Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
OECD 422
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:
guideline study
Reason / purpose:
reference to same study
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
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.
Text Table 2. Animal Identification Numbers
Dose Level
(ppm) Males Females
0 14A1468-14A1479 14A1516-14A1527
1500 14A1480-14A1491 14A1528-14A1539
4500 14A1492-14A1503 14A1540-14A1551
13500 14A1504-14A1515 14A1552-14A1563

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.


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.


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 on dams and their litters at least twice daily.
These examinations were conducted as described above.
Route of administration:
oral: feed
Vehicle:
acetone
Details on 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.
Details on mating 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 were 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.
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
Details on study schedule:
Dietary administration for both males and females began on May 28, 2014. The adult males were necropsied on July 03, 2014 (TD 34). The adult females were necropsied on July 08, 2014 to July 20, 2014 (LD 5). In the offspring, litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were assessed. Pups were euthanized on postnatal day 4 (i.e., LD 4).
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:
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.
Positive control:
no data
Parental animals: Observations and examinations:
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 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 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
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 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 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.


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]




Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
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).

There were no treatment-related effects at any dose level in any reproductive parameter.
Litter observations:
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 the 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
Postmortem examinations (parental animals):
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 recordedand organ:body weight ratios calculated.
Postmortem examinations (offspring):
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.
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).
Reproductive indices:
Reproductive indices were calculated for all dose level groups as follows:
Female mating index = (No. females with evidence of mating/No. paired) x 100
Male mating index = (No. males with evidence of mating/No. paired) x 100
Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
Male conception index = (No. males siring a litter/No. mated) x 100
Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
Male fertility index = (No. males siring a litter/No. paired) x 100
Gestation index = (No. females delivering a viable litter/No. females delivering a litter) x 100
Gestation survival index = percentage of delivered pups alive at birth
Postimplantation loss = (No. implants – No. viable offspring)/(No. implants) x 100
Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100

There were no treatment-related effects at any dose level in any reproductive parameter or pup survival index. Percent postimplantation loss (percentage of live born pups relative to total uterine implantation sites) was higher in the 1500 ppm group (6.8%) and the 13500 ppm group (8.8%) relative to the control group (4.9%). The higher percent postimplantation loss in both treated groups was considered spurious and unrelated to treatment as these differences were not statistically identified, were not dose related, and were within the laboratory’s historical control range.
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
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
Key result
Dose descriptor:
NOAEL
Effect level:
13 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
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.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects were observed
Key result
Reproductive effects observed:
no
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. 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.
Executive summary:

The purpose of this study was to evaluate the potential effects of ACCUTRACEÔS10 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.


Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
good
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

ACCUTRACE™ S10 Fuel Marker was tested following OECD Guideline No. 422 and USEPA OPPTS 870.3650. Based on these results, the no-observed-adverse-effect level (NOAEL) for generaltoxicity 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.

Similar material XU-18838.00 was tested following the OECD guideline 440 test and USEPA OPPTS 890.1600 guidelines. Based on these uterotrophic assay results, there was no indication of estrogenicity in immature female rats at doses <1000 mg/kg/day XU-18844.00 or XU-18838.00 when compared to the vehicle controls.

Short description of key information:
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. 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.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Accutrace S10 failed to produce any evidence of toxicity in an OECD 422 combined repeated dose/reproductive toxicity study up to the highest dose tested. No evidecne of reproductive toxicity was observed and hence the GHS classificatoion criteria for reproductive toxicity are not met.