Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 May 2014 - 20 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to test guidelines and in accordance with GLP
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: USEPA OPPTS 870.3650 (2000)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1-(Decyloxy)-2-(1-methylpropyl)-4-(triphenylmethyl) benzene
- Physical State: Brown viscous liquid to semi solid
- Analytical Purity: 92.6%
- Lot/Batch No.: lot ZA-03012014M

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Physical and Acclimation
During the acclimation period, each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a laboratory veterinarian, 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). The animals were housed two-three per cage in stainless steel solid bottom cages with corncob bedding, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), prior to randomization. Animals were acclimated to the laboratory for at least one week prior to the start of the study.
Housing
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 nesting material with heat-treated laboratory grade wood shavings for enrichment purposes. During breeding, one male and one female were placed in stainless steel cages with wire mesh floors that were suspended above catch pans in order to better visualize copulation and plugs. During gestation and littering {approximately gestation day (GD) 0 until LD 5}, dams (and their litters) were housed in plastic cages containing ground corn cob nesting material with heat-treated laboratory grade wood shavings. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
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 rats included the use of ground corn cob and shredded Aspen bedding (Harlan Laboratories, Inc., Indianapolis, Indiana) and open areas on the cage sides for visualization of other 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 that were placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Animal Welfare
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities are in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities used for this study were DART 01, Neurotox 01, DCO 01, Humane Endpoints 01, Blood Collection 01, Tissue Collection 01 and Animal ID 01.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
acetone
Details on oral exposure:
ACCUTRACE™ S10 Fuel Marker was initially dissolved in acetone, and this stock solution was then added to control rodent chow to prepare a concentrated test material-feed mixture (premix). The premix was left overnight in a vented area to volatilize the acetone before mixing the test diets. The high-dose diet was prepared by diluting the premix with ground feed, and lower dose diets were prepared by serial dilution of the high-dose diet with ground feed. Premixes were prepared periodically throughout the study based on stability data. Test diets were prepared as a fixed percent of test material in rodent feed. Control animals received control feed prepared using an equivalent amount of acetone representative of the amount of acetone in the premix that was used in the high-dose diet. As with the premix, control feed was left overnight in a vented area to volatilize the acetone before feeding to the animals. The concentrations of the test diets were not adjusted for purity.

Feed and Water
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were provided ad libitum. 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.

Control
Control animals received control feed prepared using an equivalent amount of acetone representative of the amount of acetone in the premix that was used in
the high-dose diet. As with the premix, control feed was left overnight in a vented area to volatilize the acetone before feeding to the animals. The concentrations of the test diets were not adjusted for purity.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration Verification and Homogeneity
Analyses of all test diets from the first mix of the main study were initiated prior to the start of dosing using high performance liquid chromatography (HPLC) with fluorescence detection (FLD) to determine target concentrations. Representative samples form the test diets were evaluated to ensure homogeneous distribution of the test material at the lowest and highest concentrations in the feed at least once during the study.

Analyses of all test diets from the first mix of the study revealed mean concentrations ranging from 97.9 to 98.5% of targeted concentrations. Analyses of the low- and high-dose diets indicated that the test material was homogeneously distributed based on relative standard deviations of ≤ 1.8%
Duration of treatment / exposure:
Males were fed test diets for at least two weeks prior to breeding and continuing throughout breeding for 33 days. Females were fed test diets for two weeks prior to breeding, and continuing through breeding (two weeks), gestation (three weeks), and lactation (four days).
Frequency of treatment:
Continuously via food
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 1500, 4500, or 13500 ppm ACCUTRACE™ S10 Fuel Marker
Basis:
nominal in diet
No. of animals per sex per dose:
Groups of 12 male and 12 female
Control animals:
yes
Details on study design:
The high-dose of 13500 ppm was selected based upon results from the previously discussed preliminary range-finding study. No toxicity was observed in the range-finding study at dietary concentrations up to and including 12000 ppm. The high-dose of 13500 ppm targeted a dose level of 1000 mg/kg/day, which represents a limit dose as defined in the Health Effects Test Guideline of the United States Environmental Protection Agency (OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test). The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high-dose group rats and to establish al NOEL.

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 that were placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Positive control:
no data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE 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 on dams and their litters at least twice daily.
These examinations were conducted as described above.

DETAILED CLINICAL OBSERVATIONS:
Clinical observations were conducted on all animals pre-exposure and on LD 0, 1, and 4
on females that delivered litters. Females were observed for signs of parturition
beginning on or about GD 20 (see litter data). 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 (DCO) were conducted on all rats pre-exposure and weekly
throughout the study. Mated females received DCO examinations on GD 0, 7, 14, and
20, and LD 3. The DCO was conducted at approximately the same time each
examination day, according to an established format. The examination included cageside,
hand-held and open-field observations, which were recorded categorically or using
explicitly defined scales (ranks).

BODY WEIGHT:
All rats were weighed pre-exposure and weekly during the pre-breeding phase. Male
body weights continued to be recorded weekly throughout the study. Presumed pregnant
females were weighed on GD 0, 7, 14, and 20. Females that delivered litters were
weighed on LD 1 and 4. Females that failed to mate or deliver a litter were not weighed
during the gestation or lactation phases.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
For males and females, feed consumed was determined weekly during the pre-breeding
phase by weighing feed crocks 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 was not measured for males. For mated females,
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)


Test material intake (TMI) was calculated for the adults using test material concentrations in the feed, actual body weights, and measured feed consumption using the following equation:

TMI=(Feed consumption [g/day])*(1000 mg/g)*(% of test material in feed/100) / [((Currrent BW [g] + Previous BW [g])/2)/1000 g/kg]


HAEMATOLOGY:
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).

CLINICAL CHEMISTRY:
Animals were fasted overnight prior to blood collection. Blood samples were obtained from the orbital sinus following anesthesia via the inhalation of a mixture of isoflurane vapors and medical oxygen at the scheduled necropsy.

URINALYSIS:
Urine samples were obtained from all males the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water was available during this procedure.
Assays
Color, appearance, specific gravity (refractometer)
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 Exam:
Urine samples were collected from each male by manual compression of the urinary bladder. The urine samples were pooled from each group, and the microsediment were characterized microscopically.



NEUROBEHAVIOURAL EXAMINATION:
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.
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 places 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
An automated system was used for motor activity (MA) data collection (Appendix D). No entry into the MA test room was allowed during the testing period. Each test session consisted of eight 8-minute epochs, totaling 64 minutes of testing per animal per test session. This duration was chosen based on the results of a validation study indicating that performance of control animals approached asymptote in 50-60 minutes in Crl:CD(SD) rats (Marty and Andrus, 2007). Activity counts for each epoch were recorded.
Motor Activity Cage Calibration: Cages to be used for testing were calibrated prior to testing each day. Calibration was performed with a rod attached to a rotary motor that broke the infrared beam at a constant speed. The duration of each beam break was calculated to ensure equivalence across chambers.
Motor Activity Cage Allocation: Rats were allocated to the motor activity cages in such a way that the counterbalancing of treatment groups and sexes across cages and test times was maximized.

Sacrifice and pathology:
Adult Necropsy
Adult males (fasted) were submitted for necropsy on TD 34. Adult females (fasted) were terminated on LD 5. The animals were anesthetized with a mixture of isoflurane vapors and medical grade oxygen. While under anesthesia, blood was collected from the orbital sinus. The animals were then 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, 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 uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain based on Kopf et al. (1964) and were examined for the presence and number of implantation sites. After evaluation, uteri was 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 PND 4 were euthanized by intraperitoneal administration of sodium pentobarbital solution, examined for gross external alterations, and then discarded. Any pups found dead or which were euthanized in moribund condition were examined to the extent possible and discarded.
Histopathology
Histopathologic examination of the tissues indicated in Table 3 was conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to those tissues that demonstrated treatment-related histologic effects at the high dose (13500 ppm) 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 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 be affected, depending on the organ/tissue involved, but generally lesions graded as moderate would not be life threatening.
Statistics:
Descriptive statistics only (means and standard deviations) were reported for globulin, albumin/globulin ratio, RBC indices, and WBC differential counts. Parental body weights and parental body weight gains, litter mean body weights, feed consumption, urine volume, urine specific gravity, coagulation, clinical chemistry data, appropriate hematologic data, and organ weights (absolute and relative) were first evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, a Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966) was performed. Feed consumption values were excluded from analysis if the feed was spilled or scratched.
Gestation length, average time to mating, and litter size were analyzed using a non-parametric ANOVA. If the ANOVA was significant, the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969) and only excluded from analysis for documented, scientifically sound reasons. The mating, conception, fertility and gestation indices were analyzed by the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni's correction. Evaluation of the neonatal sex ratio on postnatal day 1 was performed by the binomial distribution test (alpha = 0.05; Steel and Torrie, 1960).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Functional Tests

Sensory Evaluation
There were no treatment-related effects in the sensory evaluation of males or females at any dose level tested.
There were no treatment-related effects on rectal temperature. The main effect of treatment was not significant for males (p = 0.1776) or females (p = 0.7926), which indicated that treatment did not affect rectal temperature in rats.
Grip Performance
There were no treatment-related effects on grip performance. The main effect of treatment for hindlimb grip performance was not significant for males (p = 0.1478) or females (p = 0.3058), which indicated that treatment did not affect hindlimb grip performance in rats. Similarly, the main effect of treatment for forelimb grip performance was not significant for males (p = 0.6666) or females (p = 0.4156), which indicated that treatment did not affect forelimb grip performance in rats.
Motor Activity
There were no treatment-related effects on motor activity. The treatment by time interaction was not significant (males: p = 0.1735; females: p = 0.7989), which indicated that there were no treatment-related effects on motor activity at either time interval. The treatment by time by epoch interaction was also not significant (males: p = 0.6454; females: p = 0.8575), which indicated that treatment did not affect the within session distribution of motor activity counts at either time interval.

Body Weights/Body Weight Gains
There were no treatment-related effects on male body weight at any dose level tested throughout the duration of the study. Similarly, there were no treatment-related effects on female body weight or body weight gain at any dose level tested during the pre-breeding, gestation, or lactation study phases.

Feed Consumption
There were no treatment-related effects on male or female feed consumption at any dose level tested throughout the duration of the study.
Test Material Intake
Test material intake (TMI) data for male and female rats are summarized in Tables 37-38.
For the 0, 1500, 4500, and 13500 ppm groups, respective time-weighted average dose levels of ACCUTRACE™ S10 Fuel Marker during the pre-breeding phase were 0, 118, 352, or 1101 mg/kg/day for males and 0, 115, 359, or 1006 mg/kg/day for females. During the gestation and lactation phases, respective time-weighted average dose levels of ACCUTRACE™ S10 Fuel Marker for females were 0, 107, 327, or 1018 mg/kg/day and 0, 164, 486, or 1470 mg/kg/day.

Clinical Pathology
Hematology
There were no treatment-related hematologic effects in males or females at any dose level tested.

Coagulation
There were no treatment-related differences in the prothrombin times of males and females at any dose level when compared to controls.

Clinical Chemistry
Males from the 13500 ppm dose group had a treatment-related increase in mean serum cholesterol levels that was statistically identified and higher than the historical control range. This very slight increase was interpreted to be non-adverse as it was just above the historical control values from five recent reproductive/developmental studies conducted within this laboratory and occurred in isolation from associated clinical pathology or histopathology alterations.
Males in the 13500 ppm dose group had lower, statistically-identified mean sodium levels compared to the concurrent control group. This observation was of a minimal degree (>1% below the control value), did not demonstrate a dose response, occurred in isolation from associated clinical pathology or histopathology alterations, and was not considered biologically or toxicologically relevant. Therefore, the lower sodium value in the 13500 ppm group was deemed spurious and unrelated to treatment.
Text Table 5. Statistically-Identified Clinical Chemistry Values
0 ppm 1500 ppm 4500 ppm 13500 ppm
Cholesterol (mg/dL) 46 40 48 57*
Sodium (mmol/L) 144 144 144 143*
*Statistically-identified as different from control mean by Dunnett’s test, alpha=0.05.
Bold type indicates a treatment-related alteration.


Urinalysis
There were no treatment-related changes for males at any dose level tested.

Anatomic Pathology
Organ Weights
There were no treatment-related effects on organ weights at any dose level tested.

Gross Pathology
There were no treatment-related gross pathologic observations at any dose level tested. All gross pathologic observations were considered to be spontaneous alterations, unassociated with exposure to rats.

Histopathology
There were no treatment-related histopathologic observations at any dose level tested. All histopathologic observations were interpreted to be spontaneous alterations, unassociated with exposure to rats.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
13 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
1 101 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Average based on feed consumption
Dose descriptor:
NOEL
Effect level:
>= 1 006 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Average based on feed consumption

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Dietary administration of ACCUTRACE S10 Fuel Marker to Crl:CD(SD) rats resulted in no adverse treatment-related effects in any treated group on any parameter examined in males, females, or offspring. Compared to the control group, no treatment-related effects were observed in clinical observations, body weight, body weight gain, feed consumption, reproductive function, neurological function, prenatal/early neonatal growth and survival of the offspring, organ weights, gross pathology or histopathology in either sex at all dose levels tested.
A treatment-related increase in male serum cholesterol levels was observed in the 13500 ppm dose group (57 mg/dL) compared to the control group (46 mg/dL). This very slight increase was interpreted to be non-adverse as it was just above historical control values and occurred in isolation from associated clinical pathology or histopathology alterations. Based on these results, the no-observed-adverse-effect level (NOAEL) for general toxicity was 13500 ppm, the highest concentration tested.
Executive summary:

The purpose of this study was to evaluate the potential effects of ACCUTRACETM S10 Fuel Marking System following rat dietary administration on general toxicity, neurological and reproductive function, and prenatal/early neonatal growth and offspring survival. Groups of 12 male and 12 female Crl:CD(SD) rats were administered ACCUTRACE™ S10 Fuel Marking System via the diet at concentrations of 0 (control), 1500, 4500, or 13500 ppm. These dietary concentrations corresponded to time-weighted average dose levels of 0, 118, 352, or 1101 mg/kg/day for males during the pre-breeding study phase and 0, 107-164, 327-486, or
1006-1470 mg/kg/day for females during the pre-breeding, gestation, and lactation study phases. Males were fed the test diet for two weeks prior to breeding and continuing through breeding (two weeks) up until necropsy on test day (TD) 34. Females were fed the test diet for two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and through post-partum day (lactation day, LD) 4. Females were necropsied on LD 5. 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 extensive histopathologic examination of tissues. Litter size, pup survival, pup body weight, sex, and the presence of gross external abnormalities were also assessed.

Dietary administration of ACCUTRACE™ S10 Fuel Marking System to Crl:CD(SD) rats resulted in no adverse treatment-related effects in any treated group on any parameter examined in males, females, or offspring. Compared to the control group, no treatment-related effects were observed in clinical observations, body weight, body weight gain, feed consumption, reproductive function, neurological function, prenatal/early neonatal growth and survival of the offspring, organ weights, gross pathology, or histopathology in either sex at all dose levels tested.

A treatment-related increase in male serum cholesterol levels was observed in the 13500 ppm dose group (57 mg/dL) compared to the control group (46 mg/dL). This very slight increase was interpreted to be non-adverse as it was just above historical control values and occurred in isolation from associated clinical pathology or histopathology alterations.

Based on these results, the no-observed-adverse-effect level (NOAEL) for general toxicity was 13500 ppm, the highest concentration tested. The no-observed-effect level (NOEL) for neurological and reproductive toxicity and for effects on prenatal/neonatal growth and survival was 13500 ppm, the highest concentration tested.