Benzo[rst]phenanthro[10,1,2-cde]pentaphene-9,18-dione, reaction products with 1-chlorododecane

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:

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: USEPA OPPTS 870.3650 (2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzo[rst]phenanthro[10,1,2-cde]pentaphene-9,18-dione, reaction products with 1-chlorododecane

EC Number:

822-536-1

Cas Number:

2180952-76-3

Molecular formula:

na

IUPAC Name:

Benzo[rst]phenanthro[10,1,2-cde]pentaphene-9,18-dione, reaction products with 1-chlorododecane

Details on test material:

Lot #: ZA07262016

Specific details on test material used for the study:

Test Material Name: C.I. Solvent Red 175 Solid (solvent stripped)
Chemical Name: Dinaphtho(1,2,3-cd:1’,2’,3’-lm)perylene-9,18-dione, lauryl derivatives
Lot/Reference/Batch Number: ZA07262016
Purity/Characterization (Method of Analysis and Reference): The test material was determined to contain 93.9 ± 0.06% active ingredient by difference with identification by nuclear magnetic resonance spectroscopy and liquid chromatography mass spectrometry (Kiefer, 2017).
Test Material Stability Under Storage Conditions: C.I. Solvent Red 175 Solid (solvent stripped), lot ZA07262016, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under U.S. EPA OPPTS Guideline 830.6313 (Kiefer and Kerry, 2017).

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

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.

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 is 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 13. 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. Enrichment included the use of open areas on the cage sides for visualization of other rats, pair housing when applicable, and the use of nylon bones during prebreeding or paper nesting material during gestation and lactation. Males also received nylon bones after breeding was complete.

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. There were no contaminants in either the feed or water at levels that would have adversely impacted the results or interpretation of this study. Copies of these analyses are maintained in the study file.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage was the preferred route of exposure according to the relevant test guideline.
Male rats were dosed daily for 28 days prior to breeding and continuing throughout the breeding period for at least 48 days. Female rats were dosed once daily for 28 days prior to breeding, and continuing through breeding (two weeks), gestation (three weeks), and lactation (13 days).

Vehicle:

corn oil

Details on oral exposure:

Dose Preparation:
All dosing solutions of C.I. Solvent Red 175 Solid (solvent stripped) were administered in corn oil, such that a dose volume of 4 ml/kg body weight yielded 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. Dose solutions were not corrected for purity.

Vehicle:
Corn oil supplied by Sigma-Aldrich Corporation, St. Louis, Missouri was used as the vehicle for C.I. Solvent Red 175 Solid (solvent stripped).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis:
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) with fluorescence detection (HPLC/FLD).

Stability:
A previously conducted study showed C.I. Solvent Red 175 Solid (solvent stripped) to be stable for at least 24 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.

Solubility:
C.I. Solvent Red 175 Solid (solvent stripped) was determined to be soluble in corn oil at a concentration of 250 mg/ml.

Retainer Samples:
A sample of the solid test material was retained, but samples of the dose solutions were not retained.

Duration of treatment / exposure:

Groups of 12 male and 12 female Crl:CD(SD) rats were administered C.I. Solvent Red 175 Solid (solvent stripped) daily, by gavage in corn oil, at dose levels of 0 (control), 100, 300, or 1000 mg/kg/day. Males were dosed once daily for at least four weeks prior to breeding and continuing throughout breeding for a minimum of 48 days. The females were dosed daily for four weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (thirteen days).

Frequency of treatment:

Daily gavage.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12/sex/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Route, Method of Administration, Frequency, Duration and Justification:
Oral gavage was the preferred route of exposure according to the relevant test guideline.
Male rats were dosed daily for 28 days prior to breeding and continuing throughout the breeding period for at least 48 days. Female rats were dosed once daily for 28 days prior to breeding, and continuing through breeding (two weeks), gestation (three weeks), and lactation (13 days).

Dose Levels and Justification:
The high-dose level of 1000 mg/kg/day was based upon data obtained from a preliminary range-finding study (Johnson et al., 2016). The high-dose level represents the limit dose as defined in the test guidelines, and based upon the preliminary range-finding study data, C.I. Solvent Red 175 Solid (solvent stripped) exposure was expected to increase liver weights at the high-dose level. 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).

Examinations

Observations and examinations performed and frequency:

Daily In-Life Observations:
A cage-side examination was conducted at least twice daily. 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 have been 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 at least twice daily on dams and their litters during the lactation phase of the study. These examinations were performed 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. 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 males pre-exposure and weekly throughout the exposure period. Detailed clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm-positive or plug-positive) females received DCO examinations on GD 0, 7, 14, and 20. Females that delivered litters were subsequently evaluated on LD 7 and 13. Detailed clinical observations were not conducted on females that fail to mate or deliver a litter during the gestation and lactation phases of the study. The DCO 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).

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 13. Females that failed to deliver a litter did not undergo functional testing during the last week of treatment.

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, 4, 7, and 13. 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, 4-7, 7-13, and 1-13.

Feed Consumption:
Feed consumption was determined weekly during the four week pre-breeding period for all animals by weighing feed containers at the start and end of a measurement cycle. During breeding, feed consumption was not measured in males or females due to co-housing. Following breeding, feed consumption for males was not measured. For mated females, feed consumption was measured on GD 0, 7, 14, and 20. For females delivering litters, feed consumption was measured on LD 1, 4, 7, 11, and 13. Feed consumption was not measured 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)

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. Parameters evaluated were Hematology, Coagulation and Clinical Chemistry.

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. 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.

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: date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on days LD 0, 1, 4, 7 and 13, and the sex and the weight of each pup on LD 1, 4, 7, and 13. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period. In addition, pup clinical observations were recorded on days 0, 1, 4, 7, and 13 postpartum. Any pups found dead were sexed and examined grossly, if possible, for external and visual defects and then discarded.

Anogenital Distance:
Anogenital distance (AGD; absolute and relative to the cube root of PND 1 body weight) was measured in all pups on PND 1 using a digital caliper (Gallavan et al., 1999). The sequence of the data collection was counterbalanced across groups (e.g., control, high, middle, low) to the extent possible on each day to control for potential confounding influences of collection timing.

Culling:
To minimize variation in pup growth due to differences in litter size, litters were standardized to eight pups per litter on PND 4. This was accomplished by randomly ordering the pups in each litter by sex. Pups to be culled were then randomly selected using a computer generated randomization procedure, so that four males and four females remained in each litter. If it was not possible to have four pups/sex in each litter, unequal numbers of males and females were retained (e.g., five males, three females). Litters with ≤ 8 pups were not culled. Preferential culling of runts was not performed.

Contigency Sample Collection for Thyroid Analysis from Culled Pups:
Culled pups were anesthetized on PND 4 using a mixture of isoflurane vapors and medical grade oxygen, and blood was collected via cardiac puncture and then transferred into serum separator tubes. To ensure a sufficient volume for potential hormone analyses, blood was pooled by litter if possible. Blood samples were processed as described below.
While under deep isoflurane anesthesia, the pups were euthanized by decapitation. Following decapitation, thyroid glands were harvested from the same pups that were used for blood collection. The head with the trachea, with the thyroid gland attached, was removed, and placed in 10% phosphate buffered formalin for possible histopathological examination. No histopathological examination was conducted, and tissues will be discarded after the final report is issued.

Nipple/Areolae Retention:
All offspring were evaluated for the presence of nipple/areolae on PND 12 in accordance with the methods described by McIntyre et al. (2001). The average number of nipples/areolae in male and female offspring in each litter was determined. The grand mean number of nipples/areolae for males and females in each dose level was calculated from these litter means. Observers were blinded to treatment group when evaluating pups for the presence of nipples/areolae.

Sacrifice and pathology:

Anatomic Pathology:
Adult Necropsy:
Adult males (fasted) were submitted for necropsy after at least 48 days of exposure. Adult females (fasted) will be terminated on LD 14 or at least 24 days after the end of the mating period for females not producing a litter. On the morning of the scheduled necropsy, fasted rats 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, 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.
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 and examined for the presence and number of implantation sites. 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, seminal vesicles with coagulating glands (and fluids), prostate, thymus, and thyroid with parathyroids (weighed after fixation) were recorded, and organ:body weight ratios calculated.
Representative samples of tissues (See Necropsy Tissues Collected attachment) were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides that were fixed in Bouin’s or another appropriate fixative. Transponders were removed and placed in jars with the tissues.

Histopathology:
Histopathological examination of the tissues was conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to the the liver that demonstrated treatment-related histologic effects at the high dose and relevant gross lesions. Paraffin embedded tissues were sectioned approximately 6 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 are 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 have not been affected, depending on the organ/tissue involved, but generally lesions graded as moderate were not 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.

Anatomic Pathology:
Off-Spring Necropsy:
All pups surviving to PND 13 were anesthetized with a mixture of isoflurane vapors and medical oxygen. While under anesthesia, blood was collected via cardiac puncture from at least one male and one female per litter (if possible). While under anesthesia, the animals were euthanized by decapitation. Thyroids were removed from each pup that had blood collected, and fixed for possible histopathological examination. Weights of the thyroid with parathyroids (weighed after fixation) may be recorded, and organ:body weight ratios calculated. Weights and histopathological exmaination of the thyroids was not conducted.

Other examinations:

Estrous Cycle Evaluation:
Vaginal lavage samples from all females were collected daily for approximately two weeks pre-exposure and daily for two weeks immediately prior to mating and during cohabitation until each female was sperm- or plug-positive or until the two week mating period had elapsed. Lavage samples were collected by gently irrigating the vagina with water and transferring lavage fluid to a microscope slide. These vaginal lavage slides were examined microscopically, and stage of estrous was recorded. This information was evaluated to determine estrous cycle length and pattern. On the day of scheduled necropsy, the stage of the estrous cycle was also determined for all female rats via microscopic examination of the vaginal lavage sample.

Serum for Thyroid Hormone Analyses:
The sequence of the sample collection for all of the animals was counterbalanced across groups (e.g., control, high, middle, low) to the extent possible on each day of sample collection to control for potential confounding influences of collection timing. Blood was placed in serum separator tubes and placed on ice. Serum was separated from cells and stored at ~ -20°C until analysis.
Serum T3 and T4 concentrations were assessed for all samples from adult males and PND 13 pups. Serum TSH concentrations were not assessed on contingency samples from adult males and PND 13 pups. Serum T3, T4, and TSH concentrations were not assessed on contingency samples from dams and PND 4 culled pups. Samples that were not analyzed for thyroid hormones will be discarded after the final report is issued. 
Culled PND 4 Pups:
Culled pups were anesthetized on PND 4 using a mixture of isoflurane vapors and medical grade oxygen, and a terminal blood sample was collected via cardiac puncture. To ensure a sufficient volume for hormone analyses, blood was pooled by litter if possible.
PND 13 pups:
Terminal blood was collected on at least one pup/sex/litter on PND 13 (if possible) and pooled by sex to obtain appropriate volumes. Blood samples were obtained from a cardiac puncture following anesthesia with a mixture of isoflurane vapors and medical oxygen at the scheduled necropsy.PND 13 pups were not fasted prior to termination.
Adult Animals:
Blood samples were obtained from the orbital sinus following anesthesia with a mixture of isoflurane vapors and medical oxygen at the scheduled necropsy. Terminal blood samples were collected from all fasted adult males and all females that delivered a litter. Blood was not obtained from females that failed to deliver a litter.
T3 and T4 Concentrations:
Serum T3 and T4 parameters were measured using a cobas e411 Immunoanalyzer (Roche Diagnostics, Indianapolis, Indiana).
Concentrations of:
Total Triiodothyronine (T3)
Total Thyroxine (T4)

Statistics:

Statistical analysis used:
Means and standard deviations
Bartlett's test
ANOVA
Dunnett's test
Wilcoxon Rank-Sum
Bonferroni's correction
Z-test of proportions
ANCOVA
The Pillai Trace statistic
Repeated-measure design
MANOVA or ANOVA
Grubbs (1969)
Fisher exact probability test
Binomial distribution test
Wilcoxon test

More detailed explanation of statistics can be found in the "Other Information" section.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

In-Life Observations:
No treatment-related effects on behavior or demeanor were observed at any dose level during the treatment period. Treatment-related clinical observations throughout the study consisted of red feces in all treated animals and red discolored fur in several 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.

Detailed Clinical Observations:
Detailed clinical observations performed on all rats revealed no findings.

Mortality:

no mortality observed

Description (incidence):

All animals survived the study duration.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related 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. Dams given 300 mg/kg/day had a statistically significant and higher body weight gain from GD 0-7 compared to control which was considered spurious and unrelated to treatment due to the lack of a dose response and the lack of consistency of this observation over the duration of the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no treatment-related differences in the amount of feed consumed by any of the dose groups when compared to their respective controls throughout the study. Dams given 300 and 1000 mg/kg/day had statistically-identified higher feed consumption values from GD 0-7 compared to control which were considered spurious and unrelated to treatment due to the lack of consistency of this observation over the duration of the study.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology:
There were no treatment-related hematologic effects in males or females at any dose level. Females given 300 mg/kg/day (mid-dose group) had a statistically-identified lower mean platelet count relative to controls, which was interpreted to be spurious and unrelated to treatment due to the lack of a dose response.

Coagulation:
Males given 1000 mg/kg/day had a treatment-related and statistically-identified increase in mean prothrombin time that was interpreted to be non-adverse. There were no treatment-related 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 given 1000 mg/kg/day had a treatment-related and statistically-identified increase in mean urea nitrogen concentration that was interpreted to be non-adverse because of the minimal difference from the mean urea nitrogen concentration of control group males and the lack of any corresponding histopathologic effects in the urinary system of males or females at any dose level. There were no treatment-related effects on clinical chemistry parameters of males given 100 or 300 mg/kg/day or of females from any dose level.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no treatment-related alterations in urinalysis parameters for males at any dose level.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males and females given 1000 mg/kg/day had treatment-related increased mean absolute and relative liver weights (11.1% and 11.9% increase for males, respectively, and 10.7% and 7.5% increase for females, respectively) (See Organ Weight Alterations attachment). The increased mean relative liver weights of males and females given 1000 mg/kg/day were statistically identified. The increased liver weights in males given 1000 mg/kg/day corresponded to the histopathologic observation of slight hypertrophy of periportal hepatocytes in all animals from this dose level. The increased liver weights in females given 1000 mg/kg/day corresponded to the histopathologic observations of very slight hypertrophy of periportal hepatocytes in 2/12 animals and very slight vacuolization (consistent with fatty change) of periportal hepatocytes in 10/12 animals. Females given 300 mg/kg/day had a statistically-identified mean relative liver weight (6.3% higher than controls) that was interpreted to be unrelated to treatment due to the lack of any histopathologic correlates. Females given 300 mg/kg/day had statistically-identified and higher mean absolute and relative heart weights that were interpreted to be unrelated to treatment due to the lack of a dose response. Females given 1000 mg/kg/day had a statistically-identified and higher absolute kidney weight that was interpreted to be unrelated to treatment due to the lack of any corresponding effects on clinical chemistry or histopathologic parameters.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross pathologic observations.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Functional Tests:
Sensory Evaluation:
Examinations performed on males and females revealed no treatment-related findings in the sensory evaluation. There were two observations in females from the 300 mg/kg/day group that were statistically-significant when compared to control (response to tail pinch minimal, p = 0.0120; response to tail pinch moderate, p = 0.0120); however, these occurred under baseline conditions and were not deemed related to treatment.

Rectal Temperature:
There were no treatment-related effects on rectal temperature either in males (p = 0.3127) or females (p = 0.9752).

Grip Performance:
There were no treatment-related effects on forelimb grip performance either in males (p = 0.7240) or females (p = 0.4110). For hindlimb grip performance in males, the pre-exposure-by-treatment interaction was significant following the ANCOVA analysis (p = 0.0312); therefore, a repeated measures ANOVA was conducted per protocol. There were no treatment-related effects on hindlimb grip performance in males (p = 0.9312) or females (p = 0.2444).

Motor Activity:
There were no treatment- related effects on motor activity in males or females at any dose level. Treatment did not affect total motor activity (treatment-by-time interaction) in males (p = 0.7747) or females (p = 0.7769). Similarly, the distribution of the motor activity counts within session (treatment-by-time-by-interval interaction) was not affected by treatment either in males (p = 0.0825) or in females (p = 0.3958).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The liver was the target organ for histopathologic effects in males given 300 or 1000 mg/kg/day, and in females given 1000 mg/kg/day (See Histopathologic Liver Alterations attachment). All males given 1000 mg/kg/day had treatment-related slight hypertrophy of periportal hepatocytes. Very slight hypertrophy of periportal hepatocytes in 4/12 males given 300 mg/kg/day and 2/12 females given 1000 mg/kg/day was also interpreted to be treatment related. The hepatocellular hypertrophy was interpreted to be a non-adverse and adaptive effect based on the modest corresponding increases in liver weights (up to an 11.6% increase). Males given 300 or 1000 mg/kg/day (8/12 animals at each dose level) had treatment-related slight necrosis, with or without inflammation, of individual hepatocytes. Treatment-related very slight necrosis, with or without inflammation, of individual hepatocytes was present in 3/12 females given 1000 mg/kg/day. The necrotic hepatocytes were randomly scattered throughout the liver lobules in all affected animals. Nuclear characteristics of the necrotic cells consisted of pyknosis, karyorrhexis or karyolysis. The cytoplasm of the necrotic cells was darker pink as compared to normal hepatocytes. Clusters of lymphocytes and macrophages accompanied some of the necrotic hepatocytes. The necrosis of individual hepatocytes was interpreted to be an adverse effect. Females given 1000 mg/kg/day had a treatment-related increase in the incidence of very slight vacuolization, consistent with fatty change, of periportal hepatocytes as compared to controls. The periportal hepatocellular vacuolaization was microvesicular in all affected animals. The very slight fatty change was interpreted to be a non-adverse effect. Two males given 1000 mg/kg/day had very slight or slight multifocal intracytoplasmic eosinophilic inclusion bodies in hepatocytes which were interpreted to be treatment related but non-adverse. The inclusion bodies were round to oval, homogeneously light pink to red, and were variable in size with the largest inclusions having similar diameters to unaffected hepatocytes.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Levels:
There were no treatment-related effects on T3 or T4 serum concentrations of P1 males. The mean T4 serum value in P1 males given 1000 mg/kg/day was 17% higher than the control group mean value and statistically identified. The higher T4 serum value in the P1 male 1000 mg/kg/day group was interpreted to be spurious and unrelated to treatment due to a lack of a thyroid histopathological correlate, no associated treatment-related differences in relative or absolute thyroid weights, and no treatment-related effect on P1 male T3 serum concentration.

There was no evidence of an effect on estrous cyclicity at any dose level of C.I. Solvent Red 175 Solid (solvent stripped).

Reproductive Indices, Pup Survival, and Sex Ratio:
There were no treatment-related effects at any dose level on any of the reproductive parameters or pup survival indices evaluated. Pup sex ratio on LD 1 in the high-dose group was statistically-identified as different from 50% by the binomial distribution test, with a lower than expected percentage of females observed. When masculinization of genetically female rodents occurs due to treatment, concurrent alterations in AGD and nipple/areolae retention would be expected within the affected dose group (Dean et al, 2012). The change in sex ratio in the high-dose group was considered spurious and unrelated to treatment since male and female AGD and nipple/areolae retention values were similar between the control and high-dose groups.

Litter Observations:
Observations recorded in the offspring occurred at low frequency and bore no relationship to treatment.

Litter Size and Pup Body Weights:
There were no treatment-related effects on litter size or pup body weights at any dose level tested.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: NOAEL for general toxicity.

2

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: NOAEL for general toxicity.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Observations recorded in the offspring occurred at low frequency and bore no relationship to treatment. There were no treatment-related effects on litter size or pup body weights at any dose level tested.

Anogenital Distance – F1 Offspring:

There were no significant, treatment-related differences in absolute or relative anogenital distance in either male or female offspring.

Nipple/Areolae Retention – F1 Offspring:

There were no significant, treatment-related differences in nipple/areolae retention in either male or female offspring.

Thyroid Hormone Levels:

There were no treatment-related effects on T3 or T4 serum concentrations of PND 13 males and females.  

Analytical Chemistry

Analysis of all dosing solutions from the first mix revealed mean acceptable concentrations of C.I. Solvent Red 175 Solid (solvent stripped) ranging from 102.7 to 106.4% of targeted concentrations.

Analysis of aliquots for the low- and high-dose solutions indicated that the test material was homogeneously distributed based on relative standard deviations of 3.4 and 1.4 %, respectively.

Applicant's summary and conclusion

Conclusions:

Treatment-related clinical observations consisted of red feces in all treated animals and red discolored fur in several treated animals which were attributed to the dye nature of the test material and considered a biomarker of exposure.
There were no treatment-related effects on body weight, body weight gain, feed consumption, neurological or reproductive function, or prenatal neonatal survival, growth, or development of the offspring in any treated groups compared to controls.
Males given 1000 mg/kg/day had a treatment-related increase in mean prothrombin time and mean urea nitrogen concentration that were interpreted to be non-adverse.
Males and females given 1000 mg/kg/day had treatment-related higher mean absolute and relative liver weights (11.1% and 11.9% higher for males, respectively, and 10.7% and 7.5% higher for females, respectively). No treatment-related change in liver weight was observed in males or females at 100 or 300 mg/kg/day.
Males given 1000 mg/kg/day had treatment-related slight hypertrophy of periportal hepatocytes and slight necrosis (with or without inflammation) of individual hepatocytes. Females given 1000 mg/kg/day had treatment-related very slight hypertrophy of periportal hepatocytes, very slight vacuolization (consistent with fatty change) of periportal hepatocytes, and very slight necrosis (with or without inflammation) of individual hepatocytes. Males given 300 mg/kg/day had treatment-related very slight hypertrophy of periportal hepatocytes and treatment-related slight necrosis, (with or without inflammation), of individual hepatocytes. No treatment-related liver histopathology was observed at 300 mg/kg/day in females or at 100 mg/kg/day in either sex.
Based on the presence of treatment-related very slight or slight necrosis of individual hepatocytes in males given 300 or 1000 mg/kg/day and in females given 1000 mg/kg/day, the no-observed adverse effect level (NOAEL) for general toxicity was 100 mg/kg/day for males and 300 mg/kg/day for females. The NOEL for neurological and reproductive toxicity or for effects on prenatal/neonatal growth, survival, and development was 1000 mg/kg/day, the highest dose tested.

Executive summary:

The purpose of this study was to evaluate the potential effects of C.I. Solvent Red 175 Solid (solvent stripped) on general toxicity, neurological and reproductive function, and prenatal/early neonatal growth and survival of offspring in rats following repeated gavage administration. Groups of 12 male and 12 female Crl:CD(SD) rats were administered C.I. Solvent Red 175 Solid (solvent stripped) daily by gavage at dose levels of 0 (control), 100, 300, or 1000 mg/kg/day. Males were dosed for at least four weeks prior to breeding and continuing throughout breeding for a minimum of 48 days. The females were dosed for four weeks prior to breeding, continuing through breeding (up to two weeks), gestation (three weeks), and lactation (thirteen days). Effects on general systemic toxicity, neurobehavioral activity, clinical chemistry, hematology, coagulation, thyroid hormone levels, urine parameters, gonadal function, estrous cyclicity, mating behavior, conception, development of the conceptus, parturition and early postnatal growth and survival of pups were evaluated. In addition, a gross necropsy of the adults was conducted with extensive histopathologic examination of tissues. Litter size, pup survival, sex, body weight, anogenital distance, nipple retention, and gross external morphological alterations were assessed.

Treatment-related clinical observations consisted of red feces in all treated animals and red discolored fur in several treated animals which were attributed to the dye nature of the test material and considered a biomarker of exposure.

There were no treatment-related effects on body weight, body weight gain, feed consumption, neurological or reproductive function, or prenatal neonatal survival, growth, or development of the offspring in any treated groups compared to controls.

Males given 1000 mg/kg/day had a treatment-related increase in mean prothrombin time and mean urea nitrogen concentration that were interpreted to be non-adverse. 

Males and females given 1000 mg/kg/day had treatment-related higher mean absolute and relative liver weights (11.1% and 11.9% higher for males, respectively, and 10.7% and 7.5% higher for females, respectively). No treatment-related change in liver weight was observed in males or females at 100 or 300 mg/kg/day. 

Males given 1000 mg/kg/day had treatment-related slight hypertrophy of periportal hepatocytes and slight necrosis (with or without inflammation) of individual hepatocytes. Females given 1000 mg/kg/day had treatment-related very slight hypertrophy of periportal hepatocytes, very slight vacuolization (consistent with fatty change) of periportal hepatocytes, and very slight necrosis (with or without inflammation) of individual hepatocytes. Males given 300 mg/kg/day had treatment-related very slight hypertrophy of periportal hepatocytes and treatment-related slight necrosis, (with or without inflammation), of individual hepatocytes. No treatment-related liver histopathology was observed at 300 mg/kg/day in females or at 100 mg/kg/day in either sex. 

Based on the presence of treatment-related very slight or slight necrosis of individual hepatocytes in males given 300 or 1000 mg/kg/day and in females given 1000 mg/kg/day, the no-observed adverse effect level (NOAEL) for general toxicity was 100 mg/kg/day for males and 300 mg/kg/day for females. The NOEL for neurological and reproductive toxicity or for effects on prenatal/neonatal growth, survival, and development was 1000 mg/kg/day, the highest dose tested.