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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Reference substance name:
Reaction mass of 1,4-bis(methylamino)anthraquinone and 1,4-bis[(2-ethylhexyl)amino]anthraquinone and 1-[(2-ethylhexyl)amino]-4-(methylamino)anthraquinone and 9,10-Anthracenedione, 1,4-bis(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-(methylamino)-4-(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-[(2-ethylhexyl)amino]-4-(pentylamino)-, branched and linear
EC Number:
911-360-1
Molecular formula:
variable structures
IUPAC Name:
Reaction mass of 1,4-bis(methylamino)anthraquinone and 1,4-bis[(2-ethylhexyl)amino]anthraquinone and 1-[(2-ethylhexyl)amino]-4-(methylamino)anthraquinone and 9,10-Anthracenedione, 1,4-bis(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-(methylamino)-4-(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-[(2-ethylhexyl)amino]-4-(pentylamino)-, branched and linear

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and Sex
Rats (male and female)
Strain and Justification
Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Supplier and Location
Charles River Laboratories (Raleigh, North Carolina)
Age at Study Start
Approximately eight weeks of age at initiation of treatment.
Health Status and Acclimation
Upon arrival all animals were acclimated to the laboratory for approximately one week prior to the study. During the acclimation period, each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory was fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).
Housing
Upon arrival animals were housed two-three per cage in stainless steel cages. Cages had solid floors with corncob bedding. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
After assignment to study, animals were housed singly in solid bottom stainless steel cages, except during breeding and during the gestation and littering phases of the study. The solid bottom cages contained ground corn cob bedding. During breeding, one male and one female were placed in stainless steel cages with wire mesh floors that were suspended above absorbent paper in order to better visualize vaginal copulatory plugs. After the breeding phase, males were returned to solid bottom stainless steel cages with ground corn cob bedding. During gestation and littering, dams (and their litters) were housed in plastic cages provided with irradiated ground corn cob bedding from approximately GD 0 until LD 4. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
The following environmental conditions were targeted in the animal room from the day of arrival until necropsy; however, temporary excursions from these environmental conditions may have occurred on an infrequent basis. All observed ranges were documented in the study file.
Temperature: 22°C with a range of 20°C-26°C
Humidity: 50% with a range of 30-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Enrichment
Enrichment for animals was given from the day of arrival until necropsy with the exception of breeding. Enrichment included nylon bones for non-pregnant animals and paper nesting materials for pregnant rats.
Randomization and Identification
Prior to test material administration, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers. If a transponder stopped functioning or was lost, it was replaced with a new transponder that was correlated with the unique animal number.
Feed and Water
Feed and municipal water were provided ad libitum. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. Copies of these analyses are maintained in the study file.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Corn oil supplied by Sigma-Aldrich Corporation, St. Louis, Missouri was used as the vehicle for C.I. Solvent Blue 98 (3 Amine). All dosing solutions of C.I. Solvent Blue 98 (3 Amine) were administered in corn oil, such that a dose volume of 4 ml/kg body weight achieved the targeted dose. Dose volumes were adjusted using the most current body weight. Dose solutions were prepared periodically throughout the study based upon stability data. At a minimum, dose solutions were changed weekly as per TERC animal dosing policy. C.I. Solvent Blue 98 (3 Amine) dose solutions were not corrected for purity.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose Confirmation and Homogeneity
Analyses to determine concentration of the test material of all dosing solutions from the first mix were initiated prior to the start of dosing. The low- and high-dose solutions from the first mix were analyzed to confirm homogeneous distribution of the test material concurrent with dose confirmation. Analysis was conducted using high performance liquid chromatography (HPLC) using ultraviolet (UV) detection.
The dose solutions for the ENU positive controls were not analyzed for concentration, homogeneity, solubility or stability. ENU dose solutions were corrected for purity. ENU, when administered by oral gavage at doses of
20 mg/kg/day for 3 consecutive days, has been shown to induce CD59-negative erythrocytes in the Pig-a assay in rats (Dertinger et al., 2011).
Solubility
C.I. Solvent Blue 98 (3 Amine) was determined to be soluble in corn oil at a concentration of 500 mg/ml
Stability
A previously conducted study (Marty, in progress) showed C.I. Solvent Blue 98 (3 Amine) to be stable for at least 25 days in corn oil at concentrations ranging from 0.25 to 250 mg/ml. The established concentration range spanned those used in this study, and dose solutions were used within the established stability duration.
Retainer Samples
A sample of the solid test material was retained, but samples of the dose suspensions were not retained.
Duration of treatment / exposure:
Male rats were dosed daily for 14 days prior to mating and continuing throughout the mating period for at least 34 days. Female rats were dosed once daily for 14 days prior to breeding, and continuing through breeding (two weeks), gestation (three weeks), and lactation (four days).
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
The high-dose level of 1000 mg/kg/day was based upon data obtained from a preliminary range-finding study indicating adaptive changes in the liver at dose levels between 500 - 1000 mg/kg/day (Johnson et al., 2017). The high-dose level represented a limit dose as defined in the test guidelines. The intermediate- and low-dose levels were expected to provide dose response data for any treatment-related effects observed at the high-dose level and to establish a no-observed-effect level (NOEL).
Positive control:
none

Examinations

Observations and examinations performed and frequency:
A cage-side examination was conducted at least twice daily (with the exception of 26 June 2016 when only one was recorded). This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could be observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.
Cage-side examinations were also conducted on dams and their litters, at least twice daily. These examinations were conducted as described above.

Clinical Observations
Clinical observations were conducted on all animals pre-exposure and at least daily throughout the study. During the exposure period, these examinations were conducted approximately one hour after dosing. Females were observed for signs of parturition beginning on or near gestation day (GD) 20 (see Litter Data). Females that delivered litters were subsequently evaluated on lactation day (LD) 0, 1, and 4. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.
Detailed Clinical Observations
Detailed clinical observations (DCO) were conducted on all animals pre-exposure and once per week throughout the study. Mated females received DCO examinations on GD 0, 7, 14, and 20, and LD 3. In contrast to the daily cageside and clinical observations which were designed to detect acute changes, the DCO was designed to detect more persistent neurological changes and, therefore, was conducted at approximately the same time each examination day, according to an established format. The examination included cage-side, hand-held and open-field observations, which were recorded categorically or using explicitly defined scales (ranks). Categorical observations (descriptive) are summarized under the clinical observations table(s).
Details of these observations can be found in Table 2 and Appendix A.
Functional Tests
The functional tests (sensory evaluation, rectal temperature, grip performance and motor activity) were conducted pre-exposure and during the last week of the treatment period. For the females, this took place on LD 4. Females that failed to deliver did not undergo functional testing during the last week of treatment.
Sensory Evaluation
The sensory evaluation included a test for nociception (responsiveness to tail pinch) and for startle response (responsiveness to sharp noise). The evaluation was conducted in a clear plastic box. Details of the methods for each test and specific definitions of the ranks can be found in Appendix B.
Rectal Temperature
Rectal temperature was measured by carefully placing a rectal thermistor (Physitemp, Clifton, New Jersey) approximately 4 cm into the rectum for approximately 10 seconds. Temperature was then recorded. The thermistor was validated at 37C before, during, and after the study.
Grip Performance
Hindlimb grip performance was tested according to the procedure described by Mattsson et al. (1986). Briefly, the observer placed the rat’s forelegs on a plastic bench and the hindfeet were set on a horizontal screen attached to an electronic strain gauge (Chatillon, Greensboro, North Carolina). The observer then smoothly but firmly pulled backward on the tail until the rat’s grip on the screen was broken (Appendix C). An electronic strain gauge was used to record the rat’s resistance to the pull in grams. The average of three trials was used for statistical analysis. Forelimb grip performance was similarly tested. In this application, a bench was not used, and the rats were placed so that the forefeet were on the screen and the hindfeet were suspended approximately 10 cm above the plastic platform.
Instrument Validation: A standard 500-gram weight attached to a fine-gauge wire was suspended from the load cell and was checked just before and just after testing
(a 1% tolerance, i.e., 500  5 grams, was acceptable).
Motor Activity
A commercially-available automated test system (MotorMonitor, Kinder Scientific, Inc., Poway, CA) was used for motor activity (MA) data collection. Each animal was tested individually in one of 24 available square acrylic enclosures (approximately 22” X 22”) which contained a grid of infra-red beams (32 total beams, 16 X axis and 16 Y axis) near the floor of the enclosure. The MA test room was maintained under white noise conditions (55-65 dB) and with the lighting off during testing. No entry into the MA test room was allowed during the testing period. Each test session consisted of six 10-minute intervals, totaling 60 minutes of testing per animal per test session. All beam breaks (“activity counts”) for each interval were recorded.
Motor Activity Enclosure Allocation: Rats were allocated to the motor activity enclosures in such a way that the counterbalancing of treatment groups and sexes across enclosures and test times was maximized.
Body Weights/Body Weight Gains
All rats were weighed at least once during the pre-exposure period and on the first day of dosing. Male body weights continued to be recorded weekly throughout the study. Females were weighed weekly during the pre-mating and mating periods. During gestation, females were weighed on GD 0, 7, 14, 17, and 20. Females that delivered litters were weighed on LD 1 and 4. Females that failed to mate or deliver a litter were weighed at least weekly for the remainder of the study. Body weight analyses were conducted for the following days: GD 0, 7, 14, and 20. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and LD 1-4.
Feed Consumption
Feed consumed was determined weekly during the two week pre-breeding period for males and females by weighing feed crocks at the start and end of a measurement cycle. Feed consumption was not measured for males or females due to co-housing during breeding. Following breeding, feed consumption was not measured for males. For females during gestation, feed consumption was measured on GD 0, 7, 14, and 20. After parturition, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of crock - final weight of crock)
(# of days in measurement cycle)

Breeding Procedure
Breeding of the adults commenced after approximately two weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm was detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating.
Litter Data
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. All litters were examined as soon as possible after delivery. The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (LD 0), clinical observations and the number of live and dead pups on days 0, 1, and 4 postpartum, and the sex and body weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period (see animal observations). Any pups found dead or sacrificed in moribund condition were sexed and examined grossly, if possible, for external and visceral defects and then were discarded.



Sacrifice and pathology:
Clinical Pathology
Animals were fasted overnight prior to blood collection. Blood samples were obtained from the orbital sinus following anesthesia with a mixture of isoflurane vapors and medical oxygen at the scheduled necropsy. Blood samples were not obtained from females that failed to deliver a litter.
Hematology
Sample Preparation
Blood samples for a complete blood count were mixed with ethylenediamine-tetraacetic acid (EDTA). Blood smears were prepared, stained with Wright-Giemsa stain, cover-slipped and archived for potential future evaluation if warranted.
Hematologic parameters were assayed using the Advia 120 Hematology Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York).
Assays
Hematocrit (HCT)
Hemoglobin (HGB) concentration
Red blood cell (RBC) count
Total white blood cell (WBC) count
Differential WBC count
Neutrophils (NEUT)
Lymphocytes (LYMP)
Monocytes (MONO)
Eosinophils (EOS)
Basophils (BASO)
Large Unstained Cells (LUC) which include, atypical lymphocytes,
large lymphocytes, plasma cells, and blasts
Platelet (PLT) count
Reticulocyte (RET) count
RBC indices:
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin Concentration (MCHC)
Coagulation
Sample Preparation
Blood samples were collected in sodium citrate tubes, centrifuged, plasma collected, and assayed using the ACL9000 Analyzer (Instrumentation Laboratory, Bedford, Massachusetts).
Assay
Prothrombin time (PT)
Clinical Chemistry
Sample Preparation
Blood samples were collected and serum was separated from cells as soon as possible. The following serum parameters were measured using the cobas c311 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Indiana).
Enzyme Activities of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Concentrations of:
Albumin (ALB)
Albumin/Globulin Ratio (A/G) - calculated
Cholesterol (CHOL)
Creatinine (CREA)
Electrolytes
Calcium (CA)
Phosphorus (PHOS)
Sodium (NA)
Potassium (K)
Chloride (CL)
Globulin (GLOB) - calculated
Glucose (GLUC)
Total bile acids (TBA)
Total bilirubin (TBIL)
Total protein (TP)
Triglycerides (TRIG)
Urea nitrogen (UN)
Urinalysis
Urine samples were obtained from all males on test day 30. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water were available during this procedure.
Assays
Color, appearance, specific gravity, (refractometer) and urine volume
Semiquantitative analysis of the following was conducted using Siemens Multistix Reagent Strips on the Clinitek Advantus Analyzer (Siemens Healthcare Diagnostics, Tarrytown, New York):
pH
Bilirubin
Glucose
Protein
Ketones
Blood
Urobilinogen
Microscopic Examination
Urine samples were also collected from each male by manual compression of the urinary bladder. The urine samples were pooled from each group, and the microsediment was characterized microscopically.

Anatomic Pathology
Adult males (fasted) were submitted for necropsy after at least 34 days of exposure. Adult females (fasted) were terminated on LD 5 or at least 24 days after the end of the breeding period for females not producing a litter. On the morning of the scheduled necropsy, fasted rats (excluding the Pig-a assay positive control group) were weighed in the animal room and submitted alive for necropsy. The animals were anesthetized with a mixture of isoflurane vapors and medical oxygen. While under anesthesia, blood was collected from the orbital sinus (all males including the Pig-a assay positive control group and all females that littered). The animals were placed in a CO2 chamber to continue anesthesia. Under a deep plane of anesthesia, their tracheas were exposed and clamped, and the animals were euthanized by decapitation. For the Pig-a assay positive control group, no other necropsy procedures were conducted and no other tissues were saved.
A complete necropsy was conducted on all animals by a veterinary pathologist or a technician qualified to recognize lesions, assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues
were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.
The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain based on Kopf et al. (1964) for approximately one minute and were examined for the presence and number of implantation sites. After evaluation, uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.
Weights of the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus, and thyroid with parathyroids (weighed after fixation) were recorded, and organ:body weight ratios calculated.
Representative samples of tissues listed in Table 3 were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides that were fixed in Bouin’s fixative. Transponders were removed and placed in jars with the tissues.
Off-Spring Necropsy
All pups surviving to LD 4 were euthanized by an oral dose of sodium pentobarbital solution, followed by decapitation. They were examined for gross external alterations and a limited gross internal pathologic exam of the adipose tissue and stomach contents was conducted on all PND 4 pups (except for litters from #2494 (control), 2512 (mid-dose), and 2527 (high-dose)). Any pups found dead or which were euthanized in moribund condition were examined to the extent possible and discarded.
Histopathology
Histologic examination of the tissues indicated in Table 4 was conducted on all control and high-dose adult rats and one moribund female given 300 mg/kg/day. Examination of tissues from the remaining groups was limited to target tissues (liver), those tissues that demonstrated treatment-related histologic effects at the high dose (kidney, thyroid, and pancreas), and relevant gross lesions. The heart, spleen, and pancreas were embedded in the same paraffin block for each animal. Therefore, the heart and spleen were also present on the slides with the pancreas in the low and intermediate groups to aid in the blind evaluation of the pancreas, but were not examined microscopically. Paraffin embedded tissues were sectioned approximately six µm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope.
The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned at 5 µm and stained with modified periodic acid-Schiffs-hematoxylin. The presence and integrity of the stages of spermatogenesis was qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).
Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was neither expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue was adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate were not life threatening. A severe grade was used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue could have been life threatening.


Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related effects on behavior or demeanor were observed at any dose level during the lactation period. Treatment-related clinical observations consisted of blue skin/mucous membranes and/or blue feces in all treated animals which were attributed to the dye nature of the test material. All other observations occurred at low frequencies and were considered unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female given 300 mg/kg/day (#2511) was moribund on the morning of the LD 1 and was humanely euthanized (including the litter) prior to the initiation of the scheduled necropsy due to the presence of convulsions. The convulsions were caused by a malignant astrocytoma of the brain, which was interpreted to be a spontaneous neoplasm, unassociated with administration of the test material
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically-identified differences in the body weights of any treated groups for males or on body weights and body weight gains for females during their respective premating, mating, gestation, or lactation periods when compared to their respective controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically-identified differences in the amount of feed consumed by any of the dose groups when compared to their respective controls throughout the study.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the hematology parameters of males or females from any dose group. Males given 300 mg/kg/day (mid-dose group) had a statistically-identified higher mean platelet count that was interpreted to be unrelated to treatment because of the lack of a dose response.
Males given 1000 mg/kg/day had a treatment-related statistically significant increase in mean prothrombin time that was interpreted to be non-adverse. There were no treatment-related or statistically significant effects on the mean prothrombin times of males given 100 or 300 mg/kg/day or of females from any dose level.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females given 1000 mg/kg/day had treatment-related statistically identified higher mean cholesterol concentrations that were interpreted to be non-adverse. Males given 300 or 1000 mg/kg/day had statistically identified lower mean aspartate aminotransferase (AST) activities, and females given
1000 mg/kg/day had a statistically identified higher mean potassium concentration. The alterations in AST and potassium values were interpreted to be unrelated to treatment because the values were within historical control ranges.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Males given 100, 300 or 1000 mg/kg/day had purple discoloration of the urine, which was attributed to the dye nature of the test material present in the urine. Because of the discolored urine, the results of urinalysis parameters (pH, protein, glucose, ketones, bilirubin, blood, and urobilinogen) that were measured using Siemens Multistix Reagent Strips were invalid per guidance from the manufacturer (Siemens Multistix package insert 2010 Siemens Healthcare Diagnositics) and therefore excluded from interpretation. There were no treatment-related alterations in the urine volume or urine specific gravity of males or females from any dose level.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment- related effects on motor activity in males at any dose level or in females at dose levels
≤ 300 mg/kg/day. There was a treatment-related decrease (46%) in the total motor activity of females given 1000 mg/kg/day compared to controls. Total motor activity in females given 300 mg/kg/day was also decreased compared to controls following treatment, however, this difference was not considered treatment-related as it was similar to the between groups differences under baseline conditions. Treatment did not affect motor activity (treatment-by-time interaction) in males (p = 0.1403). Despite the aforementioned decrease in total motor activity of females given 1000 mg/kg/day, the treatment-by-time interaction was also not significant for females (p = 0.1446). The distribution of the motor activity counts within session (treatment-by-time-by-interval interaction) was not affected by treatment either in males (p = 0.9885) or in females (p = 0.8776).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males and females given 100, 300 or 1000 mg/kg/day had treatment-related dose-responsive increases in mean absolute and relative liver weights. The mean relative liver weights of males given 100, 300 or 1000 mg/kg/day were 5.6%, 17.4% and 35.9% higher than controls. The mean relative liver weights of females given 100, 300 or 1000 mg/kg/day were 5.3%, 16.4% and 26.9% higher than controls. The higher liver weights corresponded to hepatocellular hyperthophy in males and females from all dose groups. Males given 1000 mg/kg/day had treatment-related statistically identified increases in mean absolute and relative thyroid gland weights. The higher thyroid gland weights corresponded to slight diffuse hypertrophy of follicular cells in males given 1000 mg/kg/day. Females given
300 mg/kg/day had a statistically identified lower mean absolute brain weight that was interpreted to be unrelated to treatment because of the lack of a dose response and because the value was within the historical control group range.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
All male and female rats given 100, 300 or 1000 mg/kg/day had a blue discoloration of various tissues throughout the body due to the administration of the test material. The blue discoloration was most prominent in the adipose tissue. There were no additional treatment-related gross pathologic observations. One female given 300 mg/kg/day (#2511) was moribund on the morning of the LD 1 and was euthanized due to the presence of convulsions. The convulsions were caused by a malignant astrocytoma of the brain, which was interpreted to be a spontaneous neoplasm, unassociated with administration of the test material.
Neuropathological findings:
not examined
Description (incidence and severity):
A treatment-related histopathologic effect in the liver consisted of very slight to slight hypertrophy of hepatocytes, with altered tinctorial properties (increased cytoplasmic eosinophilia) . The hypertrophy was present in the centrilobular/midzonal regions of the hepatic lobule in all affected males given 100 mg/kg/day, 6/12 affected males given 300 mg/kg/day and all affected females given 100, 300 or 1000 mg/kg/day. The hypertrophy was panlobular, involving the entire hepatic lobule, in 6/12 males given 300 mg/kg/day and in all males given 1000 mg/kg/day. The hepatocellular hypertrophy was interpreted to an adverse effect in males and females given 1000 mg/kg/day because the corresponding increases in relative liver weights were more than 25% higher compared to controls. Histopathologic effects in the pancreas involved the exocrine tissue, not the islets of Langerhans, and consisted of a treatment-related, slight increase in apoptosis/necrosis of acinar cells of males given 100, 300 or 1000 mg/kg/day and females given 300 or 1000 mg/kg/day compared to the controls. These effects were characterized by an increase in the frequency of apoptotic/necrotic acinar cells and the occurrence of autophagic/vacuolated individual acinar cells. The slight apoptosis/necrosis of pancreatic acinar cells was interpreted to be an adverse effect in all animals with this alteration. A treatment-related histopathologic effect in the kidneys consisted of a slight increase in the accumulation of hyaline protein droplets in the cytoplasm of tubular epithelial cells of the proximal convoluted tubules in males given 1000 mg/kg/day compared to the controls. One male given 300 mg/kg/day and one male given 1000 mg/kg/day had a moderate amount of hyaline droplets in the proximal convoluted tubules; however, the majority of these droplets were different in appearance, in that the droplets were much larger, stained faintly and seemed to be in the tubular lumen rather then the cytoplasm of affected epithelial cells. These moderate accumulations of hyaline droplets were not interpreted to be treatment related due to the differences in appearance of these droplets. The occurrence of slight accumulations of hyaline droplets in the tubules of males given
1000 mg/kg/day was interpreted to be a non-adverse effect because this alteration was not accompanied by degeneration or necrosis of the tubular epithelial cells and because hyaline droplet nephropathy is a male rat specific lesion with no significant relevance to humans. A histopathologic effect also occurred in the thyroid glands of males given
1000 mg/kg/day and consisted of a treatment-related slight hypertrophy (increase in size) of the follicular epithelial cells compared to the controls. This hypertrophy of the epithelial cells may have been caused by the induction of liver microsomal enzymes responsible for the biliary excretion of thyroid hormones, with resultant increased thyroid stimulating hormone production causing chronic stimulation of the thyroid gland.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
pancreas
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Histopathologic Liver Effects – Males and Females

Sex

Males

Females

Dose (mg/kg/day)

0

100

300

1000

0

100

300

1000

Liver (Number Examined)

12

12

12

12

12

12

12

12

Hypertrophy, with altered tinctorial properties, hepatocytes, panlobular, slight

0

0

6

12

0

0

0

0

Hypertrophy, with altered tinctorial properties, hepatocytes, centrilobular/midzonal,         slight

3

9

6

0

0

0

11

12

Hypertrophy, with altered tinctorial properties, hepatocytes, centrilobular/midzonal, very slight

0

0

0

0

1

11

0

0

Bolded values interpreted to be treatment-related effects.

Histopathologic Pancreatic Effects – Males and Females

Sex

Males

Females

Dose (mg/kg/day)

0

100

300

1000

0

100

300

1000

Pancreas (Number Examined)

12

12

12

12

12

12

12

12

Apoptosis/necrosis, individual cells, acinus, multifocal, very slight

11

4

3

1

10

10

10

6

Apoptosis/necrosis, individual cells, acinus, multifocal, slight

1

7

9

11

0

0

2

6

Bolded values interpreted to be treatment-related effects.

Histopathologic Kidney Effects – Males

Dose (mg/kg/day)

0

100

300

1000

Kidneys (Number Examined)

12

12

12

12

Hyaline droplet formation, cortex, proximal convoluted tubules, bilateral, multifocal, very slight

8

7

7

4

Hyaline droplet formation, cortex, proximal convoluted tubules, bilateral, multifocal, slight

2

1

2

7

Hyaline droplet formation, cortex, proximal convoluted tubules, bilateral, multifocal, moderate

0

0

1

1

Bolded values interpreted to be treatment-related effects.

Histopathologic Thyroid Gland Effect – Males

Dose (mg/kg/day)

0

100

300

1000

Thyroid gland (Number Examined)

12

12

12

12

Hypertrophy, follicular cell, diffuse, very slight

10

7

12

5

Hypertrophy, follicular cell, diffuse, slight

0

0

0

7

Bolded values interpreted to be treatment-related effects

Applicant's summary and conclusion

Conclusions:
A no-observed-adverse-effect level (NOAEL) could not be determined for males because of the increase in necrosis/apoptosis of pancreatic acinar cells in males from all treated groups. The NOAEL for females was 100 mg/kg/day based upon the increase in necrosis/apoptosis of pancreatic acinar cells at 300 mg/kg/day.
Executive summary:

The purpose of this study was to evaluate the potential effects of C.I. Solvent Blue 98 (3Amine) on general systemic toxicity, reproductive and neurological function, and prenatal/early neonatal growth and survival of the offspring. Furthermore, the integrated in vivo gene mutation Pig-aassay evaluated the mutagenic potential of the test material.

Groups of 12 male and 12 female Crl:CD(SD) rats were administered C.I. Solvent Blue 98 (3Amine) daily, by gavage at dose levels of 0 (control), 100, 300, or 1000 mg/kg/day. Females were dosed once daily for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through postpartum day 4. Females were necropsied on postpartum day 5. Males were dosed two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 34 or 35). Effects on reproductive and neurological function as well as general toxicity were evaluated. In addition, postmortem examinations included a gross necropsy of the adults with collection of organ weights and histopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed. Anintegrated in vivo gene mutation Pig-a assay was also conducted on six males/group to evaluate the mutagenic potential of the test material. The Pig-a assay included apositive control group (6 males) that was dosed with 20 mg/kg N-Nitroso-N-ethylureaon test days (TD) 1-3. Mutation frequency in reticulocytes (RET) and red blood cells (RBC) of each designated male was determined by flow cytometry quantification of CD59-negative RET and RBC (RETCD59-and RBCCD59-).

Treatment-relatedclinical observations consisted of blue skin/mucous membranes and/or blue feces in all treated animals which were attributed to the dye nature of the test material.

There were no treatment-related effects on body weight, body weight gain, or feed consumption in males or females in any treated groups compared to controls. 

There were no treatment-related effects on reproductive function in either males or females or prenatal/early neonatal growth and survival of offspring in any treated groups compared to controls.

There were no treatment-related effects on sensory evaluation, rectal temperature or grip performance in males or females from any dose group. There were no treatment-related effects on motor activity in males at any dose level or in females at dose levels
≤ 300 mg/kg/day. There was a treatment-related decrease in the total motor activity of females given 1000 mg/kg/day when compared to control females. There were no treatment-related changes in the hematology parameters of males or females in any dose group. Males given 1000 mg/kg/day had a treatment-related increase in mean prothrombin time that was interpreted to be non-adverse. Males and females given 1000 mg/kg/day had treatment-related higher mean cholesterol concentrations that were interpreted to be non-adverse. Males given 100, 300 or 1000 mg/kg/day had purple discoloration of the urine, which was attributed to the dye nature of the test material present in the urine. 

Males and females given 100, 300 or 1000 mg/kg/day had treatment-related increases in mean absolute and relative liver weights. The higher liver weights corresponded to hepatocellular hyperthophy in males and females in all dose groups. Males given
1000 mg/kg/day had treatment-related increases in mean absolute and relative thyroid gland weights. The higher thyroid gland weights corresponded to slight diffuse hypertrophy of follicular cells in males given 1000 mg/kg/day. 

All male and female rats given 100, 300 or 1000 mg/kg/day had a blue discoloration of various tissues throughout the body attributed to the presence of the test material. The blue discoloration was most prominent in adipose tissue. Limited gross pathological observations on postpartum day 4 pups revealed blue adipose tissue and stomach contents in pups from all treated groups.  This discoloration was attributed to the dye nature of the test material and demonstrated systemic exposure to the pups via the milk. 

A treatment-related histopathologic effect in the liver consisted of very slight to slight hypertrophy of hepatocytes with increased cytoplasmic eosinophilia. The hypertrophy was present in the centrilobular/midzonal regions of the hepatic lobule in the male 100 and
300 mg/kg/day groups and all treated female groups.  In some males from the
300 mg/kg/day group and all males in the 1000 mg/kg/day group, the hypertrophy was panlobular. The hepatocellular hypertrophy was interpreted to be an adverse effect in the male and female 1000 mg/kg/day groups because the corresponding increases in mean relative liver weights were at least 25% higher than the control group mean.

A treatment-related histopathologic effect occurred in the thyroid glands of males given
1000 mg/kg/day and consisted of a slight hypertrophy of the follicular epithelial cells. This was interpreted to be a non-adverse adaptive response likely secondary to the induction of liver microsomal enzymes.

Treatment-related histopathologic effects in the pancreas involved the exocrine tissue, not the islets of Langerhans, and consisted of a slight increase in apoptosis/necrosis of acinar cells and in individual autophagic/vacuolated acinar cells. These treatment-related effects were present in all male dose groups and in the female 300 and 1000 mg/kg/day dose groups. The apoptosis/necrosis of pancreatic acinar cells was interpreted to be an adverse effect.

A treatment-related histopathologic effect in the kidneys consisted of a slight increase in the accumulation of hyaline protein droplets in the cytoplasm of tubular epithelial cells of the proximal convoluted tubules in males given 1000 mg/kg/day. This observation was interpreted to be a non-adverse effect because it was not accompanied by degeneration or necrosis of the tubular epithelial cells and because hyaline droplet nephropathy is a male rat specific lesion with no significant relevance to humans.

Results for thePig-aassay showed no significant change in the frequency of RETCD59-or RBCCD59-in animals treated with the test material. Therefore, C.I. Solvent Blue 98 (3 Amine) was negative in thisin vivogene mutationPig-aassay.

A no-observed-effect level (NOEL) for general toxicity could not be determined for male or female rats due to the occurrence of treatment-related effects at all dose levels. A no-observed-adverse-effect level (NOAEL) could not be determined for males because of the increase in necrosis/apoptosis of pancreatic acinar cells in males from all treated groups. The NOAEL for females was 100 mg/kg/day based upon the increase in necrosis/apoptosis of pancreatic acinar cells at 300 mg/kg/day. The NOEL for neurological toxicity was
300 mg/kg/day in females and 1000 mg/kg/day in males, the highest dose level tested. The NOEL for reproductive toxicity or for effects on prenatal/neonatal growth and survival was 1000 mg/kg/day, the highest dose level tested.


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