol. of contents	0	3	5	5	0	2	5	5
Colon Discol. of contents	0	1	5	5	0	1	5	5
Jejunum Discol. of contents	0	2	5	5	0	3	5	5

Histopathology

Lungs	Male animals				Female animals
Test group	0	1	2	3	0	1	2	3
(mg/kg bw/d)	0	(40)	(200)	(1000)	0	(40)	(200)	(1000)
No. of animals	5	5	5	5	5	5	5	5
Inflammation, (multi)focal	0	0	0	5	0	1	0	5
·   Grade 2				2				4
·   Grade 3				3				1
·   Grade 4						1

Kidneys	Male animals
Test group (mg/kg bw/d)	0 (0)	1 (40)	2 (200)	3 (1000)
No. of animals	5	5	5	5
Eosinophilic droplets in tubules	5	5	5	5
·   Grade 1	5	5	5
·   Grade 2				5

Applicant's summary and conclusion

Conclusions:

The oral administration of the test article by gavage to male and female Wistar rats over a period of 4 weeks resulted in signs of systemic toxicity which were determined at a dose level of 200 mg/kg bw/d and above. Therefore, the no observed adverse effect level (NOAEL) was considered to be 32 mg/kg bw/d in male and female Wistar rats (only 80% of the nominal dose level of 40 mg/kg bw/d were found during concentration control analysis).