Registration Dossier
Registration Dossier
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EC number: 940-734-7 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
A two-generation reproductive toxicity study (OECD 416) was conducted with GTL Gasoil (C8-C26, branched and linear). In this oral gavage study (RTI, 2011a), there were no effects on any reproductive parameters up to the highest dose tested, 750 mg/kg bw/day. In a similar study with GTL Base Oil Distillates (C18-C50, branched, cyclic and linear), there were no effects up to the highest dose tested, 1000 mg/kg bw/day (RTI, 2011b).
Link to relevant study records
- Endpoint:
- two-generation reproductive toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA Health Effects Test Guideline OPPTS 870.3800 and OECD Guidelines for the Testing of Chemicals, No, 416 "Two-Generation Reproduction Toxicity Study".
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: F0 animals were approximately 6 to 7 weeks (42 to 46 days) of age at the initiation of test item administration.
- Weight at study initiation: 142.4 to 190.6 g; female group mean range 137.4 to 165.1 g.
- Fasting period before study: Not applicable.
- Housing: During the acclimation period (F0 animals only) and throughout the study, all F0 and F1 parental animals were housed individually, except during the mating period, in solid-bottom polycarbonate cages with stainless steel wire lids. The rats were paired for mating in the home cage of the male. Following positive evidence of mating (or at the completion of the 2-week mating period), the females were transferred to individual solid-bottom polycarbonate cages with stainless steel wire lids. Beginning on pre-natal Day 21, the selected F1 pups were individually housed until the start of the mating period.
- Diet: Purina Certified Pelleted Rodent Diet® (No. 5002, PMI Feeds, Inc., St. Louis, MO) was available ad libitum in the wire cage lids. Available information on the diet did not indicate the presence of any substance at a concentration likely to have influenced the outcome of the study.
- Water (tap water) was available ad libitum via plastic water bottles with butyl rubber stoppers and stainless steel sipper tubes. Available information on the water did not indicate the presence of any substance at a concentration likely to have influenced the outcome of the study.
- Acclimation period: Seven days
ENVIRONMENTAL CONDITIONS
- Temperature: (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): At least ten air changes per hour
- Photoperiod (hr dark / hrs light):12 hours continuous light and 12 hours darkness
IN-LIFE DATES: First day of treatment: 13 October 2009 - Final necropsy day was 07 July 2010. - Route of administration:
- oral: gavage
- Type of inhalation exposure (if applicable):
- not specified
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared as solutions at concentrations of 0 (vehicle), 25, 100, and 375 mg/mL were formulated 10 times (approximately every 4 weeks), dispensed into 30-mL amber glass bottles with Teflon-lined lids for use as daily dose aliquots, and stored at room temperature protected from light. Concentrations of 25 and 375 mg/mL in the same vehicle have been shown to be homogeneous and stable for 42 days when stored refrigerated or at room temperature protected from light (Analytical Chemistry report. Report date: 10 March 2010).
The test item formulations (but not the vehicle formulation) were stirred continuously throughout dose administration.
Prior to use on study, samples (~25 mL each) for homogeneity determination were collected from the top, middle, and bottom strata of each test item formulation from 3 of the 10 formulation dates (06 October 2009, 26 January 2010, and 15 June 2010). Samples (~25 mL each) for concentration analysis were also collected from the middle stratum of each dosing formulation (including the vehicle formulation) from these same 3 formulation dates. All analyses were conducted using a validated gas chromatography method with flame ionization detection (AM-0212131-002.0). Details about the methodology and results of these analyses are presented in Appendix 1, and summarized as follows:
Based on these results, the analyzed dosing formulations were found to contain the amount of test item prescribed in the protocol (±10% of nominal) and were homogeneous. Therefore, the protocol-specified dosages of test item were administered to the animals.
-DIET PREPARATION
Not applicable
- Rate of preparation of diet (frequency):
Not applicable
- Mixing appropriate amounts with (Type of food):
Not applicable
- Storage temperature of food:
No data
- VEHICLE
Corn oil
- Justification for use and choice of vehicle (if other than water):
Not applicable
- Concentration in vehicle:
0, 25, 100, and 375 mg/mL
- Amount of vehicle (if gavage):
2 ml/kg
- Lot/batch no. (if required):
Not applicable - Details on mating procedure:
- - M/F ratio per cage:
1/1 (Animals were paired on a 1 male: 1 female basis within each dose group).
- Length of cohabitation:
Following positive evidence of mating (or at the completion of the 2-week mating period).
- Proof of pregnancy:
Vaginal smears were performed daily on each F0 and F1 female for 21 days prior to pairing and continuing from the day after pairing until evidence of mating was observed or until the end of the mating period. The slides from the 21 days prior to mating were evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female according to test facility SOPs. Vaginal smears were also performed on the day of necropsy to determine the stage of estrus at demise.
- After a set number of days of unsuccessful pairing replacement of first male by another male with proven fertility.:
Not applicable
- Further matings after two unsuccessful attempts:
Not applicable
- After mating each pregnant female was returned to individual caging:
Mated females were housed individually during the period of gestation and lactation.
- Any other deviations from standard protocol:
Not applicable - Details on analytical verification of doses or concentrations:
- SUMMARY
The analytical method has been satisfactorily validated and details about the methodology and results of these analyses are presented in detail.
The results are summarized as follows.
Three sets of dose formulation samples were analyzed for concentration verification and homogeneity assessment for the test item in corn oil formulations had previously been evaluated for homogeneity , in long-term storage, and animal room dosing stabilities in the concentration range from 25 to 375 mg/mL and the detailed analytical procedures were reported in an Analytical Chemistry Report, Report Date 10 March 2010.
The formulation analyses reported here were performed following an validated analytical method to verify the test item concentration and homogeneity in corn oil formulations prepared on October 6, 2009 and January 26 and June 15, 2010. All dose formulations analyzed were found to be within ±10% of the nominal concentration and were homogeneous.
ANALYTICAL METHOD
The validated analytical method used to analyze study samples is outlined as follows:
For each dose formulation sample, triplicate 1 mL aliquots were in 10-mL volumetric flasks and diluted to volume with n-hexane. Processed formulation samples were then analysed on a gas chromatograph with a flame ionization detector along with a series of vehicle standards used to generate a calibration curve. Vehicle standards were prepared by mixing blank formulations samples. Test item concentrations were calculated using a least square s linear regression equation that fit the relationship between the nominal concentration s of vehicle standard and the detector response (ie the sum of integrated areas of selected peaks within the chromatogram). The formulation sample concentrations were determined in mg/g and converted to mg/mL measuring the formulation densities. - Duration of treatment / exposure:
- Duration of treatment / exposure
All male and female for the F0 generation and F1 generation were administered the test item or vehicle control, daily for at least 70 consecutive days prior to mating.
The test item was administered to offspring selected to become the F1 parental generation following weaning, beginning on postnatal day (PND) 22.
The F0 and F1 males continued to receive the test item throughout mating and through the day prior to euthanasia. The F0 and F1 females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia.
A complete gross necropsy was conducted after death for any parental (F0 and F1) or offspring (F1 and F2) animals found in moribund condition or dead on study.
Organs were not weighed for moribund animals.
Histopathological examination of study specified organs were performed for all high dose and control parental (F0 and F1). Additionally, reproductive organs of the low and mid dose animals suspected of reduced fertility were subjected to histopathological evaluation.
For any organ(s) demonstrating possible treatment-related changes in the high dose group versus the organ(s) of interest were evaluated histopathologically from successively lower concentration dose groups until a clear no observed adverse effect level (NOAEL) was established. However, at the request of the Sponsor, no further evaluation of the lungs from lower dose groups was conducted. - Frequency of treatment:
- The test and vehicle control item formulations were administered to the F0 and F1 males and females and once daily for a minimum of 70 consecutive days prior to mating and for males continued throughout mating to the day prior to termination.
The F0 and F1 females continued to be dosed throughout mating, gestation, and lactation, through to the day prior to termination.
The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) were potentially exposed to the test article in utero and through nursing (Pre Natal Day 0 to 21).
The F1 pups selected as parents for the F2 generation (25/sex/group) were administered the test item following weaning (beginning on Pre Natal Day 22). - Details on study schedule:
- Three groups of male and female Crl:CD(SD) rats (25 and 28/sex/group for the F0 and F1 generations, respectively) were administered the test item (CAS No. 848301-67-7), daily by oral gavage for at least 70 consecutive days prior to mating. A concurrent control group of 25 and 28/sex for the F0 and F1 generations, respectively, received the vehicle, corn oil (CAS No. 8001-30-7) on a comparable regimen. The F0 and F1 females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia.
The test item was administered to offspring selected to become the F1 parental generation following weaning, beginning on postnatal day (PND) 22. The F0 and F1 males continued to receive the test item throughout mating and through the day prior to euthanasia.
The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) were potentially exposed to the test article in utero and through nursing (Pre Natal Day 0 to 21). The F1 pups selected as parents for the F2 generation (25/sex/group) were administered the test item following weaning (beginning on Pre Natal Day 22).
The first day of dosing was designated as Day 1 of the study.
Animals were paired on a 1 male: 1 female basis within each dose group.
The rats were paired for mating in the home cage of the male.
Following positive evidence of mating (or at the completion of the 2-week mating period), the females were transferred to individual solid-bottom polycarbonate cages with stainless steel wire lids. - Remarks:
- Doses / Concentrations:
50 mg/kg/day (25 mg/ml)
Basis:
actual ingested - Remarks:
- Doses / Concentrations:
200 mg/kg/day (100 mg/ml)
Basis:
actual ingested - Remarks:
- Doses / Concentrations:
750 mg/kg/day (375 mg/ml)
Basis:
actual ingested - No. of animals per sex per dose:
- F0 generation
0 mg/kg/day (Control) - 25 per sex
50 mg/kg/day - 25 per sex
200 mg/kg/day - 25 per sex
750 mg/kg/day - 25 per sex
F1 generation
0 mg/kg/day (Control) - 28 per sex
50 mg/kg/day - 28 per sex
200 mg/kg/day - 28 per sex
750 mg/kg/day - 28 per sex - Details on study design:
- - Dose selection rationale:
Dosage levels were selected based on repeat-dose studies on similar substances and a 90-day repeat-dose study on 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' and 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear'. In addition to repeat-dose studies on conventional petroleum-based diesel, a number of repeat-dose studies on individual components of the test substance were available. These studies do not give any indication of unexpected effects and are in line with the observations in 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' and 'Naphtha (Fischer-Tropsch), light, C4-10 - branched and linear' studies. The selected route of administration was oral (gavage) by the Sponsor because this is a possible route of human exposure. The number of animals selected for this study was the minimum required to yield statistically and scientifically meaningful data and was consistent with regulatory agency expectations.
- Rationale for animal assignment (if not random):
Random
- Rationale for selecting satellite groups:
Not applicable
- Post-exposure recovery period in satellite groups:
Not applicable
- Section schedule rationale (if not random):
Random - Positive control:
- Not applicable
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS:
- See attached Summary Tables 6, 14, 20, 25, 41, 49, 55, 60 and Individual Data: Appendix 2, Tables 4, 10, 17, 35, 41, 45 and 48
- Mortality
Observations were made twice daily at least 6 hours apart.
- Clinical examinations
- Time schedule:
Clinical observations were conducted and recorded at least once daily, approximately 1-2 hours post dosing, throughout the course of the study.
BODY WEIGHT:
- See attached Summary Tables 2, 3, 10, 11, 16, 17, 21, 22, 37, 38, 45, 46, 51, 52, 56 and 57 and Appendix 2, Tables 2, 8, 15, 31, 32, 33, 39, 43 and 46.
- Time schedule for examinations:
Individual body weights of the male F0 and F1 rats were determined and recorded on the first day of dosing and weekly through the pre breed exposure and mating periods.
The body weights of male F0 and F1 rats were recorded in the same manner after the mating period until scheduled euthanasia.
The body weights of female F0 and F1 rats were recorded in the same manner until evidence of mating was observed.
During gestation, sperm-positive (presumed pregnant) females were weighed on gestational days (GD) 0, 7, 14, and 20.
Dams producing litters were weighed on lactational days 0, 4, 7, 14, and 21.
FOOD CONSUMPTION AND EFFICIENCY:
- See attached Summary Tables 4, 5, 12, 13, 18, 19, 23, 24, 39, 40, 47, 48, 53, 54, 58 and 59 and Individual Data: Appendix 2, Tables 3, 9, 16, 34, 40, 44and 47.
Individual F0 and F1 male and female feed consumption was measured weekly until mating.
Feed consumption was not recorded during the 2-week mating period.
Following mating, male feed consumption was measured on a weekly basis until the scheduled necropsy.
During pregnancy of F0 and F1 females, feed consumption was recorded on gestation days 0, 7, 14, and 20.
During lactation of F1 and F2 litters, maternal feed consumption was measured on 0, 4, 7, 14, and 21.
- Food efficiency:
Percent food efficiency (body weight gained as a percentage of food consumed) was also calculated
WATER CONSUMPTION:
Not applicable
OPHTHALMOSCOPIC EXAMINATION:
Not applicable
HAEMATOLOGY AND CLINICAL CHEMISTRY:
Not applicable
- Time schedule for collection of blood:
Not applicable
- Anaesthetic used for blood collection:
Not applicable.
- Animals fasted:
Not applicable.
- How many animals:
Not applicable.
URINALYSIS:
Not applicable
NEUROBEHAVIOURAL EXAMINATION:
Not applicable.
- Time schedule for examinations:
Not applicable.
Behavioural assessment)
Not applicable.
(Functional Performance Tests)
Not applicable.
(Sensory Reactivity)
Not applicable.
- Dose groups that were examined:
Not applicable.
OTHER:
- MATING
Animals were paired on a 1 male: 1 female basis
Vaginal smears were performed daily on each F0 and F1 female for 21 days prior to pairing and continuing from the day after pairing until evidence of mating was observed or until the end of the mating period. The slides from the 21 days prior to mating were evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female according to test facility SOPs. Vaginal smears were also performed on the day of necropsy to determine the stage of estrus at demise.
- see attached Summary Tables 15, 32 and 50 and Individual Data: Appendix 2, Tables 11, 28, 42 and 59
PREGNANCY AND PARTURITION
During the period of expected parturition, the females were observed at least twice daily for initiation and completion of parturition. On the day parturition was completed (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded.
Individual gestation length was calculated using the date delivery completed. - Oestrous cyclicity (parental animals):
- A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.
- Sperm parameters (parental animals):
- Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0 and F1 males as appropriate.
- Litter observations:
- Clinical observations, body weights, and sexes for F1 and F2 pups were recorded at appropriate intervals. For both generations (F1 and F2), 10 pups per litter (5 per sex, when possible) were arbitrarily selected on PND 4 to reduce the variability among the litters. Offspring (28/sex/group) from the pairing of the F0 animals were selected on PND 21 to constitute the F1 generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats.
- Postmortem examinations (parental animals):
- Each F0 and F1 parental animal received a complete detailed gross necropsy (the surviving female parental animals were necropsied on LD 21), and selected organs were weighed. Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0 and F1 males as appropriate, and ovarian primordial follicle counts and the corpora lutea counts were recorded for all F1 females in the control and high-dose groups. Designated tissues from the F0 and F1 parental animals were examined microscopically.
- Postmortem examinations (offspring):
- Non selected F1 pups were necropsied on PND 21, and F2 pups were necropsied on PND 21. Selected organs (brain, spleen, and thymus) were weighed from 1 pup/sex/litter (when possible) from both F1 and F2 pups that were necropsied on PND 21.
- Statistics:
- STATISTICAL METHODS
See Attachment “"Report Text" pages 34 to 35. - Reproductive indices:
- For the Mating, fertility, and gestational indices see attached ""Report Text" page 25.
- Offspring viability indices:
- For the Offspring viability indices see attached "Report Text" page 26.
- Clinical signs:
- effects observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- > 750 mg/kg bw/day
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects observed up to the highest concentration tested
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Sexual maturation:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- > 750 mg/kg bw/day
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects observed up to the highest concentration tested
- Dose descriptor:
- NOAEL
- Generation:
- F2
- Effect level:
- > 750 mg/kg bw/day
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects observed up to the highest concentration tested
- Reproductive effects observed:
- not specified
- Conclusions:
- SUMMARY OF RESULTS
Administration of the test substance to male and female rats at dosages up to 750 mg/kg/day had no effect on reproductive performance or gestation length and parturition of both the F0 and F1 parental generations. In addition, there were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition/mortality, anogenital distance, and pup body weights. Vaginal patency of F1 females was unaffected by test item administration. There was a statistically significant, test article-related increase in the mean age and adjusted age of attainment of balanopreputial separation in F1 males given 750 mg/kg/day. However, this change was not considered adverse, as the age and adjusted age of attainment fell within the historical control data range for this parameter. Reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day, although slightly lower than the control group, were not statistically significantly different from the control group and also fell within the historical control data range. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group, with the longest delay in attainment of balanopreputial separation, were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse. Sperm morphology assessments of F1 males showed a statistically significant increase in the percent of abnormal sperm in those males given 750 mg/kg/day; however, this change was not observed in F0 males and resulted primarily from a single male, and was therefore considered equivocal and non adverse since the percent of abnormal sperm seen fell within the historical control data range for this parameter, reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day were unaffected, and there were no microscopic findings in the testes.
Overall, there were 19 unscheduled deaths of parental animals (4 F0 males, 6 F0 females, 5 F1 males, and 4 F1 females). None of the deaths were directly attributed to the test item. Based on macroscopic (and in some cases microscopic) findings, 11 of the deaths were likely due to gavage error and/or aspiration of the dose formulation into the lungs. With the low viscosity nature of the test item and the use of corn oil as the vehicle, aspiration events were not unexpected. Two F0 females were injured by their mating partner and euthanized for humane reasons, and 1 F0 male was euthanized for humane reasons due to lesions on the neck, likely caused by scratching at the ear tag. One F0 female given 750 mg/kg/day was euthanized due to dystocia, and 1 F1 female given 750 mg/kg/day was found dead the day after dystocia was observed. The cause of death for 3 animals (1 F1 male given 50 mg/kg/day, 1 F1 male given 750 mg/kg/day, and 1 F1 female given 750 mg/kg/day) could not be definitively determined at necropsy.
There were no adverse test item-related effects on F0 and F1 parental body weights, body weight changes, feed consumption, and food efficiency. Test item-related histopathological lesions were identified in the lungs of both males and females of the F0 and F1 generations and the kidneys (males only) of the F1 generation. There was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F0 and F1 males and females given 750 mg/kg/day; this microscopic finding correlated with macroscopic observations in F0 and F1 males and F1 females, as well as increased absolute and relative lung weights in F0 and F1 males and females. As these lung lesions were considered to be secondary to aspiration of the dose formulation, and the chronic interstitial/alveolus inflammation finding was not unanticipated based on a previous 90-day, repeat-dose study with a similar test item, the lungs from the lowand
mid-dose animals were not evaluated. In the F1 males, test item-related slight increases in renal tubule degeneration/necrosis and renal tubule hyaline droplets, suggestive of hydrocarboninduced α2μ-globulin male rat nephropathy, were seen in the males given 750 mg/kg/day.
Special staining of kidneys from males in the control and high-dose group confirmed this lesion to be hydrocarbon-induced α2μ-globulin male rat nephropathy, an anticipated outcome of this study; therefore, the kidneys of the low- and mid-dose animals were not evaluated. In the F0 males, renal tubule mineralization was noted. Since this finding may be an artifactual change resulting from tissue fixation and/or processing, and since mineralized tubules were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal and non-adverse.
There were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition, mortality, pup body weights and anogenital distance. Equivocal, non-adverse decreases (not statistically significant) in absolute and relative spleen weights were observed in the F1 and F2 male and female PND 21 pups in the group given 750 mg/kg/day, compared with the control group. Macroscopic and microscopic findings in PND 21 pups were not test item related.
CONCLUSIONS
The test substance [0 (vehicle), 50, 200, or 750 mg/kg/day)] was given orally to F0 and F1 parental male and female rats for at least 70 days prior to mating and throughout the 14-day mating, post mating holding (for males), and gestation and lactation (for females) periods. For F1 males given 750 mg/kg/day, there was a test item-related, non-adverse effect on preputial separation (slight delay) and an equivocal, non-adverse effect on sperm morphology (slight increase in percent abnormal
sperm); the slight changes in these parameters fell within the historical control data range. Test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0 and F1 generations with corresponding macroscopic findings and increased lung weights and the kidneys (renal tubule degeneration/necrosis and renal tubule hyaline droplets confirmed to be α2μ-globulin) of F1 males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. Additional equivocal, non-adverse findings included renal tubule mineralization in the kidneys of F0 males
given 750 mg/kg/day and slightly decreased spleen weights in F1 and F2 pups. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, and the absence of adverse findings directly attributable to the test item in non reproductive tissues, a dosage level of 750 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity. - Executive summary:
SUMMARY OF RESULTS AND CONCLUSION
Administration of the test substance to male and female rats at dosages up to 750 mg/kg/day had no effect on reproductive performance or gestation length and parturition of both the F0 and F1 parental generations. In addition, there were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition/mortality, anogenital distance, and pup body weights. Vaginal patency of F1 females was unaffected by test item administration. There was a statistically significant, test article-related increase in the mean age and adjusted age of attainment of balanopreputial separation in F1 males given 750 mg/kg/day. However, this change was not considered adverse, as the age and adjusted age of attainment fell within the historical control data range for this parameter. Reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day, although slightly lower than the control group, were not statistically significantly different from the control group and also fell within the historical control data range. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group, with the longest delay in attainment of balanopreputial separation, were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse. Sperm morphology assessments of F1 males showed a statistically significant increase in the percent of abnormal sperm in those males given 750 mg/kg/day; however, this change was not observed in F0 males and resulted primarily from a single male, and was therefore considered equivocal and non adverse since the percent of abnormal sperm seen fell within the historical control data range for this parameter, reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day were unaffected, and there were no microscopic findings in the testes. Overall, there were 19 unscheduled deaths of parental animals (4 F0 males, 6 F0 females, 5 F1 males, and 4 F1 females). None of the deaths were directly attributed to the test item. Based on macroscopic (and in some cases microscopic) findings, 11 of the deaths were likely due to gavage error and/or aspiration of the dose formulation into the lungs. With the low viscosity nature of the test item and the use of corn oil as the vehicle, aspiration events were not unexpected. Two F0 females were injured by their mating partner and euthanized for humane reasons, and 1 F0 male was euthanized for humane reasons due to lesions on the neck, likely caused by scratching at the ear tag. One F0 female given 750 mg/kg/day was euthanized due to dystocia, and 1 F1 female given 750 mg/kg/day was found dead the day after dystocia was observed. The cause of death for 3 animals (1 F1 male given 50 mg/kg/day, 1 F1 male given 750 mg/kg/day, and 1 F1 female given 750 mg/kg/day) could not be definitively determined at necropsy. There were no adverse test item-related effects on F0 and F1 parental body weights, body weight changes, feed consumption, and food efficiency. Test item-related histopathological lesions were identified in the lungs of both males and females of the F0 and F1 generations and the kidneys (males only) of the F1 generation. There was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F0 and F1 males and females given 750 mg/kg/day; this microscopic finding correlated with macroscopic observations in F0 and F1 males and F1 females, as well as increased absolute and relative lung weights in F0 and F1 males and females. As these lung lesions were considered to be secondary to aspiration of the dose formulation, and the chronic interstitial/alveolus inflammation finding was not unanticipated based on a previous 90-day, repeat-dose study with a similar test item, the lungs from the lowand mid-dose animals were not evaluated. In the F1 males, test item-related slight increases in renal tubule degeneration/necrosis and renal tubule hyaline droplets, suggestive of hydrocarboninduced α2μ-globulin male rat nephropathy, were seen in the males given 750 mg/kg/day. Special staining of kidneys from males in the control and high-dose group confirmed this lesion to be hydrocarbon-induced α2μ-globulin male rat nephropathy, an anticipated outcome of this study; therefore, the kidneys of the low- and mid-dose animals were not evaluated. In the F0 males, renal tubule mineralization was noted. Since this finding may be an artifactual change resulting from tissue fixation and/or processing, and since mineralized tubules were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal and non-adverse. There were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition, mortality, pup body weights and anogenital distance. Equivocal, non-adverse decreases (not statistically significant) in absolute and relative spleen weights were observed in the F1 and F2 male and female PND 21 pups in the group given 750 mg/kg/day, compared with the control group. Macroscopic and microscopic findings in PND 21 pups were not test item related.
CONCLUSIONS
The test substance [0 (vehicle), 50, 200, or 750 mg/kg/day)] was given orally to F0 and F1 parental male and female rats for at least 70 days prior to mating and throughout the 14-day mating, post mating holding (for males), and gestation and lactation (for females) periods. For F1 males given 750 mg/kg/day, there was a test item-related, non-adverse effect on preputial separation (slight delay) and an equivocal, non-adverse effect on sperm morphology (slight increase in percent abnormal sperm); the slight changes in these parameters fell within the historical control data range. Test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0 and F1 generations with corresponding macroscopic findings and increased lung weights and the kidneys (renal tubule degeneration/necrosis and renal tubule hyaline droplets confirmed to be α2μ-globulin) of F1 males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. Additional equivocal, non-adverse findings included renal tubule mineralization in the kidneys of F0 males given 750 mg/kg/day and slightly decreased spleen weights in F1 and F2 pups. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, and the absence of adverse findings directly attributable to the test item in non reproductive tissues, a dosage level of 750 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity.
- Endpoint:
- two-generation reproductive toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Between 20 April 2010 and 06 January 2011.
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Read-across is considered to be reliability 2.
- Qualifier:
- according to guideline
- Guideline:
- other: US EPA Health Effects Test Guideline OPPTS 870.3800 and OECD Guidelines for the Testing of Chemicals, No, 416 "Two-Generation Reproduction Toxicity Study".
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: The males and females were approximately 35-37 days and 36-39 days old, respectively, upon receipt.
- Weight at study initiation: 153.4 to 197.0 g; female group mean range 135.4 to 171.1 g.
- Fasting period before study: Not applicable.
- Housing: During the acclimation period (F0 animals only) and throughout the study, all F0 and F1 parental animals were housed individually, except during the mating period, in solid-bottom polycarbonate cages with stainless steel wire lids. The rats were paired for mating in the home cage of the male. Following positive evidence of mating (or at the completion of the 2-week mating period), the females were transferred to individual solid-bottom polycarbonate cages with stainless steel wire lids. Beginning on pre-natal Day 21, the selected F1 pups were individually housed until the start of the mating period.
- Diet: Purina Certified Pelleted Rodent Diet® (No. 5002, PMI Feeds, Inc., St. Louis, MO) was available ad libitum in the wire cage lids. Available information on the diet did not indicate the presence of any substance at a concentration likely to have influenced the outcome of the study.
- Water (tap water) was available ad libitum via plastic water bottles with butyl rubber stoppers and stainless steel sipper tubes. Available information on the water did not indicate the presence of any substance at a concentration likely to have influenced the outcome of the study.
- Acclimation period: Seven days
ENVIRONMENTAL CONDITIONS
- Temperature: (°C): 18 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): At least ten air changes per hour
- Photoperiod (hr dark / hrs light):12 hours continuous light and 12 hours darkness
IN-LIFE DATES: First day of treatment: Between 20 April 2010 - Final necropsy day was 06 January 2011 - Route of administration:
- oral: gavage
- Type of inhalation exposure (if applicable):
- not specified
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared as solutions in corn oil at concentrations of 0 (vehicle), 25, 125, and 500 mg/mL were formulated 11 times, dispensed into 30-mL amber glass bottles with Teflon-lined lids for use as daily dose aliquots, and stored at room temperature protected from light. Concentrations of 25 and 500 mg/mL in the same vehicle have been shown to be homogeneous and stable for 133 days when stored refrigerated or at room temperature protected from light (Analytical Chemistry report. RTI-1092-AN. Stability and Homogeneity Evaluation for 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' in Corn Oil Formulation. Report date: 22 March 2011).
The test item formulations (but not the vehicle formulation) were stirred continuously throughout dose administration.
Prior to use on study, samples (~25 mL each) for concentration and homogeneity determination were collected from the top, middle, and bottom strata of each test item formulation from 4 of the 11 formulation dates (14 April 2010, 28 April 2010, 04 August 2010, and 01 December 2010). Additional samples (~25 mL each) for concentration analysis were also collected from 2 other formulation dates (12 and 26 May 2010). All analyses were conducted by using a validated 13C nuclear magnetic resonance (NMR) spectroscopy method. Details about the methodology and results of these analyses are presented in Appendix 1 and summarized as follows:
Based on these results, the analyzed dosing formulations were found to contain the amount of test item prescribed in the protocol (±20% of nominal) and were homogeneous. Therefore, the protocol-specified dosages of test item were administered to the animals.
-DIET PREPARATION
Not applicable
- Rate of preparation of diet (frequency):
Not applicable
- Mixing appropriate amounts with (Type of food):
Not applicable
- Storage temperature of food:
No data
- VEHICLE
Corn oil
- Justification for use and choice of vehicle (if other than water):
Not applicable
- Concentration in vehicle:
0, 25, 125, and 500 mg/mL
- Amount of vehicle (if gavage):
2 ml/kg
- Lot/batch no. (if required):
Not applicable
- Purity:
Not applicable - Details on mating procedure:
- - M/F ratio per cage:
1/1 (Animals were paired on a 1 male: 1 female basis within each dose group).
- Length of cohabitation:
Following positive evidence of mating (or at the completion of the 2-week mating period).
- Proof of pregnancy:
Vaginal smears were performed daily on each F0 and F1 female for 21 days prior to pairing and continuing from the day after pairing until evidence of mating was observed or until the end of the mating period. The slides from the 21 days prior to mating were evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female according to test facility SOPs. Vaginal smears were also performed on the day of necropsy to determine the stage of estrus at demise.
- After a set number of days of unsuccessful pairing replacement of first male by another male with proven fertility.:
Not applicable
- Further matings after two unsuccessful attempts:
Not applicable
- After mating each pregnant female was returned to individual caging:
Mated females were housed individually during the period of gestation and lactation.
- Any other deviations from standard protocol:
Not applicable - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The analytical method has been satisfactorily validated and details about the methodology and results of these analyses are presented in detail in Appendix 1.
The results are summarized as follows.
Prior to use on study, samples (~25 mL each) for concentration and homogeneity determination were collected from the top, middle, and bottom strata of each test item formulation from 4 of the 11 formulation dates. Additional samples for concentration analysis were also collected from two other formulation dates. All analyses were conducted by Analytical Chemistry and Pharmaceutics, RTI International.
ANALYTICAL METHOD
The validated analytical method "Analysis of the test item in Corn Oil Formulations" used to analyze study samples is outlined as follows:
The formulation samples were removed from the ambient storage locations on the day of analysis and stirred for 30 minutes prior to sampling. The transferred aliquot weights were recorded then transferred to the NMR lab for analysis. Formulation samples with a concentration over 250 mg/mL were diluted in order to fall within the calibration curve range. The dilution was performed by weighing 2 mL (-1.7 g) of the sample into a dear scintillation vial and adding corn oil to a total weight of 4 g. The diluted sample was mixed for an extra 30 minutes prior to aliquotting.
Spiked vehicle calibration standards were prepared by directly mixing the test item with corn oil.
The amount of the test material (in mg) in each formulation sample was calculated using a regression equation generated from the actual amount of the substance (in ng) versus the intensity of the selected 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' signal (35.0-35.5 ppm) when the signal of 1,4-dioxane (64.5-65.5 ppm) was set to 100. The amount of the test item (in mg) determined in each formulation sample was then converted to concentration (in mg/mL) based on the weight of the sample aliquot and its density.
Statistical analyses were performed on the integrated :areas of the NMR data.
FORMULATION ANALYSIS
The formulation analyses were performed following the analytical method described above to verify the test item concentration in corn oil formulations.
For each formulation date, four formulations were tested for concentration; the nominal concentrations of these four formulations were 0 (corn oil vehicle), 25,125, and 500 mg/mL. An exception to this was the samples prepared on May 12,2010, where only the low (25 mg/mL) and high (500 mg/mL) samples were tested. The three test item formulations were tested for homogeneity (the 0 mg/mL formulation was not tested for homogeneity). For concentration verification and homogeneity assessment, the mean found concentration at each sampling location (top, middle, and bottom) for each formulation was evaluated for accuracy and precision of duplicate determinations, as well as precision of all six determinations.
CONCLUSION
All dose formulations analyzed were found to be within 20 percent of the nominal concentration and were homogeneous. The relative standard deviation (RSD) for each replicate determination was ≤ 20 percent). - Duration of treatment / exposure:
- All male and female for the F0 generation and F1 generation were administered the test item or vehicle control, daily for at least 70 consecutive days prior to mating.
The test item was administered to offspring selected to become the F1 parental generation following weaning, beginning on postnatal day (PND) 22.
The F0 and F1 males continued to receive the test item throughout mating and through the day prior to euthanasia. The F0 and F1 females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia.
A complete gross necropsy was conducted after death for any parental (F0 and F1) or offspring (F1 and F2) animals found in moribund condition or dead on study.
Organs were not weighed for moribund animals.
Histopathological examination of study specified organs were performed for all high dose and control parental (F0 and F1). Additionally, reproductive organs of the low and mid dose animals suspected of reduced fertility were subjected to histopathological evaluation.
For any organ(s) demonstrating possible treatment-related changes in the high dose group versus the organ(s) of interest were evaluated histopathologically from successively lower concentration dose groups until a clear no observed adverse effect level (NOAEL) was established. However, at the request of the Sponsor, no further evaluation of the lungs from lower dose groups was conducted. - Frequency of treatment:
- The test and vehicle control item formulations were administered to the F0 and F1 males and females and once daily for a minimum of 70 consecutive days prior to mating and for males continued throughout mating to the day prior to termination.
The F0 and F1 females continued to be dosed throughout mating, gestation, and lactation, through to the day prior to termination.
The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) were potentially exposed to the test article in utero and through nursing (Pre Natal Day 0 to 21).
The F1 pups selected as parents for the F2 generation (25/sex/group) were administered the test item following weaning (beginning on Pre Natal Day 22). - Details on study schedule:
- Four groups of male and female rats for the F0 generation (25 or 26/sex/group) and F1 generation (25/sex/group for the) were administered the test item or control vehicle , daily for at least 70 consecutive days prior to mating and thereafter males continued to be treated through to the day prior to termination. The F0 and F1 females were dosed throughout mating, gestation, and lactation to the day prior to termination.
The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) were potentially exposed to the test article in utero and through nursing (Pre Natal Day 0 to 21).
The F1 pups selected as parents for the F2 generation (25/sex/group) were administered the test item following weaning (beginning on Pre Natal Day 22).
The first day of dosing was designated as Day 1 of the study.
Animals were paired on a 1 male: 1 female basis within each dose group.
The rats were paired for mating in the home cage of the male.
Following positive evidence of mating (or at the completion of the 2-week mating period), the females were transferred to individual solid-bottom polycarbonate cages with stainless steel wire lids. - Remarks:
- Doses / Concentrations:
50 mg/kg/day (25 mg/ml)
Basis:
actual ingested - Remarks:
- Doses / Concentrations:
250 mg/kg/day (125 mg/ml)
Basis:
actual ingested - Remarks:
- Doses / Concentrations:
1000 mg/kg/day (500 mg/ml)
Basis:
actual ingested - No. of animals per sex per dose:
- F0 generation
0 mg/kg/day (Control) - 25 or 26 per sex
50 mg/kg/day - 25 or 26 per sex
250 mg/kg/day - 25 or 26 per sex
1000 mg/kg/day - 25 or 26 per sex
F1 generation
0 mg/kg/day (Control) - 25 per sex
50 mg/kg/day - 25 per sex
250 mg/kg/day - 25 per sex
1000 mg/kg/day - 25 per sex - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Dosage levels were selected based on a 90-day, repeat-dose study on the same test substance by oral gavage. Doses tested were 0, 50, 200, and 1000 mg/kg/day. The NOEL was 50 mg/kg/day and the NOAEL was considered to be 1000 mg/kg for both males and females. Since 50 mg/kg/day was a clear NOEL in the 90-day, repeat-dose study;, this dose was selected as the low dose for this reproductive toxicity study. The intermediate dose was 250 mg/kg/day to provide an assessment of dose-response relationships.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Random - Positive control:
- Not applicable
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS:
- See attached Summary Tables 6, 14, 20, 25, 41, 49, 55, 60 and Individual Data: Appendix 2, Tables 4, 10, 17, 35, 41, 45 and 48
- Mortality
Observations were made twice daily at least 6 hours apart.
- Clinical examinations
- Time schedule:
Clinical observations were conducted and recorded at least once daily, approximately 1-2 hours post dosing, throughout the course of the study.
BODY WEIGHT:
- See attached Summary Tables 2, 3, 10, 11, 16, 17, 21, 22, 37, 38, 45, 46, 51, 52, 56 and 57 and Appendix 2, Tables 2, 8, 15, 31, 32, 33, 39, 43 and 46.
- Time schedule for examinations:
Individual body weights of the male F0 and F1 rats were determined and recorded on the first day of dosing and weekly through the pre breed exposure and mating periods.
The body weights of male F0 and F1 rats were recorded in the same manner after the mating period until scheduled euthanasia.
The body weights of female F0 and F1 rats were recorded in the same manner until evidence of mating was observed.
During gestation, sperm-positive (presumed pregnant) females were weighed on gestational days (GD) 0, 7, 14, and 20.
Dams producing litters were weighed on lactational days 0, 4, 7, 14, and 21.
FOOD CONSUMPTION AND EFFICIENCY:
- See attached Summary Tables 4, 5, 12, 13, 18, 19, 23, 24, 39, 40, 47, 48, 53, 54, 58 and 59 and Individual Data: Appendix 2, Tables 3, 9, 16, 34, 40, 44and 47.
Individual F0 and F1 male and female feed consumption was measured weekly until mating.
Feed consumption was not recorded during the 2-week mating period.
Following mating, male feed consumption was measured on a weekly basis until the scheduled necropsy.
During pregnancy of F0 and F1 females, feed consumption was recorded on gestation days 0, 7, 14, and 20.
During lactation of F1 and F2 litters, maternal feed consumption was measured on 0, 4, 7, 14, and 21.
- Food efficiency:
Percent food efficiency (body weight gained as a percentage of food consumed) was also calculated
WATER CONSUMPTION:
Not applicable
OPHTHALMOSCOPIC EXAMINATION:
Not applicable
HAEMATOLOGY AND CLINICAL CHEMISTRY:
Not applicable
- Time schedule for collection of blood:
Not applicable
- Anaesthetic used for blood collection:
Not applicable.
- Animals fasted:
Not applicable.
- How many animals:
Not applicable.
URINALYSIS:
Not applicable
NEUROBEHAVIOURAL EXAMINATION:
Not applicable.
- Time schedule for examinations:
Not applicable.
Behavioural assessment)
Not applicable.
(Functional Performance Tests)
Not applicable.
(Sensory Reactivity)
Not applicable.
- Dose groups that were examined:
Not applicable.
OTHER:
- MATING
Animals were paired on a 1 male: 1 female basis
Vaginal smears were performed daily on each F0 and F1 female for 21 days prior to pairing and continuing from the day after pairing until evidence of mating was observed or until the end of the mating period. The slides from the 21 days prior to mating were evaluated to assess the regularity and duration of the estrous cycles of each F0 and F1 female according to test facility SOPs. Vaginal smears were also performed on the day of necropsy to determine the stage of estrus at demise.
- see attached Summary Tables 15, 32 and 50 and Individual Data: Appendix 2, Tables 11, 28, 42 and 59
PREGNANCY AND PARTURITION
During the period of expected parturition, the females were observed at least twice daily for initiation and completion of parturition. On the day parturition was completed (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded.
Individual gestation length was calculated using the date delivery completed. - Oestrous cyclicity (parental animals):
- A vaginal smear was prepared for each female and the stage of the oestrous cycle was recorded.
- Sperm parameters (parental animals):
- Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0 and F1 males as appropriate.
- Litter observations:
- Clinical observations, body weights, and sexes for F1 and F2 pups were recorded at appropriate intervals. For both generations (F1 and F2), 10 pups per litter (5 per sex, when possible) were arbitrarily selected on PND 4 to reduce the variability among the litters. Offspring (25/sex/group) from the pairing of the F0 animals were selected on PND 21 to constitute the F1 generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats.
- Postmortem examinations (parental animals):
- Each F0 and F1 parental animal received a complete detailed gross necropsy (the surviving female parental animals were necropsied on LD 21), and selected organs were weighed. Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0 and F1 males as appropriate, and ovarian primordial follicle counts and the corpora lutea counts were recorded for all F1 females in the control and high-dose groups. Designated tissues from the F0 and F1 parental animals were examined microscopically.
- Postmortem examinations (offspring):
- Non selected F1 pups were necropsied on PND 21, and F2 pups were necropsied on PND 21. Selected organs (brain, spleen, and thymus) were weighed from 1 pup/sex/litter (when possible) from both F1 and F2 pups that were necropsied on PND 21.
- Statistics:
- STATISTICAL METHODS
See Attachment “"Report Text" pages 33 to 35. - Reproductive indices:
- For the Mating, fertility, and gestational indices see attached ""Report Text" page 25.
- Offspring viability indices:
- For the Offspring viability indices see attached "Report Text" page 26.
- Clinical signs:
- effects observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Other effects:
- not specified
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse test item-related effects in nonreproductive tissues, on in-life parameters (such as body weight, feed consumption, and clinical observations) or on the integrity and performance of the male and female reproductive systems.
- Critical effects observed:
- no
- Dose descriptor:
- NOAEL
- Effect level:
- > 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects were observed in nonreproductive tissues. No adverse, test item-related findings on reproductive performance or gestation length and parturition were observed.
- Critical effects observed:
- no
- Clinical signs:
- effects observed, treatment-related
- Mortality / viability:
- mortality observed, treatment-related
- Body weight and weight changes:
- effects observed, treatment-related
- Sexual maturation:
- effects observed, treatment-related
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings:
- effects observed, treatment-related
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: see 'Remark'
- Critical effects observed:
- no
- Dose descriptor:
- NOAEL
- Generation:
- F2
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: there were no test item-related effects on F2 litter parameters, postnatal survival, physical condition/mortality, or pup body weights.
- Reproductive effects observed:
- not specified
- Conclusions:
- The substance [0 (vehicle), 50, 250, or 1000 mg/kg/day)] was given orally to F0 and F1 parental male and female rats for at least 70 days prior to mating and throughout the 14-day mating, postmating holding (for males), and gestation and lactation (for females) periods. There was a test item-related, nonadverse decrease in AGD in F1 females born from dams given 250 and 1000 mg/kg/day. Test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0 and F1 generations with corresponding macroscopic findings and/or increased lung weights and the kidneys (renal tubulehyaline droplets likely representing α2μ-globulin) of the F1 males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. An equivocal, nonadverse slight decrease in F2 pup brain weights was noted. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, the absence of adverse findings directly attributable to the test item in nonreproductive tissues, and the absence of adverse effects on in-life parameters (such as body weight, feed consumption, and clinical observations), a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity.
- Executive summary:
SUMMARY OF RESULTS AND CONCLUSION
Administration of the test material 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' to male and female rats at dosages up to 1000 mg/kg/day had no effect on reproductive performance or gestation length and parturition of both the F0and F1parental generations. In addition, there were no test item-related effects on F1and F2litter parameters, postnatal survival, physical condition/mortality, or pup body weights. Vaginal patency of F1females and balanopreputial separation in F1males were unaffected by test item administration. Andrology parameters were unaffected by test item administration.
Overall, there were 8 unscheduled deaths of parental animals (1 F0female, 4 F1males, and 3 F1females). None of the deaths were directly attributed to the test item. Based on macroscopic (and in some cases microscopic) findings, 3 of the deaths (all F1males; 1 in Group 3 and 2 in Group 4) were likely due to gavage error and/or aspiration of the dose formulation into the lungs.
With the relatively low viscosity nature of the test item (kinematic viscosity at 40 ºC of 22.7 cSt) and the use of corn oil as the vehicle, aspiration events were not unexpected. One F1female (Group 3) was injured by her mating partner and euthanized for humane reasons. One F1female (Group 3) was euthanized following a self-inflicted injury (it had caught its head/jaw in the cage lid). One F0(Group 2) and one F1(Group 1) females with dystocia were also euthanized. The cause of death for one F1male (Group 4) was unable to be definitively determined at necropsy.
There were no adverse test item-related effects on F0and F1parental body weights, body weight changes, feed consumption, and food efficiency. Test item-related histopathological lesions were identified in the lungs of both males and females of the F0and F1generations. There was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F0and F1males and females given 1000 mg/kg/day; this microscopic finding correlated with macroscopic observations in F0males and females, as well as increased absolute and/or relative lung weights in F0and F1males and females. As these lung lesions were considered to be secondary to aspiration of the dose formulation, and the chronic interstitial/alveolus inflammation finding was not unanticipated based on a previous 90-day, repeat-dose study with this test item, the lungs from the low- and mid-dose animals were not evaluated. A test item-related, slight increase in the incidence of renal tubule hyaline droplets was seen in the F1males given 1000 mg/kg/day; however, only kidneys with gross lesions were examined in this study. The morphologic appearance of these droplets suggested hydrocarbon-inducedα2μ-globulin male rat nephropathy, an anticipated outcome of this study. No degenerative/necrotic renal tubule changes were observed in association with the hyaline droplets.
Anogenital distance (AGD) for female F1, but not F2, pups was statistically significantly decreased to a similar level in all test item-treated groups and in groups given 250 and 1000 mg/kg/day when adjusted for body weight compared with the control group. However, these changes were not considered adverse, as the AGD and adjusted AGD values for all groups spanned a range similar to the historical control range for AGD in F2pups and fell within the range noted for the F2females on this study. (Note: F1pup AGD is a nonstandard endpoint, not required by the OECD 416 guidelines; therefore, no historical control ranges exist at this test facility.) Other female parameters such as vaginal patency and estrous cyclicity were unaffected by test item administration. A decrease in AGD values in females is generally not considered toxicologically relevant. In addition, the biological meaning of this small change in AGD is not clear, as hyperfeminization was not apparent based on other endpoints, and since there were no adverse effects on the F1 reproductive parameters or the F2 pup parameters. An equivocal, nonadverse, slight decrease in F2pup brain weights was noted. Macroscopic and microscopic findings in PND 21 pups were not test item related.
CONCLUSIONS
The substance [0 (vehicle), 50, 250, or 1000 mg/kg/day)] was given orally to F0and F1parental male and female rats for at least 70 days prior to mating and throughout the 14-day mating, postmating holding (for males), and gestation and lactation (for females) periods. There was a test item-related, nonadverse decrease in AGD in F1females born from dams given 250 and 1000 mg/kg/day. Test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0and F1generations with corresponding macroscopic findings and/or increased lung weights and the kidneys (renal tubulehyaline droplets likely representingα2μ-globulin) of the F1males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. An equivocal, nonadverse slight decrease in F2pup brain weights was noted. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, the absence of adverse findings directly attributable to the test item in nonreproductive tissues, and the absence of adverse effects on in-life parameters (such as body weight, feed consumption, and clinical observations), a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity.
Referenceopen allclose all
PREBREED EXPOSURE PERIOD
Summary Data: Tables 1, 6, 8, 14, 33, Individual Data: Appendix 2, Tables 1, 4, 7, 10, 29.
There were no deaths attributed to test substance administration noted through the F0 prebreed exposure period (Days 0 through 70). Seven unscheduled deaths (3 found dead and 4 euthanized for humane reasons) occurred on study through Day 70.
Male No. 153 (Group 4) and Female No. 152 (Group 4) were found dead on Day 1 and Day 68, respectively. The cause of death for these animals was not conclusively determined from the gross necropsy. Red staining around the nose and left eye (Male No. 153) or nose and mouth (Female No. 152), mottled (Male No. 153, correlated with alveolus hemorrhage) or discolored/dark (Female No. 152) lungs, and reddish, dark fluid in the intestines (Female No. 152 only) indicate that aspiration of the test item is the most likely cause of death of these animals. Female No. 154 (Group 4) was found dead on Day 27, approximately 1.2 hours postdose, due to an apparent dosing error, as evidenced by red fluid around the nose and mouth, lungs that failed to collapse and were discolored (dark), and fluid expelled from the trachea with slight pressure applied to lungs.
Male No. 7 (Group 1), Male No. 185 (Group 4), and Female No. 124 (Group 3) were euthanized on Days 56, 66, and 70, respectively. Gross observations at necropsy (mottled/discolored lungs, red/dark fluid in stomach and gastrointestinal tract, red staining around mouth, and/or red fluid in mouth) indicate most likely aspiration of the test item, leading to the clinical signs that prompted humane euthanasia of these animals; microscopic lung findings for Male No. 7 and 185 further support this conclusion. In addition, Male No. 143 (Group 3) was euthanized for humane reasons on Day 16 due to bilateral lesions on the neck that worsened despite veterinary intervention. All other animals survived to Day 70.
There were no test item-related clinical observations from Day 0 to 70. All clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Two males (Nos. 115 and 127, Group 3) exhibited neck lesions that required veterinary intervention; the neck lesions healed by Day 70.
MATING, MALE POSTMATING, AND FEMALE GESTATION AND LACTATION PERIODS
Summary Data: Tables 6, 14, 20, 25, 33 Individual Data: Appendix 2, Tables 4, 10, 14, 17, 29.
For the F0 males, there were no deaths during the mating/postmating period (Days 70 through 107). There was an increased incidence of rooting in animals given 750 mg/kg/day and of chromodacryorrhea (eye) in animals given the 200 and 750 mg/kg/day compared with the control group. All other clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Male No. 127 (Group 3; terminated as scheduled on Day 106) exhibited a neck lesion that required veterinary intervention on Days 97 to 105; this same animal had previous neck lesions requiring veterinary intervention that had healed by Day 72.
For the F0 females, there were no deaths attributed to the test substance administration during the mating, gestation, and lactation periods (Days 70 to 128); there were 3 unscheduled F0 female deaths during this time. Female Nos. 38 (Group 1) and 180 (Group 4) were severely injured by their mating partner and were therefore euthanized on Day 72 for humane reasons (both were sperm negative on Day 72 prior to euthanasia). There were no macroscopic necropsy findings for these 2 females other than the wound inflicted by the mating partner. Female No. 166 (Group 4) was euthanized for humane reasons on Day 95 (GD 22) due to dystocia with concomitant hypothermia. Macroscopic necropsy findings for Female No. 166 included pale, mottled, mildly enlarged adrenal glands and mottled lungs (all lobes), and there were 18 visible implant sites that corresponded to 18 pups (3 live and 15 dead) in the uterus.
From Day 71 through Day 113, there were no test item-related clinical observations in the F0 females that remained sperm negative; all clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
For sperm-positive F0 females, there were occasional observations of struggling during dosing in animals given 200 and 750 mg/kg/day, and there were very occasional observations of rooting post dosing and salivating prior to dosing in up to 2 females given 750 mg/kg/day during the gestation period (GD 0 through 23). Likewise, there were occasional observations of animals struggling during dosing in the groups given 200 and 750 mg/kg/day during the lactation period.
One dam (No. 118) given 200 mg/kg/day had an eye injury and swollen nose from LD 17 to 21 that were not test item-related. All other clinical findings during gestation and lactation in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals or single occurrences, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
REPRODUCTIVE PERFORMANCE
Summary Data: Tables 15, 26 Individual Data: Appendix 2, Tables 6, 11, 18, 19.
No test item-related effects on F0 reproductive performance were observed at any dosage level.
There were no statistically significant differences on male and female mating indices and male and female fertility indices. Male mating indices were 69.6%, 80.0%, 95.8%, and 90.9% and female mating indices were 70.8%, 80.0%, 95.8%, and 90.9% in the control, 50, 200, and 750 mg/kg/day groups, respectively. Historical control data for male and female mating indices ranged from 87.5% to 100% and 88% to 100%, respectively. Following a thorough investigation, which included a detailed review of all the environmental data during the study and discussions with the animal vendor, the reason for the low mating indices in the control and low-dose groups remained unknown; however, 2 males (Nos. 3 and 41) were infertile based on andrology parameters, thus partially accounting for the lower mating indices in the control group. Male fertility indices were 100.0%, 90.0%, 95.7%, and 95.0% and female fertility indices were 100.0%, 90.0%, 95.7%, and 95.0% in the same respective groups. All fertility indices were within the historical control data ranges of 75.9% to 100% for F0 males and 76.7% to 100% for
F0 females.
All F0 females evaluated during the final 3 weeks of the 10-week prebreed period were noted to be cycling, with a similar number and percent of females with abnormal or irregular cycles noted across all groups, including the control. There was no difference in mean cycle length (3.8 to 4.0 days) or number of cycles (4.4 to 4.6) in test item-treated groups compared with control values (3.9 days and 4.4, respectively). The mean number of days between pairing and coitus (i.e., the precoital interval) in the test item-treated groups was similar to the control group values of 2.4 days.
GESTATION LENGTH AND PARTURITION
Summary Data: Table 26 Individual Data: Appendix 2, Table 19.
No test item-related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Mean F0 gestation lengths in the test item-treated groups were similar to the control group value. The mean gestation lengths in the groups given 50, 200, and 750 mg/kg/day were 22.4, 22.2, and 22.4 days, respectively, compared to the mean gestation length of 22.3 days in the concurrent control group. Gestation indices were 94.4%, 95.5%, and 89.5% in the groups given 50, 200, and 750 mg/kg/day, respectively, and were not statistically significantly different from the control group value of 100.0% and were within the historical control data range of 87% to 100%. There were corresponding increases and decreases in the stillbirth index and live birth index, respectively, for groups given 200 and 750 mg/kg/day.
These slight changes in the live born gestation index, stillbirth index, and live birth index in the group given 750 mg/kg/day were due to Female No. 166 being euthanized on GD 22 due to dystocia and Female No. 200 having an all-stillborn litter. Likewise, the slight changes in these same indices for the group given 200 mg/kg/day were due to Female No. 128 having a presumed all-stillborn litter, and the slight decrease in live born gestation index for the group given 50 mg/kg/day was due to Female No. 86 not delivering any pups, though having 5 visible implant sites at necropsy. No statistically significant differences were noted between the control and test item-treated groups in the number or percentage of females completing delivery and females completing delivery with stillborn pups or with all stillborn. No statistically significant differences in the mean number of implantation sites, percent post implantation loss, pups delivered (live, dead, and total), and number of live pups per litter on PND 0 and PND 4 for litters having at least 1 live pup in the group were noted.
BODY WEIGHTS, FOOD CONSUMPTION, AND FOOD EFFICIENCY
PREBREED EXPOSURE, MATING, AND POSTMATING PERIODS
Summary Data: Tables 2, 3, 4, 5, 10, 11, 12, 13, Individual Data: Appendix 2, Tables 2, 3, 8, 9.
There were no adverse test item-related effects on body weights, body weight changes, or feed consumption during the prebreed exposure period (Days 0 to 70). Slight, nondose-related increases in feed consumption (g/day and g/kg/day) were noted for males and females at several intervals (occasionally statistically significant) for all test item-treated groups when compared with the control group; however, these changes in feed consumption did not result in or correlate with statistically significant changes in body weights, body weight changes, or food efficiency compared with the controls.
There were no adverse test item-related effects on F0 male body weights and body weight changes from Days 70 to 105 and feed consumption from Days 84 to 105. A statistically significant increase in feed consumption (g/day and g/kg/day) for one interval (Days 84 to 91) was noted for the F0 males given 750 mg/kg/day; however, this change in feed consumption did not result in or correlate with statistically significant effects on body weights, body weight changes, or food efficiency compared with the controls.
In general, there were no notable test item-related effects on body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency from Days 84 to 112 for those F0 females that were never found sperm positive or that never delivered a live litter.
Occasional statistically significant changes were observed in these parameters, but were not considered biologically significant as they were sporadic and not dose related.
GESTATION
Summary Data: Tables 16, 17, 18, 19, Individual Data: Appendix 2, Tables 12, 13.
During gestation, there were no adverse test item-related changes in F0 female body weights and feed consumption (g/day and g/kg/day). Compared with the concurrent control group, there was a statistically significant decrease (~17%) in percent food efficiency from GD 7 to 14 for the females given 750 mg/kg/day, which correlated with a decrease (~16%) in body weight change that was not statistically significant for this same group during the same interval. The following interval (GD 14 to 20) also showed a slight, though not statistically significant, decrease in
percent food efficiency and body weight change (~10% and ~8%, respectively) for the females given 750 mg/kg/day compared with the concurrent control group. Thus, percent food efficiency and body weight change were slightly reduced (~8% and ~7%, respectively) between GD 0 and 20 but did not achieve statistical significance for the females given 750 mg/kg/day compared with the control group; thus, these changes were considered incidental to treatment and not adverse.
LACTATION
Summary Data: Tables 21, 22, 23, 24, Individual Data: Appendix 2, Tables 15, 16.
During lactation (LD 0 to 21), there were no notable test item-related or statistically significant changes in F0 female body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group.
ANDROLOGY
Summary Data: Table 7, Individual Data: Appendix 2, Table 6.
No test item-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility and morphology) in males at any dosage concentration. Differences from the control group were slight and were not statistically significant. As per the protocol, assessments using the frozen right testis, as well as sperm morphology evaluations, were conducted initially on the control and high-dose groups only. As there were no test item-related effects in the group given 750 mg/kg/day, evaluation of the lower dose groups was not conducted for these endpoints.
ANATOMIC PATHOLOGY – SCHEDULED DEATHS
MACROSCOPIC EXAMINATION
Summary Data: Tables 7, 8, 26, 32, 33, Individual Data: Appendix 2, Tables 5, 7, 19, 28, 29.
The only test item-related macroscopic finding in F0 parental animals was noted in the lungs of the males given 200 and 750 mg/kg/day. At the F0 male necropsy, 7 males given 750 mg/kg/day were noted as having pale lungs (cranial lobe, left or bilateral), and this gross finding correlated with an increase in both absolute and relative lung weights in this dose group. Two males given 200 mg/kg/day also had pale lungs (cranial lobe, left); however, there was no significant increase in lung weights in this dose group. The thymus of 2 males (Nos. 187 and 199) given 750 mg/kg/day had multiple, pinpoint, red foci. There was an increased incidence of mottled kidneys (bilateral) in the males given 200 and 750 mg/kg/day (10 of 24 and 9 of 23 animals, respectively) compared with the control and low-dose groups (5 of 24 and 5 of 25 animals, respectively). The testes of 2 control males (Nos. 3 and 41) were found to be reduced in size (bilateral) and flaccid (bilateral), and the epididymides of Male No. 3 were also reduced in size. Neither of these males (Nos. 3 and 41) sired a litter and both had few motile or progressively motile sperm (thus the reason for the larger standard error in these parameters for the control group compared with the test item-treated groups). All other macroscopic gross findings were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
There were no test item-related macroscopic findings for the F0 females necropsied on LD (≡PND) 21. All macroscopic gross findings for F0 females at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
No test item-related effects were observed on the number of implantation sites. The number of females in proestrus, estrus, metestrus, and diestrus at demise were similar for all groups. The differences between the control and test item-treated groups were slight and not statistically significant.
ORGAN WEIGHTS
Summary Data: Tables 7, 32, Individual Data: Appendix 2, Tables 5, 27.
There was an increase in both absolute and relative lung weights (38% and 33%, respectively) in F0 males given 750 mg/kg/day. Absolute and relative lung weights were also statistically significantly increased (14.0% and 12.8%, respectively) in F0 females given 750 mg/kg/day compared with the control group. The increased lung weights correlated with the macroscopic (males) and microscopic (males and females) lung findings in rats given 750 mg/kg/day.
Although there was a statistically significant increase (approximately 10%) in the absolute lung weight for F0 males given 50 mg/kg/day, there was no significant increase in relative lung weight and no macroscopic lung findings in this same group, as well as no significant changes in absolute and relative lung weights for F0 females given 50 mg/kg/day and F0 males and females given 250 mg/kg/day; therefore, the change in absolute lung weight for F0 males given 50 mg/kg/day was considered spurious.
MICROSCOPIC EXAMINATION
Summary Data: Tables 9, 34, Individual Data: Appendix 3.
A test item-related histopathological lesion was identified in the lungs. Chronic interstitial/alveolus inflammation was increased in incidence and severity in the lungs of F0 males and females given 750 mg/kg/day compared with the controls. The increased lung weights correlated with the chronic interstitial/alveolus inflammation observed microscopically.
This lesion was characterized by a spectrum of lesions which were severity dependent and included the presence of foamy to highly vacuolated macrophages within alveolar spaces, thickened alveolar septae due to the infiltration of primarily mononuclear inflammatory cells and hypertrophy/hyperplasia of alveolar epithelial cells, edema, slight congestion and/or hemorrhage, occasional infiltration of polymorphonuclear inflammatory cells, fibrosis, increased perivascular cuffing by mononuclear inflammatory cells and a variable hyperplasia of the bronchiolar associated lymphoid tissue (BALT). This inflammatory change was graded on a 5-grade severity scale where 1 was the most minimal change noted and 5 was the most severe. Most test itemexposed animals had a severity of mild to moderately severe (2-4). The overall lung severity grade was an average from both the left and right lobes. Inflammatory changes were usually focal to multifocal. Only when the severity was grade 3 to 4 did the inflammatory foci tend to merge and become confluent. The left lung tended to have more lesions and more severe lesions than the right lobe. Lesions, in general, were highly airway-centric, located near the terminal portions of the airways. This appearance was suggestive of chemical exposure through aspiration. In other areas, this determination was more difficult but may have been influenced by sectioning. There did not appear to be any significant difference between male and female lungs with regard to the appearance of interstitial/alveolus inflammation.
Another similar pulmonary finding (alveolar macrophage aggregation, alveolus) was noted in control male and female lungs and was characterized by the presence of foamy appearing macrophages in alveolar spaces without any other significant changes associated with it. This finding can be seen as a spontaneous change in the lungs of rats, though one cannot rule out the possibility that this finding is related to dosing with the corn oil vehicle.
An equivocal, non-adverse histopathological lesion was identified in the kidneys. Renal tubule mineralization was observed in the kidneys of 7 of 23 males given 750 mg/kg/day. This lesion was characterized by the presence of multifocal mineralized renal tubules (proximal convoluted tubules) in the outer cortex. Similar appearing tubules were not seen in any of the 24 male F0 controls. The renal tubules were characterized as basophilic tubules with mineralization of both the nucleus and cytoplasm affected. Some tubules were strongly mineralized and others less.
There was no apparent cause for the mineralization and no histopathological reaction to the mineralized tubules was noted. In the study pathologist’s experience, these tubules appeared similar to artifactual changes occasionally observed as a result of tissue fixation and/or processing. Since mineralized tubules may be an artifactual change and were only observed in the F0 males given 750 mg/kg/day, with no similar mineralized tubules seen in the F0 control males, F0 females given 750 mg/kg/day, and F1 males and females given vehicle or 750 mg/kg/day, this finding was considered equivocal.
Other changes in the kidneys such as chronic progressive nephropathy and renal tubule regeneration were not clearly increased in either incidence or in severity and were therefore not deemed test item-related.
The microscopic examination of reproductive tissues for F0 animals suspected of reduced fertility in the low- and mid-dose groups did not have any relationship to test item administration.
A number of other histopathological findings were noted in a number of tissues which were typical of the spontaneous type of microscopic pathology that may be observed in this strain and age of rat.
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: Table 26, Individual Data: Appendix 2, Tables 19, 20.
The mean number of pups delivered (live, dead, and total); number of live pups per litter on PND 0, 4, 7, 14, and 21(for litters having at least 1 live pup); percentage of males per litter on PND 0, 4, 7, 14, and 21; lactational index; and postnatal survival indices (4-, 7-, 14-, and 21-day) were unaffected by the test item at all dosage concentrations. Differences from the control group were slight and were not statistically significant. The slight reduction (which was not statistically significant) in the 4-day survival index per litter in the group given 750 mg/kg/day was because the entire litter for Female No. 198 was found dead (3 pups) or euthanized (12 pups) prior to PND 2 due to poor clinical condition (including no milk band, cold to touch, reduced activity, and loss of skin elasticity) due to the lack of care for the pups by the dam.
GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: Table 28, Individual Data: Appendix 2, Tables 20, 23.
The numbers of F1 pups found dead, euthanized, and/or missing, as well as the general physical condition of all F1 pups in this study, were unaffected by test item administration. Pups that were found dead or euthanized (prior to scheduled euthanasia on PND 21) numbered 4, 6, 12, and 19 in the control, 50, 200, and 750 mg/kg/day groups, respectively. One litter (Dam No. 198) accounted for 15 of the 19 pups in the group given 750 mg/kg/day. In addition, 3, 3, 2 and 0 pups in the same respective groups were either missing (and presumed dead) or cannibalized.
ANOGENITAL DISTANCE
Summary Data: Table 27, Individual Data: Appendix 2, Table 21.
The anogenital distances (absolute and adjusted for body weight as the covariate) in the F1 male and female pups delivered from dams given 50, 200, and 750 mg/kg/day were similar to the respective control group values. Differences from the control group were slight and not statistically significant.
OFFSPRING BODY WEIGHTS
.
Summary Data: Table 27, Individual Data: Appendix 2, Table 22.
Mean male and female pup body weights and body weight changes in the groups given 50, 200, and 750 mg/kg/day were unaffected by test item administration throughout the postnatal period.
No statistically significant differences from the control group were noted.
NECROPSIES OF PUPS STILLBORN, FOUND DEAD, OR EUTHANIZED
Summary Data: Tables 28, 29, Individual Data: Appendix 2, Tables 23, 24.
The numbers of pups (litters) stillborn, found dead, or euthanized from PND 0 through the selection of the F1 generation on PND 21 numbered 4(4), 8(5), 12(6), and 19(5) in the control, 50, 200, and 750 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration to the test item were noted at the necropsies of pups that were stillborn, found dead, or euthanized.
The combined incidence of bilateral and unilateral hydronephrosis in stillborn pups was not different from the control group, nor was the incidence of hydronephrosis seen in pups necropsied as scheduled on PND 21 different in test item-treated groups compared with the control group; therefore, the increased pup and litter incidence of bilateral hydronephrosis in stillborn pups in both the 200 and 750 mg/kg/day dose groups compared with the control group was not considered test item-related.
There were several F1 pups found dead and euthanized moribund in the 200 mg/kg/day and/or 750 mg/kg/day groups, with observations of no milk in the stomach, indicating the pups were not nursing. There were no F1 pups found dead in the control group and no F1 pups euthanized moribund in the control and 200 mg/kg/day dose groups, so a direct comparison of the incidence of this macroscopic finding across groups in unscheduled death pups is not possible. The one low-dose pup euthanized moribund also had no milk in the stomach and loss of skin elasticity noted at necropsy. Of the 12 pups euthanized moribund in the high-dose group (all from Dam No. 198), 3 had distended ureter (either unilateral or bilateral); however, the incidence of this finding in the culled pups from all dose groups was similar, and thus this finding was not considered to be test item-related.
NECROPSIES OF WEANLINGS - PND 21
MACROSCOPIC EXAMINATION
Summary Data: Table 31, Individual Data: Appendix 2, Table 26.
At the PND 21 necropsy of F1 weanlings, there were no findings in the test item-treated groups that were not also observed with similar or higher incidence in the control group.
ORGAN WEIGHTS
Summary Data: Table 30, Individual Data: Appendix 2, Table 25.
There were no statistically significant changes in F1 pup final body weights and organ weights compared with the control group. Slight decreases in absolute and relative spleen weights (approximately 9% to 10% and 4% to 5%, respectively) were observed in F1 pups in the group given 750 mg/kg/day compared with the control group. The spleen weights in F1pups from the high-dose group in this study fall within the historical control data ranges for this parameter and were therefore considered an equivocal, non-adverse finding.
MICROSCOPIC EXAMINATION
Summary Data: Table 31, Individual Data: Appendix 3.
There were no microscopic findings in the F1 PND 21 weanlings in the test item-treated groups that were not also observed with similar or higher incidence in the control group.
F1 DEVELOPMENTAL LANDMARKS
BALANOPREPUTIAL SEPARATION
Summary Data: Table 36, Individual Data: Appendix 2, Table 31.
The test item-related increase in attainment of balanopreputial separation in males given 750 mg/kg/day was not considered adverse, as the day of attainment falls within the historical control ranges for this parameter. The mean ages of attainment of balanopreputial separation were 43.3, 43.2, and 43.2 days in the groups given 50, 200, and 750 mg/kg/day, respectively, compared with 41.6 in the control group, and were all statistically significantly longer than the control group, though in a nondose-responsive manner and within the historical control range for the mean age of attainment of balanopreputial separation of 40.8 to 43.6 days. Mean body weights at the age of attainment were 254.0, 249.8, and 245.4 g in the same respective groups, compared with 238.9 g in the control group, and were not statistically significant. When adjusted with body weight as a covariate, the adjusted mean ages of attainment of balanopreputial separation were 43.0, 43.1, and 43.3 days in the groups given 50, 200, and 750 mg/kg/day, respectively, compared with 42.0 in the control group, and although dose-responsive, only the group given 750 mg/kg/day was statistically significantly longer than the control group. This test item-related increase in adjusted age of attainment of balanopreputial separation was not considered adverse, as 43.3 days falls within the historical control ranges for this parameter of 40.7 to 43.8 days. In addition, andrology parameters and sperm morphology data for the individual F1 males in the high-dose group with the longest delay in attainment of balanopreputial separation were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse.
VAGINAL PATENCY
Summary Data: Table 36, Individual Data: Appendix 2, Table 32.
Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test item administration. The mean ages of attainment of vaginal patency were 31.3, 30.6, and 30.0 days in the groups given 50, 200, and 750 mg/kg/day, respectively compared with 29.9 days in the control group. Mean body weights at the age of attainment were 117.5, 111.0, and 105.5 g in the same respective groups compared with 108.6 g in the control group. The mean age for the group given 50 mg/kg/day was statistically significant, though was not considered test item-related as there was no dose-response and the adjusted mean age was not statistically significantly different from the control group.
GENERATION F1
CLINICAL OBSERVATIONS AND MORTALITY
PREBREED EXPOSURE PERIOD
Summary Data: Tables 35, 41, 43, 49, 68, Individual Data: Appendix 2, Tables 30, 35, 38, 41, 60.
There were no deaths directly attributed to the test substance administration noted through the F1 weaning and prebreed exposure period (Days -20 through 70). Seven unscheduled deaths (6 found dead and 1 euthanized for humane reasons) occurred through Day 70.
Male No. 1147 (Group 2, 50 mg/kg/day) was found dead on Day -2 (PND 36) after being noted as lethargic the previous day. Male No. 1305 (Group 4, 750 mg/kg/day) was also found dead on Day -2 (PND 38). The cause of death for these 2 males could not be determined upon necropsy; there were no obvious signs of gavage error and some autolysis was present. Group 4 Male Nos. 1329 and 1351 were found dead on Days 2 (PND 41) and 3 (PND 40), respectively. Both males had hydronephrosis of the right kidney and dark red lungs. The lungs from Male No. 1351 failed to collapse and had pale areas around the edges of all lobes. Slight pressure on the lungs released bubbles from the trachea, suggesting the cause of death of Male No. 1351 was a gavage error. Based on the appearance of the lungs at necropsy, Male No. 1329 may have died due to aspiration of the dose formulation. Group 4 Female Nos. 1346 and 1354 were found dead on Days 18 (PND 56) and 30 (PND 67), respectively. Female No. 1346 had struggled during dosing the day prior to being found dead. At necropsy, the only finding was red substance in the nose and mouth. The cause of death of Female No. 1346 could not be determined upon necropsy. Female No. 1354 was noted as lethargic at the postdose observation time and died a short time later. At necropsy, slight hydronephrosis of the right kidney was seen, and the lungs were found to be red and failed to collapse fully. Slight pressure on the lungs released fluid and bubbles from the trachea, suggesting the cause of death of Female No. 1354 was gavage error.
Female No. 1348 (Group 4, 750 mg/kg/day) was euthanized for humane reasons on Day 54 (PND 91) on welfare grounds. At necropsy, reddish fluid from nose and mouth, reddish fluid in the intestines, very little food present in the stomach, and dark red lungs that did not fully collapse were noted, suggesting the apparent cause of death was gavage error. All other animals survived to Day 70.
There was a slightly increased incidence of rooting in male and female F1 animals given 750 mg/kg/day and of chromodacryorrhea (eye) in females given 200 mg/kg/day and 750 mg/kg/day compared with the control group. These observations are not adverse. All other clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were limited to unscheduled death animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
MATING, MALE POSTMATING, AND FEMALE GESTATION AND
LACTATION PERIODS
Summary Data: Tables 41, 43, 49, 55, 60, 68, Individual Data: Appendix 2, Tables 35, 38, 41, 45, 48, 60.
For the F1 males, there was one death during the mating/postmating period (Days 70 through 107). Male No. 1325 (Group 4, 750 mg/kg/day) was noted as lethargic and gasping for breath prior to dying on Day 78. At necropsy, the lungs were mottled and failed to fully collapse; when slight pressure was applied to the lungs, bubbles were released from the trachea. The apparent cause of death of this animal was either a direct dosing error or inadvertent aspiration of the test item leading to a chronic aspiration pneumonia.
All clinical findings in the F1 male, test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
For the F1 females, there were no deaths directly attributed to the test substance administration during the mating, gestation, and lactation periods. Female No. 1352 (Group 4, 750 mg/kg/day) was found dead on Day 96 (LD 1) with the macroscopic necropsy finding of dark red substance present in the uterus. On the previous day (LD 0), clinical signs for Animal No. 1352 included piloerection, dystocia, labored breathing, lethargy, and excess red substance present; as the animal was not hypothermic and was actively delivering pups at the time of evaluation by the veterinarian, the veterinarian recommendation was for continued monitoring.
From Day 71 through Day 113, there were no test item-related clinical observations in the F1 females that remained sperm negative; all clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
For sperm-positive F1 females, there were no test item-related clinical observations during the gestation period (GD 0 through 23) or lactation period (LD 0 through 21). One dam (No. 1316) given 750 mg/kg/day a mass/sore on the chest for the entire lactation period and from days 9 (mass) or 18 (sore) through 21 of gestation. The area was not seen at terminal necropsy and thus was not saved for evaluation. All other clinical findings during gestation and lactation in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals or single occurrences, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
REPRODUCTIVE PERFORMANCE
Summary Data: Tables 50, 61
Individual Data: Appendix 2, Tables 42, 49, 50
No test item-related effects on F1 reproductive performance were observed at any dosage level.
There were no statistically significant differences on male and female mating indices and male and female fertility indices. Male mating indices were 92.9%, 92.6%, 85.7%, and 88.0% and female mating indices were 92.9%, 92.9%, 85.7%, and 88.0% in the control, 50, 200, and 750 mg/kg/day groups, respectively. Historical control data male and female F1 mating indices ranged from 70.0% to 100% and 50% to 100%, respectively. Male fertility indices were 96.2%, 92.0%, 95.8% and 81.8% and female fertility indices were 96.2%, 92.3%, 95.8% and 81.8% in the same respective groups. Although the fertility index for males and females was lower in the group given 750 mg/kg/day, all fertility indicies were within the historical control data ranges of 76.2% to 100% for F1 males and 70.8% to 100% for F1 females.
All test item-treated F1 females evaluated during the final 3 weeks of the 10-week pre breed period were noted to be cycling compared with 89.3% of the control females noted to be cycling.
There was no statistically significant difference in the number and percent of test item-treated females with abnormal or irregular cycles compared with the control group. There were no differences in mean cycle length (4.1 to 4.3 days) and number of cycles (4.0 to 4.4) in test item treated groups compared with control values (4.0 days and 4.6, respectively). The mean numbers of days between pairing and coitus (i.e., the precoital interval) in the test item-treated groups were similar to the control group values of 2.8 days.
GESTATION LENGTH AND PARTURITION
Summary Data: Table 61, Individual Data: Appendix 2, Table 50.
No test item-related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration.
Gestation indices were 100%, 100%, and 88.9% in the groups given 50, 200, and 750 mg/kg/day, respectively, and were not statistically significantly different from the control group value of 96.0%. The historical control data range for F1 gestational index is 94.1% to 100%. The slight, though not statistically significant, decrease in the liveborn gestation index in the group given 750 mg/kg/day was due to Female Nos. 1324 and 1344 having implantation sites but not delivering a litter. There was also one control female (No. 1024) that had implantation sites but did not deliver a litter. Therefore, these observations are not considered test item related. No statistically significant differences were noted between the control and test item-treated groups in the number or percentage of females completing delivery and females completing delivery with stillborn pups or with all stillborn. No statistically significant differences in the mean number of implantation sites, percent postimplantation loss, pups delivered (live, dead, and total), stillbirth index, live birth index, and number of live pups per litter on PND 0 and PND 4 for litters having at least 1 live pup in the group were noted.
BODY WEIGHTS, FOOD CONSUMPTION, AND FOOD EFFICIENCY
PREBREED EXPOSURE, MATING, AND POSTMATING PERIODS
Summary Data: Tables 37, 38, 39, 40, 45, 46, 47, 48, Individual Data: Appendix 2, Tables 33, 34, 39, 40.
There were no adverse, test item-related effects on F1 body weights or feed consumption during the prebreed exposure period (Days 0 to 70). The increased (7% to 11%) body weights for F1 females given 50 mg/kg/day, though statistically significant at every interval from Day 0 to Day 70, was not considered test item-related since there was no dose relationship and only occasional corresponding significant increases in body weight change, food consumption (g/day only), and food efficiency. Although other occasional statistically significant changes in body weight, body weight change, food consumption, and food efficiency were noted when compared with the control group, including the slightly decreased (4.4% and 5.4%) percent food efficiency for the overall interval from Days 0 to 70 for F1 males given 50 and 750 mg/kg/day, respectively, these changes were not considered the result of test item administration since these changes were sporadic, slight, not dose related, and generally had no effect on the other body weight and/or food consumption parameters.
There were no adverse test item-related effects on F1 male body weights and body weight changes from Days 70 to 105 and feed consumption from Days 84 to 105. Statistically significant decreases in body weight change and percent food efficiency for one interval (Days 91 to 98) were noted for the F1 males given 750 mg/kg/day; however, these changes did not result in or correlate with statistically significant effects on body weight or feed consumption (g/day and g/kg/day) compared with the controls for this same interval and were thus considered nonadverse. The statistically significant increases in body weight change and/or percent food efficiency during the postmating holding period, for F1 males given the lower dose levels, were sporadic and not dose related, and therefore not considered test item related.
There were no statistically significant changes in body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency from Days 84 to 112 for those F1 females that were never found sperm positive or that never delivered a live litter.
GESTATION
Summary Data: Tables 51, 52, 53, 54, Individual Data: Appendix 2, Tables 43, 44.
There were no test item-related changes in body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group during gestation. Although body weights for females given 50 mg/kg/day were statistically significantly increased at each interval, there were no corresponding statistically significant increased in body weight change, feed consumption, or percent food efficiency. The change in body weights was not considered test item-related as there was no dose response.
LACTATION
Summary Data: Tables 56, 57, 58, 59, Individual Data: Appendix 2, Tables 46, 47.
During lactation (LD 0 to 21), there were no notable test item-related changes in F1 female body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group. The one statistically significant change (an increase in body weight for females given 50 mg/kg/day on LD 21) was not considered test item-related as the change was sporadic, showed no dose response, and did not correlate with any changes in any other body weight or food parameters.
ANDROLOGY
Summary Data: Table 42, Individual Data: Appendix 2, Table 37
There was an equivocal, non-adverse, statistically significant increase in the percent of abnormal sperm in the F1 males given 750 mg/kg/day compared with the control group; however, the historical control data range of 1.29% to 6.1% encompasses the 2.29% seen in the high dose in this study. There was an increase in the mean number of sperm with blunt heads (indicating the absence of the acrosome), sperm with small heads, and sperm with heads only or tails only. One particular male in the high dose group (Animal No. 1339) had a very high percentage of sperm with heads only; this breakage was possibly test item-related and Female No. 1336, mated with this male, failed to become pregnant.
No test item-related effects were observed on the F1 spermatogenesis endpoints of mean testicular and epididymal sperm numbers and sperm production rate, motility, and progressive motility in males at any dosage concentration. Differences from the control group were slight
and were not statistically significant for these parameters.
ANATOMIC PATHOLOGY – SCHEDULED DEATHS
MACROSCOPIC EXAMINATION
Summary Data: Tables 42, 43, 61, 67, 68, Individual Data: Appendix 2, Tables 36, 38, 50, 58, 59, 60.
The only test item-related macroscopic findings in F1 parental animals were observed in the lungs of males and females given 750 mg/kg/day. At the F1 male scheduled necropsy, a total of 11 males given 750 mg/kg/day were noted as having lung findings, including small dark red spots and mottled (alone or with other findings such as firm, tan, and/or small dark spots), and these gross findings correlated with an increase in both absolute and relative lung weights in this dose group. There was an increased incidence of mottled kidneys (bilateral) in the males given 750 mg/kg/day (10 of 24) compared with the control group (4 of 28). All other macroscopic gross findings in F1 males were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
At the scheduled necropsies on LD (≡PND) 21, a total of 4 F1 females given 750 mg/kg/day were noted as having lung findings including mottled, firm, tan, for 2 animals and left cranial lobe, tan, meaty for 2 animals; these gross findings correlated with an increase in absolute lung
weights in this dose group. There were 2 F1 females given 750 mg/kg/day that had a fluid-filled cyst in the uterus at necropsy; the relationship to test item administration of this finding is unknown. All other macroscopic gross findings for F0 females at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
No test item-related effects were observed on the number of implantation sites. The number of females in proestrus, estrus, metestrus, and diestrus at demise were similar for all groups. The differences between the control and test item-treated groups were slight and not statistically significant.
ORGAN WEIGHTS
Summary Data: Tables 42, 67, Individual Data: Appendix 2, Tables 36, 58.
There was an increase in both absolute and relative lung weights (39.5% and 37.7%, respectively) in F1 males given 750 mg/kg/day. Absolute and relative lung weights were also increased [12.9% (statistically significant) and 6.9% (not statistically significant), respectively] in F1 females given 750 mg/kg/day compared with the control group. The increased lung weights correlated with the macroscopic and microscopic lung findings in rats given 750 mg/kg/day.
MICROSCOPIC EXAMINATION
Summary Data: Tables 44, 67, 69, Individual Data: Appendix 3.
Like the F0 males and females, chronic interstitial/alveolus inflammation was increased in incidence and severity in the lungs of F1 males and females given 750 mg/kg/day compared with the controls. All 24 F1 males and 24 F1 females given 750 mg/kg/day that survived until scheduled euthanasia had this test item-related finding. The increased lung weights correlated with the chronic interstitial/alveolus inflammation observed microscopically.
Also like the F0 animals, alveolar macrophage aggregation, alveolus was noted in F1 control male and female lungs and was characterized by the presence of foamy appearing macrophages in alveolar spaces without any other significant changes associated with it. This finding can be seen as a spontaneous change in the lungs of rats, though one cannot rule out the possibility that this finding is related to dosing with the corn oil vehicle.
Test item-related slight increases in incidence of renal tubule degeneration/necrosis and renal tubule hyaline droplets were seen in the F1 males given 750 mg/kg/day. Degeneration/necrosis was characterized by the presence of multifocal, cortical proximal convoluted tubules which were lined by epithelial cells that were slightly vacuolated with nuclear pyknosis and containing occasional sloughed necrotic cells. This change was very subtle and recorded as minimal. In a few instances, lightly staining eosinophilic droplets were increased in proximal convoluted tubules of the same animals. This morphologic appearance suggested hydrocarbon-induced α2μ-globulin male rat nephropathy. Hyaline droplets were not observed in the treated females.
Kidneys from selected males given the vehicle or 750 mg/kg/day from both the F0 and F1 generations (10 males/group/generation) were stained with Mallory Heidenhain stain which is used to characterize α2μ-globulin in renal tubules. As expected due to the age of these rats, renal tubules from all of these selected animals contained variable numbers of tubule droplets which stained positive and had the characteristic distribution and features of α2μ-globulin (male rats of this age contain α2μ-globulin). The droplets were subjectively scored on a scale of 1 to 5 +. No apparent increase in positive tubule droplets was present in the F0 males given 750 mg/kg/day compared with the F0 control males. However, the mean average score of the positive tubule droplets was increased in the F1 males given 750 mg/kg/day compared with the F1 control males, confirming that these kidney findings were the result of α2μ -globulinergic nephropathy, which is known to occur in male rats following exposure to hydrocarbons.
Other changes in the kidneys, such as chronic progressive nephropathy and renal tubule regeneration, were not clearly increased in either incidence or in severity and were therefore not deemed test item-related.
For F1 males and females in the low- and mid-dose groups suspected of reduced fertility, there were no test item-related microscopic findings in the reproductive organs/tissues examined. Two males given 50 mg/kg/day and 3 males given 200 mg/kg/day had inflammation of the prostate gland; however, 7 males given the vehicle control and 9 males given the high dose of 750 mg/kg/day had a similar finding; thus, this finding was not considered test item-related. One male given 200 mg/kg/day showed seminiferous tubule atrophy in the testis. One female given 50 mg/kg/day had dilatation of the uterus and one other had an ovarian cyst while one female given 200 mg/kg/day had ovarian atrophy. These microscopic findings were not observed in F1 males and females given the high dose of 750 mg/kg/day and may have contributed to the reduced fertility for these particular animals.
A number of other histopathological findings were noted in a number of tissues which were typical of the spontaneous type of microscopic pathology that may be observed in this strain and age of rat.
Ovarian follicle counts were not statistically significantly different in the F1 females given 750 mg/kg/day compared with the control group; therefore, counts were not conducted on the lower dose groups.
F2 LITTER DATA
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: Table 61, Individual Data: Appendix 2, Tables 50, 51.
The mean number of pups delivered (live, dead, and total); number of live pups per litter on PND 0, 4, 7, 14, and 21 (for litters having at least 1 live pup); percentage of males per litter on PND 0, 4, 7, 14, and 21; lactational index; and postnatal survival indices (4-, 7-, 14-, and 21-day) were unaffected by the test item at all dosage concentrations. Differences from the control group were slight and were not statistically significant.
GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: Table 63, Individual Data: Appendix 2, Tables 51, 54.
The numbers of F2 pups found dead, euthanized, and/or missing, as well as the general physical condition of all F2 pups in this study, were unaffected by test item administration. Pups that were found dead or euthanized (prior to scheduled euthanasia on PND 21) numbered 17, 17, 25, and 17 in the control, 50, 200, and 750 mg/kg/day groups, respectively. One litter (Dam No. 1352 found dead on LD 1) accounted for 10 of the 17 pups in the group given 750 mg/kg/day. In addition, 3, 3, 3 and 0 pups in the same respective groups were missing (and presumed dead).
ANOGENITAL DISTANCE
Summary Data: Table 62, Individual Data: Appendix 2, Table 52.
The anogenital distances (absolute and adjusted for body weight as the covariate) in the F2 male and female pups delivered from dams given 50, 200, and 750 mg/kg/day were similar to the respective control group values. Differences from the control group were slight and not statistically significant.
OFFSPRING BODY WEIGHTS
Summary Data: Table 62, Individual Data: Appendix 2, Table 53.
Mean male and female pup body weights and body weight changes in the 50, 200, and 750 mg/kg/day groups were unaffected by parental test item administration throughout the postnatal period. No statistically significant differences from the control group were noted.
NECROPSIES OF PUPS STILLBORN, FOUND DEAD, OR EUTHANIZED
Summary Data: Tables 63, 64, Individual Data: Appendix 2, Tables 54, 55.
The number of F2 pups (litters) stillborn, found dead, or euthanized from PND 0 through PND 21 were 17(11), 16(13), 25(13), and 17(4) in the control, 50, 200, and 750 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration to the test item were noted at the necropsies of F2 pups that were stillborn, found dead, or euthanized.
Findings in the stillborn, found dead, or euthanized pups from test item-treated dams were similar to the findings in stillborn, found dead, or euthanized pups from vehicle-treated dams, culled PND 4 pups, and/or scheduled death PND 21 F2 pups.
NECROPSIES OF WEANLINGS - PND 21
MACROSCOPIC EXAMINATION
Summary Data: Table 66, Individual Data: Appendix 2, Table 57.
At the PND 21 necropsy of F2 weanlings, there were no test item-related findings.
Hydronephrosis of the right kidney was noted at a similar incidence in males in the control and test item-treated groups.
ORGAN WEIGHTS
Summary Data: Table 65, Individual Data: Appendix 2, Table 56.
There were no statistically significant changes in F2 pup final body weights and organ weights compared with the control group. Slight decreases in absolute and relative spleen weights (approximately 7% to 8% and 4% to 7%, respectively) were observed in the F2 male and female pups in the group given 750 mg/kg/day compared with the control group. Given the small magnitude of the change, the lack of statistical significance, and since the weights fall within the historical control data ranges, these changes were considered equivocal and not adverse.
MICROSCOPIC EXAMINATION
Summary Data: Table 66, Individual Data: Appendix 3.
Dilatation of the pelvis of the kidney was noted for all male pups with a macroscopic finding of hydronephrosis; thus, this finding was noted at a similar incidence in the control and test item treated groups and was therefore not considered test item-related.
OBJECTIVE AND METHODS
The objective of this study was to characterize the effects of the test substance on the integrity and performance of the male and female reproductive systems, including gonadal function, the estrus cycle, mating behavior, conception, gestation, parturition, lactation, and weaning of the F1generation, as well as the neonatal survival, growth, and development of the F1and F2 offspring.
In addition, the systemic toxicity of the test substance was partially characterized in this study by evaluation of several non-reproductive tissues.
Three groups of male and female Crl:CD(SD) rats (25 and 28/sex/group for the F0and F1generations, respectively) were administered the test item (CAS No. 848301-67-7), daily by oral gavage for at least 70 consecutive days prior to mating. Dosage levels were 50, 200, and 750 mg/kg/day for the F0and F1generations. A concurrent control group of 25 and 28/sex for the F0and F1 generations, respectively, received the vehicle, corn oil (CAS No. 8001-30-7) on a comparable regimen. F0 animals were approximately 6 to 7 weeks (42 to 46 days) of age at the initiation of test item administration. The test item was administered to offspring selected to become the F1 parental generation following weaning, beginning on postnatal day (PND) 22.
The F0 and F1 males continued to receive the test item throughout mating and through the day prior to euthanasia. The F0 and F1 females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia.
Animals were observed at least twice daily for mortality. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for males throughout the study and for females prior to mating and during gestation and lactation. Vaginal smears were performed daily for determination of estrous cycles beginning 21 days prior to pairing. All F0 and F1 females were allowed to deliver and rear their pups until weaning on lactation day (LD≡PND) 21. Clinical observations, body weights, and sexes for F1and F2pups were recorded at appropriate intervals. For both generations (F1and F2), 10 pups per litter (5 per sex, when possible) were arbitrarily selected on PND 4 to reduce the variability among the litters.
Offspring (28/sex/group) from the pairing of the F0 animals were selected on PND 21 to constitute the F1generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1 rats. Nonselected F1 pups were necropsied on PND 21, and F2 pups were necropsied on PND 21. Selected organs (brain, spleen, and thymus) were weighed from 1 pup/sex/litter (when possible) from both F1 and F2 pups that were necropsied on PND 21. Each F0 and F1 parental animal received a complete detailed gross necropsy (the surviving female parental animals were necropsied on LD 21), and selected organs were weighed. Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0 and F1 males as appropriate, and ovarian primordial follicle counts and the corpora lutea counts were recorded for all F1 females in the control and high-dose groups. Designated tissues from all F0 and F1 parental animals were examined microscopically.
PREBREED EXPOSURE PERIOD
Summary Data: Tables 1, 6, 8, 14, 33, Individual Data: Appendix 2, Tables 1, 4, 7, 10, 29
There were no deaths attributed to 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' administration noted through the F0 prebreed exposure period (Days 0 through 70). There were no test item-related clinical observations from Day 0 to 70. All clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Many of the findings of alopecia and sores were likely due to the animals scratching at the ear tag and surrounding area(s).
MATING, MALE POSTMATING, AND FEMALE GESTATION AND LACTATION PERIODS
Summary Data: Tables 6, 14, 20, 25, 33, Individual Data: Appendix 2, Tables 4, 10, 14, 17, 29
For the F0 males, there were no deaths during the mating/postmating period (Days 70 through 107). There were no test item-related clinical observations from Days 70 to 107.
All clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Many of the findings of alopecia and sores were likely due to the animals scratching at the ear tag and surrounding area(s).
For the F0 females, there were no deaths attributed to 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' administration during the mating, gestation, and lactation periods (Days 70 to 128). Female No. 60 (Group 2) was euthanized for humane reasons on Day 93 (LD 0) due to dystocia after delivering 6 dead pups.
Clinical observations for this animal prior to death included labored breathing, piloerection, pale eyes, and stretching/straining during labor. All other animals survived to study termination.
Macroscopic findings for Female No. 60 included pale kidneys, adrenal glands, lungs, and brain; pale areas in all lobes of the liver; and dark small and large intestines. There were 13 visible implant sites and 8 pups (2 live and 6 dead) in the uterus at the time of necropsy.
From Day 71 through Day 113, there were no test item-related clinical observations in the F0 females that remained sperm negative; all clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
REPRODUCTIVE PERFORMANCE
Summary Data: Tables 15, 26, Individual Data: Appendix 2, Tables 6, 11, 18, 19
No test item-related effects on F0 reproductive performance were observed at any dosage level.
There were no statistically significant differences on male and female mating indices and male and female fertility indices. Male mating indices were 100.0%, 96.0%, 100.0%, and 100.0% and female mating indices were 100.0%, 96.0%, 100.0%, and 100.0% in the control, 50, 250, and 1000 mg/kg/day groups, respectively. Historical control data male and female mating indices range from 87.5% to 100% and 88% to 100%, respectively. Male fertility indices were 84.6%, 91.7%, 84.0% and 80.8% and female fertility indices were 84.6%, 91.7%, 84.0%, and 80.8% in the same respective groups. All fertility indicies were within the historical control data ranges of 75.9% to 100% for F0 males and 76.7% to 100% for F0 females.
All F0 females evaluated during the final 3 weeks of the 10-week prebreed period were noted to be cycling, with a similar number and percent of females with abnormal or irregular cycles noted across all groups, including the control. There was no difference in mean cycle length (4.0 to 4.3 days) or number of cycles (4.0 to 4.4) in test item-treated groups compared with control values (4.0 days and 4.5, respectively). The mean numbers of days between pairing and coitus (i.e., the precoital interval) in the test item-treated groups were similar to the control group value of 3.1 days.
GESTATION LENGTH AND PARTURITION
Summary Data: Table 26, Individual Data: Appendix 2, Table 19
No test item-related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Mean F0 gestation lengths in the test item-treated groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the groups given 50, 250, and 1000 mg/kg/day were 22.0, 22.2, and 22.3 days, respectively, compared to mean gestation length of 22.3 days in the concurrent control group. Gestation indices were 90.9%, 100.0%, and 95.2% in the groups given 50, 250, and 1000 mg/kg/day, respectively, and were not statistically significantly different from the control group value of 100.0% and were within the historical control data range (87% to 100%). No statistically significant differences were noted between the control and test item-treated groups in the number or percentage of females completing delivery and females completing delivery with stillborn pups or with all stillborn. No statistically significant differences in the mean number of implantation sites, percent postimplantation loss, pups delivered (live, dead, and total), stillbirth
index, live birth index, and number of live pups per litter on PND 0 and PND 4 for litters having at least 1 live pup in the group were noted.
BODY WEIGHTS, FOOD CONSUMPTION, AND FOOD EFFICIENCY
PREBREED EXPOSURE, MATING, AND POSTMATING PERIODS
Summary Data: Tables 2, 3, 4, 5, 10, 11, 12, 13, Individual Data: Appendix 2, Tables 2, 3, 8, 9.
There were no adverse test item-related effects on body weights, body weight changes, or feed consumption during the prebreed exposure period (Days 0 to 70). Occasional statistically significant changes in these parameters occurred, though none of the changes were adverse.
Males given 1000 mg/kg/day showed statistically significant increases in feed consumption (g/kg/day; 6.3% to 6.9%) at several intervals, beginning with the Day 35 to 42 interval, such that the overall feed consumption (Day 0 to 70) for this group was increased significantly (4.5%) compared with the control group. A corresponding, statistically significant increase (29.7%) in body weight change was seen only at the Day 49 to 56 interval, with no significant changes in body weight. Statistically significant decreases in percent food efficiency (12.1% and 15.7%) occurred at two intervals (Days 28 to 35 and 42 to 49, respectively) for males given 1000 mg/kg/day. Other slight changes were noted for males given 50 mg/kg/day [decreased feed consumption (g/kg/day) at one interval] and males given 250 mg/kg/day (changes in percent food efficiency at two intervals and increased body weight changes at two intervals). Females given 1000 mg/kg/day showed corresponding, statistically significant increases in body feed consumption (g/day and g/kg/day; 13.8% and 16.8%, respectively) and weight change (4.7%) for the Day 14 to 21 interval, with no corresponding statistically significant change in percent food efficiency or body weight compared with the control group. Feed consumption (g/kg/day) was also increased significantly (10.5%) for females given 1000 mg/kg/day for the Day 28 to 35 interval.
There were no adverse test item-related effects on F0 male body weights or body weight changes from Days 70 to 105 and feed consumption or percent food efficiency from Days 84 to 105.
Although statistically significant increases in feed consumption (g/day and/or g/kg/day) for one or more intervals from Days 84 to 105 were noted for the F0 males given 1000 mg/kg/day, with an overall increase in feed consumption of 9% (g/day) and 8% (g/kg/day) for the postmating holding period, these changes in feed consumption did not result in or correlate with statistically significant effects on body weights, body weight changes, or percent food efficiency compared with the controls. A sporadic, nondose-related, statistically significant increase in body weight change in F0 males given 250 mg/kg/day was noted for 1 interval (Days 98 to 105), which did not correlate with any statistically significant changes in body weight, feed consumption, or
percent food efficiency.
In general, there were no notable test item-related effects on body weights and body weight changes from Days 70 to 112 and on feed consumption (g/day and g/kg/day) and percent food efficiency from Days 70 to 112 for those F0 females that were never found sperm positive or that never delivered a live litter. Occasional statistically significant changes were observed in these parameters, but were not considered test item-related as they were sporadic and not dose related.
GESTATION
Summary Data: Tables 16, 17, 18, 19, Individual Data: Appendix 2, Tables 12, 13.
During gestation (GD 0 to 22), there were no test item-related changes in F0 female body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group.
LACTATION
Summary Data: Tables 21, 22, 23, 24, Individual Data: Appendix 2, Tables 15, 16.
During lactation (LD 0 to 21), there were no test item-related changes in F0 female body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group.
ANDROLOGY
Summary Data: Table 7, Individual Data: Appendix 2, Table 6
No test item-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility and morphology) in males at any dosage concentration. The statistically significant reduction (10%) in percent progressively motile sperm in males given 50 mg/kg/day was mainly driven by one male (No. 67) with 7% progressively motile sperm; this male did not sire a litter. This finding in the low dose group was not considered test item-related as no dose response was observed.
ANATOMIC PATHOLOGY – SCHEDULED DEATHS
MACROSCOPIC EXAMINATION
Summary Data: Tables 7, 8, 26, 32, 33, Individual Data: Appendix 2, Tables 5, 7, 19, 28, 29.
The only test item-related macroscopic finding in F0 parental animals was noted in the lungs of the males and females given 1000 mg/kg/day. At the F0 male necropsy, males given 1000 mg/kg/day were noted as having an increased incidence of lung findings (mottled, discolored, thickened, firm, and/or multiple foci in 5 of 26 animals) compared with controls (0 of 26 animals with lung findings) and animals given 50 and 250 mg/kg/day (1 of 25 animals with lung findings in each of these groups); this gross finding correlated with an increase in both absolute and relative lung weights in the high-dose group. One male given 1000 mg/kg/day had a mass adjacent/attached to the thoracic cavity side of the diaphragm; the relationship of this finding to test article administration is unknown. In addition, the left testes and epididymis of one male (No. 57) given 50 mg/kg/day were found to be reduced in size and the left testis was flaccid; however, this male did sire a litter. These findings in this one low-dose male were considered sporadic and not test item-related as there was no dose response observed. All other macroscopic findings for the F0 males at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
At the F0 female necropsies conducted on LD (≡PND) 21, lung findings of discoloration (tan, grey, or white) or mottled (with or without nodules present) were noted with greater incidence in the females given 1000 mg/kg/day (8 of 26 animals) compared with the controls (1 of 26 animals). There was a low incidence of dilatation of the pelvis of the right kidney and the presence of a nodule on the kidney(s) in females given 250 and 1000 mg/kg/day. Although these findings showed a dose response, nodules on kidneys were also noted in F0 males in all groups, including controls. Based on the low incidence of kidney nodules in the females, which was similar to the incidence in control males, this finding was not considered test item-related. All other macroscopic gross findings for F0 females at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
No test item-related effects were observed on the number of implantation sites. The number of females in proestrus, estrus, metestrus, and diestrus at demise were similar for all groups. The differences between the control and test item-treated groups were slight and not statistically significant.
ORGAN WEIGHTS
Summary Data: Tables 7, 32, Individual Data: Appendix 2, Tables 5, 27.
There was a statistically significant increase in both absolute and relative lung weights (approximately13% and 11%, respectively) in F0 males given 1000 mg/kg/day. Absolute and relative lung weights were also statistically significantly increased (approximately14% and 14%, respectively) in F0 females given 1000 mg/kg/day compared with the control group.
MICROSCOPIC EXAMINATION
Summary Data: Tables 9, 34, Individual Data: Appendix 3
A test item-related histopathological lesion was identified in the lungs. Chronic interstitial/alveolus inflammation was increased in incidence and severity in the lungs of F0 male and females given 1000 mg/kg/day compared with the controls. This change was also apparent in control animals but at a much lower incidence and severity, and therefore was considered to be a test item-related finding. The increased lung weights correlated with the chronic interstitial/alveolus inflammation observed microscopically. This lesion was characterized by a spectrum of lesions which were severity dependent and included the presence of foamy to highly vacuolated macrophages within alveolar spaces, thickened alveolar septae due to the infiltration of primarily mononuclear inflammatory cells and hypertrophy/hyperplasia of alveolar epithelial cells, edema, slight congestion and/or hemorrhage, occasional infiltration of polymorphonuclear inflammatory cells, fibrosis, increased perivascular cuffing by mononuclear inflammatory cells and a variable hyperplasia of the bronchiolar associated lymphoid tissue (BALT). This inflammatory change was graded on a 5-grade severity scale where 1 was the most minimal change noted and 5 was the most severe. Most test item-exposed animals had a severity of mild to moderately severe (2-4). The overall lung severity grade was an average from both the left and right lobes. Inflammatory changes were usually focal to multifocal. Only when the severity was grade 3 to 4 did the inflammatory foci tend to merge and become confluent. The left lung tended to have more lesions and more severe lesions than the right lobe. Lesions, in general, were highly airway-centric, located near the terminal portions of the airways. This appearance was suggestive of chemical exposure through aspiration. In other areas, this determination was more difficult but may have been influenced by sectioning. There did not appear to be any significant difference between male and female lungs with regard to the appearance of chronic interstitial/alveolus inflammation.
This change was noted mainly in control male and female lungs and was characterized by the presence of foamy appearing macrophages in alveolar spaces without any other significant changes associated with it. This non-adverse finding, although it can be seen as a spontaneous change in the lungs of rats, was most likely related to dosing with the corn oil vehicle and was also observed in the 90-day oral repeated dose toxicity study with CLINICAL OBSERVATIONS AND MORTALITY
PREBREED EXPOSURE PERIOD
Summary Data: Tables 1, 6, 8, 14, 33, Individual Data: Appendix 2, Tables 1, 4, 7, 10, 29
There were no deaths attributed to 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' administration noted through the F0 prebreed exposure period (Days 0 through 70). There were no test item-related clinical observations from Day 0 to 70. All clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Many of the findings of alopecia and sores were likely due to the animals scratching at the ear tag and surrounding area(s).
MATING, MALE POSTMATING, AND FEMALE GESTATION AND LACTATION PERIODS
Summary Data: Tables 6, 14, 20, 25, 33, Individual Data: Appendix 2, Tables 4, 10, 14, 17, 29
For the F0 males, there were no deaths during the mating/postmating period (Days 70 through 107). There were no test item-related clinical observations from Days 70 to 107.
All clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Many of the findings of alopecia and sores were likely due to the animals scratching at the ear tag and surrounding area(s).
For the F0 females, there were no deaths attributed to 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' administration during the mating, gestation, and lactation periods (Days 70 to 128). Female No. 60 (Group 2) was euthanized for humane reasons on Day 93 (LD 0) due to dystocia after delivering 6 dead pups.
Clinical observations for this animal prior to death included labored breathing, piloerection, pale eyes, and stretching/straining during labor. All other animals survived to study termination.
Macroscopic findings for Female No. 60 included pale kidneys, adrenal glands, lungs, and brain; pale areas in all lobes of the liver; and dark small and large intestines. There were 13 visible implant sites and 8 pups (2 live and 6 dead) in the uterus at the time of necropsy.
From Day 71 through Day 113, there were no test item-related clinical observations in the F0 females that remained sperm negative; all clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
REPRODUCTIVE PERFORMANCE
Summary Data: Tables 15, 26, Individual Data: Appendix 2, Tables 6, 11, 18, 19
No test item-related effects on F0 reproductive performance were observed at any dosage level.
There were no statistically significant differences on male and female mating indices and male and female fertility indices. Male mating indices were 100.0%, 96.0%, 100.0%, and 100.0% and female mating indices were 100.0%, 96.0%, 100.0%, and 100.0% in the control, 50, 250, and 1000 mg/kg/day groups, respectively. Historical control data male and female mating indices range from 87.5% to 100% and 88% to 100%, respectively. Male fertility indices were 84.6%, 91.7%, 84.0% and 80.8% and female fertility indices were 84.6%, 91.7%, 84.0%, and 80.8% in the same respective groups. All fertility indicies were within the historical control data ranges of 75.9% to 100% for F0 males and 76.7% to 100% for F0 females.
All F0 females evaluated during the final 3 weeks of the 10-week prebreed period were noted to be cycling, with a similar number and percent of females with abnormal or irregular cycles noted across all groups, including the control. There was no difference in mean cycle length (4.0 to 4.3 days) or number of cycles (4.0 to 4.4) in test item-treated groups compared with control values (4.0 days and 4.5, respectively). The mean numbers of days between pairing and coitus (i.e., the precoital interval) in the test item-treated groups were similar to the control group value of 3.1 days.
GESTATION LENGTH AND PARTURITION
Summary Data: Table 26, Individual Data: Appendix 2, Table 19
No test item-related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Mean F0 gestation lengths in the test item-treated groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the groups given 50, 250, and 1000 mg/kg/day were 22.0, 22.2, and 22.3 days, respectively, compared to mean gestation length of 22.3 days in the concurrent control group. Gestation indices were 90.9%, 100.0%, and 95.2% in the groups given 50, 250, and 1000 mg/kg/day, respectively, and were not statistically significantly different from the control group value of 100.0% and were within the historical control data range (87% to 100%). No statistically significant differences were noted between the control and test item-treated groups in the number or percentage of females completing delivery and females completing delivery with stillborn pups or with all stillborn. No statistically significant differences in the mean number of implantation sites, percent postimplantation loss, pups delivered (live, dead, and total), stillbirth
index, live birth index, and number of live pups per litter on PND 0 and PND 4 for litters having at least 1 live pup in the group were noted.
BODY WEIGHTS, FOOD CONSUMPTION, AND FOOD EFFICIENCY
PREBREED EXPOSURE, MATING, AND POSTMATING PERIODS
Summary Data: Tables 2, 3, 4, 5, 10, 11, 12, 13, Individual Data: Appendix 2, Tables 2, 3, 8, 9.
There were no adverse test item-related effects on body weights, body weight changes, or feed consumption during the prebreed exposure period (Days 0 to 70). Occasional statistically significant changes in these parameters occurred, though none of the changes were adverse.
Males given 1000 mg/kg/day showed statistically significant increases in feed consumption (g/kg/day; 6.3% to 6.9%) at several intervals, beginning with the Day 35 to 42 interval, such that the overall feed consumption (Day 0 to 70) for this group was increased significantly (4.5%) compared with the control group. A corresponding, statistically significant increase (29.7%) in body weight change was seen only at the Day 49 to 56 interval, with no significant changes in body weight. Statistically significant decreases in percent food efficiency (12.1% and 15.7%) occurred at two intervals (Days 28 to 35 and 42 to 49, respectively) for males given 1000 mg/kg/day. Other slight changes were noted for males given 50 mg/kg/day [decreased feed consumption (g/kg/day) at one interval] and males given 250 mg/kg/day (changes in percent food efficiency at two intervals and increased body weight changes at two intervals). Females given 1000 mg/kg/day showed corresponding, statistically significant increases in body feed consumption (g/day and g/kg/day; 13.8% and 16.8%, respectively) and weight change (4.7%) for the Day 14 to 21 interval, with no corresponding statistically significant change in percent food efficiency or body weight compared with the control group. Feed consumption (g/kg/day) was also increased significantly (10.5%) for females given 1000 mg/kg/day for the Day 28 to 35 interval.
There were no adverse test item-related effects on F0 male body weights or body weight changes from Days 70 to 105 and feed consumption or percent food efficiency from Days 84 to 105.
Although statistically significant increases in feed consumption (g/day and/or g/kg/day) for one or more intervals from Days 84 to 105 were noted for the F0 males given 1000 mg/kg/day, with an overall increase in feed consumption of 9% (g/day) and 8% (g/kg/day) for the postmating holding period, these changes in feed consumption did not result in or correlate with statistically significant effects on body weights, body weight changes, or percent food efficiency compared with the controls. A sporadic, nondose-related, statistically significant increase in body weight change in F0 males given 250 mg/kg/day was noted for 1 interval (Days 98 to 105), which did not correlate with any statistically significant changes in body weight, feed consumption, or
percent food efficiency.
In general, there were no notable test item-related effects on body weights and body weight changes from Days 70 to 112 and on feed consumption (g/day and g/kg/day) and percent food efficiency from Days 70 to 112 for those F0 females that were never found sperm positive or that never delivered a live litter. Occasional statistically significant changes were observed in these parameters, but were not considered test item-related as they were sporadic and not dose related.
GESTATION
Summary Data: Tables 16, 17, 18, 19, Individual Data: Appendix 2, Tables 12, 13.
During gestation (GD 0 to 22), there were no test item-related changes in F0 female body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group.
LACTATION
Summary Data: Tables 21, 22, 23, 24, Individual Data: Appendix 2, Tables 15, 16.
During lactation (LD 0 to 21), there were no test item-related changes in F0 female body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency in the test item-treated groups compared with the control group.
ANDROLOGY
Summary Data: Table 7, Individual Data: Appendix 2, Table 6
No test item-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility and morphology) in males at any dosage concentration. The statistically significant reduction (10%) in percent progressively motile sperm in males given 50 mg/kg/day was mainly driven by one male (No. 67) with 7% progressively motile sperm; this male did not sire a litter. This finding in the low dose group was not considered test item-related as no dose response was observed.
ANATOMIC PATHOLOGY – SCHEDULED DEATHS
MACROSCOPIC EXAMINATION
Summary Data: Tables 7, 8, 26, 32, 33, Individual Data: Appendix 2, Tables 5, 7, 19, 28, 29.
The only test item-related macroscopic finding in F0 parental animals was noted in the lungs of the males and females given 1000 mg/kg/day. At the F0 male necropsy, males given 1000 mg/kg/day were noted as having an increased incidence of lung findings (mottled, discolored, thickened, firm, and/or multiple foci in 5 of 26 animals) compared with controls (0 of 26 animals with lung findings) and animals given 50 and 250 mg/kg/day (1 of 25 animals with lung findings in each of these groups); this gross finding correlated with an increase in both absolute and relative lung weights in the high-dose group. One male given 1000 mg/kg/day had a mass adjacent/attached to the thoracic cavity side of the diaphragm; the relationship of this finding to test article administration is unknown. In addition, the left testes and epididymis of one male (No. 57) given 50 mg/kg/day were found to be reduced in size and the left testis was flaccid; however, this male did sire a litter. These findings in this one low-dose male were considered sporadic and not test item-related as there was no dose response observed. All other macroscopic findings for the F0 males at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
At the F0 female necropsies conducted on LD (≡PND) 21, lung findings of discoloration (tan, grey, or white) or mottled (with or without nodules present) were noted with greater incidence in the females given 1000 mg/kg/day (8 of 26 animals) compared with the controls (1 of 26 animals). There was a low incidence of dilatation of the pelvis of the right kidney and the presence of a nodule on the kidney(s) in females given 250 and 1000 mg/kg/day. Although these findings showed a dose response, nodules on kidneys were also noted in F0 males in all groups, including controls. Based on the low incidence of kidney nodules in the females, which was similar to the incidence in control males, this finding was not considered test item-related. All other macroscopic gross findings for F0 females at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
No test item-related effects were observed on the number of implantation sites. The number of females in proestrus, estrus, metestrus, and diestrus at demise were similar for all groups. The differences between the control and test item-treated groups were slight and not statistically significant.
ORGAN WEIGHTS
Summary Data: Tables 7, 32, Individual Data: Appendix 2, Tables 5, 27.
There was a statistically significant increase in both absolute and relative lung weights (approximately13% and 11%, respectively) in F0 males given 1000 mg/kg/day. Absolute and relative lung weights were also statistically significantly increased (approximately14% and 14%, respectively) in F0 females given 1000 mg/kg/day compared with the control group.
MICROSCOPIC EXAMINATION
Summary Data: Tables 9, 34, Individual Data: Appendix 3
A test item-related histopathological lesion was identified in the lungs. Chronic interstitial/alveolus inflammation was increased in incidence and severity in the lungs of F0 male and females given 1000 mg/kg/day compared with the controls. This change was also apparent in control animals but at a much lower incidence and severity, and therefore was considered to be a test item-related finding. The increased lung weights correlated with the chronic interstitial/alveolus inflammation observed microscopically. This lesion was characterized by a spectrum of lesions which were severity dependent and included the presence of foamy to highly vacuolated macrophages within alveolar spaces, thickened alveolar septae due to the infiltration of primarily mononuclear inflammatory cells and hypertrophy/hyperplasia of alveolar epithelial cells, edema, slight congestion and/or hemorrhage, occasional infiltration of polymorphonuclear inflammatory cells, fibrosis, increased perivascular cuffing by mononuclear inflammatory cells and a variable hyperplasia of the bronchiolar associated lymphoid tissue (BALT). This inflammatory change was graded on a 5-grade severity scale where 1 was the most minimal change noted and 5 was the most severe. Most test item-exposed animals had a severity of mild to moderately severe (2-4). The overall lung severity grade was an average from both the left and right lobes. Inflammatory changes were usually focal to multifocal. Only when the severity was grade 3 to 4 did the inflammatory foci tend to merge and become confluent. The left lung tended to have more lesions and more severe lesions than the right lobe. Lesions, in general, were highly airway-centric, located near the terminal portions of the airways. This appearance was suggestive of chemical exposure through aspiration. In other areas, this determination was more difficult but may have been influenced by sectioning. There did not appear to be any significant difference between male and female lungs with regard to the appearance of chronic interstitial/alveolus inflammation.
This change was noted mainly in control male and female lungs and was characterized by the presence of foamy appearing macrophages in alveolar spaces without any other significant changes associated with it. This non-adverse finding, although it can be seen as a spontaneous change in the lungs of rats, was most likely related to dosing with the corn oil vehicle and was also observed in the 90-day oral repeated dose toxicity study with 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' (see IUCLID section 7.5.1).
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: Table 26, Individual Data: Appendix 2, Tables 19, 20
The mean number of pups delivered (live, dead, and total); number of live pups per litter on PND 0, 4, 7, 14, and 21(for litters having at least 1 live pup); percentage of males per litter on PND 0, 4, 7, 14, and 21; lactational index; and postnatal survival indices (4-, 7-, 14-, and 21-day) were unaffected by the test item at all dosage concentrations. Differences from the control group were slight and were not statistically significant.
GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: Table 28, Individual Data: Appendix 2, Tables 20, 23
The numbers of F1 pups found dead, euthanized, and/or missing, as well as the general physical condition of all F1 pups in this study were unaffected by test item administration. Pups that were found dead or missing (and presumed dead) prior to scheduled euthanasia on PND 21 numbered 10, 17, 9, and 9 in the control, 50, 250, and 1000 mg/kg/day groups, respectively.
ANOGENITAL DISTANCE
Summary Data: Table 27, Individual Data: Appendix 2, Table 21
Absolute AGD was statistically significantly decreased in the F1 female pups delivered from dams given 50, 250, and 1000 mg/kg/day (-20%, -27%, and -19%, respectively) when compared with the control group. When adjusted for body weight as the covariate, AGD was decreased by -16%, -26%, and -19%, respectively, in the 50, 250 and 1000 mg/kg/day groups (not statistically significant for the group given 50 mg/kg/day). These decreases in F1 female AGD and adjusted AGD were considered equivocal and not adverse. F1 pup AGD is a nonstandard endpoint not required by the OECD 416 guidelines therefore, no historical control ranges exist at this test facility. However, the historical control range for AGD and adjusted AGD in F2 females is 0.73 to 1.10 mm and 0.76 to 1.10 mm, respectively. The AGD values for the F1 females in this study fell within these historical control ranges and within the range of values noted for the F2 females on this study (which were comparable to the control values).
Other female parameters such as vaginal patency and estrous cyclicity were unaffected by test item administration. A decrease in AGD values in females is generally not considered toxicologically relevant. In addition, the biological meaning of this small change in AGD is not clear as hyperfeminization was not apparent based on other endpoints and since there were no adverse effects on the F1 reproductive parameters or the F2 pup parameters. .
OFFSPRING BODY WEIGHTS
Summary Data: Table 27, Individual Data: Appendix 2, Table 22
Mean male and female pup body weights and body weight changes in the groups given 50, 250, and 1000 mg/kg/day were unaffected by test item administration throughout the postnatal period.
No statistically significant differences from the control group were noted.
NECROPSIES OF PUPS STILLBORN, FOUND DEAD, OR EUTHANIZED
Summary Data: Tables 28, 29, Individual Data: Appendix 2, Tables 23, 24
The numbers of pups (litters) stillborn, found dead, or euthanized from PND 0 through the selection of the F1 generation on PND 21 numbered 10(6), 18(6), 8(6), and 7(6) in the control, 50, 250 and 1000 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration of the test item were noted at the necropsies of pups that were stillborn, found dead, or euthanized.
NECROPSIES OF WEANLINGS - PND 21
MACROSCOPIC EXAMINATION
Summary Data: Table 31, Individual Data: Appendix 2, Table 26
At the PND 21 necropsy of F1 weanlings, there were no findings in the test item-treated groups that were not also observed with similar or higher incidence in the control group.
ORGAN WEIGHTS
Summary Data: Table 30, Individual Data: Appendix 2, Table 25
There were no statistically significant changes in F1 pup final body weights and organ weights compared with the control group.
MICROSCOPIC EXAMINATION
Summary Data: Table 31, Individual Data: Appendix 3
There were no microscopic findings in the F1 PND 21 weanlings in the test item-treated groups that were not also observed with similar or higher incidence in the control group.
DEVELOPMENTAL LANDMARKS F1
BALANOPREPUTIAL SEPARATION
Summary Data: Table 36, Individual Data: Appendix 2, Table 31
Mean age and adjusted age of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test item administration. The mean ages of attainment of balanopreputial separation were 43.9, 43.9, and 42.6 days in the 50, 250, and 1000 mg/kg/day groups, respectively, when compared to 43.0 days in the control group. Mean body weights at the age of attainment were 247.3, 249.5, and 238.4 g in the same respective groups when compared to 247.4 g in the control group. None of the differences from the control group were statistically significant
VAGINAL PATENCY
Summary Data: Table 36, Individual Data: Appendix 2, Table 32
Mean age and adjusted age of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test item administration. The mean ages of attainment of vaginal patency were 31.4, 31.9, and 30.8 days in the groups given 50, 250, and 1000 mg/kg/day, respectively compared with 31.1 days in the control group. Mean body weights at the age of attainment were 113.3, 119.1, and 109.6 g in the same respective groups compared with 115.2 g in the control group. None of the differences from the control group were statistically significant.
GENERATION F1
CLINICAL OBSERVATIONS AND MORTALITY
PREBREED EXPOSURE PERIOD
Summary Data: Tables 35, 41, 43, 49, 68, Individual Data: Appendix 2, Tables 30, 35, 38, 41, 60
There were no deaths directly attributed to 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' administration noted through the F1 weaning and prebreed exposure period (Days -13 through 70). Four unscheduled deaths (1 found dead and 3 euthanized) occurred through Day 70.
Male No. 1321 (Group 4, 1000 mg/kg/day) was euthanized moribund on Day 12 (PND 44) with clinical observations of gasping, pale ear(s), and cold to touch. At necropsy, the lungs from Male No. 1321 failed to collapse and slight pressure on the lungs released fluid from the trachea, suggesting the cause of death of Male No. 1321 was a gavage error. This animal also had dilitation of the right kidney pelvis, a common finding in rats that was noted in some of the concurrent control rats on this study. Male No. 1317 (Group 4, 1000 mg/kg/day) was found dead on Day 13 (PND 46), with no previous clinical signs noted. At necropsy, all lobes of the lungs were red and mottled, suggesting possible aspiration of the dose formulation. Female No. 1212 (Group 3, 250 mg/kg/day) was euthanized for humane reasons on Day 28 (PND 61). This animal had caught its head in the cage lid the previous day and was observed to have a swollen snout and chromodaccyorrhea of the eye. The following day, additional observations of this animal included lethargy, head shaking, sunken eye, and mouth open (lower jaw hanging).
There were no gross lesions observed at necropsy for Animal No. 1212. Male No. 1221 (Group 3, 250 mg/kg/day) was euthanized for humane reasons on Day 60 (PND 92) due to clinical observations of labored breathing and discharge from the mouth. The lungs from Animal No. 1221 were mottled (all lobes) and failed to collapse at necropsy, and slight pressure on the lungs released bubbles from the trachea. The esophagus was filled with a yellow/brown substance.
Hydronephrosis of the right kidney was also noted. Necropsy findings for Animal No. 1221 suggest the cause of death was gavage error or aspiration of the dose formulation into the lungs.
All other animals survived to Day 70.
There were no test item-related clinical observations. All clinical findings in the test item-treatedgroups were noted with similar incidence in the control group, were limited to single animals, were limited to unscheduled death animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
MATING, MALE POSTMATING, AND FEMALE GESTATION AND
LACTATION PERIODS
Summary Data: Tables 41, 43, 49, 55, 60, 68, Individual Data: Appendix 2, Tables 35, 38, 41, 45, 48, 60
For the F1 males, there was one death during the mating/postmating period (Days 70 through 107). Animal No. 1303 (Group 4, 1000 mg/kg/day) was euthanized moribund on Day 93 with clinical observations of lethargic, pale ear(s), and rough coat. At necropsy, Male No. 1303 was noted with several findings including an enlarged liver and spleen; distended small intestines;
pale adrenal glands, brain, and lungs; and a depression in the brain with a dark mass nearby. The cause for these various macroscopic findings and the cause of death could not be definitively determined at necropsy. All clinical findings in the surviving F1 male test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Many of the findings of alopecia and sores were likely the result of the animals scratching at the ear tag and surrounding area(s).
For the F1 females, there were no deaths directly attributed to 'Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear' administration during the mating, gestation, and lactation periods (Days 70 to 129); there were two unscheduled F1 female deaths during this time. Female No. 1010 (Group 1, 0 mg/kg/day) was euthanized on Day 94 (GD 23) due to dystocia. Female No. 1208 (Group 3, 250 mg/kg/day) was euthanized on Day 71 due to a laceration inflicted during mating.
From Day 71 through Day 113, there were no test item-related clinical observations in the F1 females that remained sperm negative; all clinical findings in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
For sperm-positive F1 females, there were no test item-related clinical observations during the gestation period (GD 0 through 23). Likewise, there were no test item-related clinical observations during the lactation period (LD 0 through 21). All clinical findings during gestation and lactation in the test item-treated groups were noted with similar incidence in the control group, were limited to single animals or single occurrences, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain. Many of the findings of alopecia and sores were likely the result of the animals scratching at the ear tag and surrounding area(s).
REPRODUCTIVE PERFORMANCE
Summary Data: Tables 50, 61, Individual Data: Appendix 2, Tables 42, 49, 50
No test item-related effects on F1 reproductive performance were observed at any dosage level.
There were no statistically significant differences on male and female mating indices and male and female fertility indices. Male mating indices were 92.0%, 96.0%, 73.9%, and 87.0% and female mating indices were 92.0%, 96.0%, 73.9%, and 88.0% in the control, 50, 250 and 1000 mg/kg/day groups, respectively. Although the mating index for males and females was lower in the group given 250 mg/kg/day, all F1 mating indicies were within the historical control data ranges of 70.0% to 100% and 50% to 100% for males and females, respectively. This change was not considered test item-related since no dose response was observed. Male fertility indices were 95.7%, 95.8%, 82.4% and 95.0% and female fertility indices were 95.7%, 95.8%, 82.4% and 95.5% in the same respective groups. Although the fertility index for males and females was lower in the group given 250 mg/kg/day, all fertility indicies were within the historical control data ranges of 76.2% to 100% for F1 males and 70.8% to 100% for F1 females. This change was not considered test item-related since no dose response was observed.
All F1 females (control and test item-treated) evaluated during the final 3 weeks of the 10-week prebreed period were noted to be cycling. There was no statistically significant difference in the number and percent of test item-treated females with abnormal or irregular cycles compared with the control group. There was no difference in mean cycle length (4.0 to 4.1 days) and number of cycles (4.4) in test item-treated groups compared with control values (4.1 days and 4.5, respectively). The mean numbers of days between pairing and coitus (i.e., the precoital interval) in the test item-treated groups were similar to the control group value of 2.4 days.
GESTATION LENGTH AND PARTURITION
Summary Data: Table 61, Individual Data: Appendix 2, Table 50
No test item-related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Mean F1 gestation lengths in the test item-treated groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the groups given 50, 250, and 1000 mg/kg/day were 22.2, 22.3 and 22.4 days, respectively, compared with a mean gestation length of 22.0 days in the concurrent control group. Gestation indices were 100%, 85.7%, and 100.0% in the groups given 50, 250, and 1000 mg/kg/day, respectively, and were not statistically significantly different from the control group value of 95.5%. The historical control data range for F1 gestational index is 94.1% to 100%. The slight, though not statistically significant, decrease in the live born gestation index in the group given 250 mg/kg/day was due to Female No. 1244 having implantation sites but not delivering a litter and Female No. 1224 delivering a litter of all dead pups. The decrease in gestation index was not considered test item-related since no dose response was observed. No statistically significant differences were noted between the control and test item-treated groups in the number or percentage of females completing delivery and females completing delivery with stillborn pups or with all stillborn. No statistically significant differences in the mean number of implantation sites, percent postimplantation loss, pups delivered (live, dead, and total), stillbirth index, live birth index, and number of live pups per litter on PND 0 and PND 4 for litters having at least 1 live pup in the group were noted.
BODY WEIGHTS, FOOD CONSUMPTION, AND FOOD EFFICIENCY
PREBREED EXPOSURE, MATING, AND POSTMATING PERIODS
Summary Data: Tables 37, 38, 39, 40, 45, 46, 47, 48, Individual Data: Appendix 2, Tables 33, 34, 39, 40
There were no adverse test item-related effects on body weights, body weight changes, or feed consumption during the F1 prebreed exposure period (Days 0 to 70) for males or females. A statistically significant lower body weight change for one interval (Days 70 to 77) was noted for the F1 males given 250 mg/kg/day; however, this change did not result in or correlate with statistically significant effects on body weights, body weight changes, or percent food efficiency compared with the controls. Although statistically significant increases were noted in feed consumption (g/day or g/kg/day) for one or more intervals from Days 49 to 70 for the F1 males and Days 14 to 56 for F1 females given 1000 mg/kg/day, with overall increases in feed consumption (g/kg/day) of 5% and 6% for males and females, respectively, these changes in feed consumption did not result in or correlate with statistically significant effects on body weights, body weight changes, or percent food efficiency compared with the controls.
There were no adverse test item-related effects on F1 male body weights or body weight changes from Days 70 to 105. Although statistically significant increases were noted in feed consumption (g/day and/or g/kg/day) for one or both intervals from Days 91 to 105 for the F1 males given 50, 250, and 1000 mg/kg/day, these changes in feed consumption did not result in or correlate with statistically significant effects on body weights, body weight changes, or percent food efficiency compared with the controls.
There were no statistically significant changes in body weights, body weight changes, feed consumption (g/day and g/kg/day), and percent food efficiency from Days 70 to 112 for those F1 females that were never found sperm positive or that never delivered a live litter.
GESTATION
Summary Data: Tables 51, 52, 53, 54, Individual Data: Appendix 2, Tables 43, 44
There were no test item-related effects on body weights or body weight changes during
gestation. There were no adverse test item-related effects on feed consumption during gestation.
Although statistically significant increases were noted in feed consumption (g/day and g/kg/day) from Days 0 to 7 and Days 7 to 14 for the F1 females given 1000 mg/kg/day, with overall increases in feed consumption of 15% and 11% (g/day and g/kg/day, respectively) for Days 0 to 20, these changes in feed consumption did not result in or correlate with statistically significant effects on body weights, body weight changes, or percent food efficiency compared with the controls.
LACTATION
Summary Data: Tables 56, 57, 58, 59, Individual Data: Appendix 2, Tables 46, 47
There were no test item-related effects on body weights, body weight changes, or feed consumption during lactation. A statistically significant increased percent food efficiency from Days 4 to 7 was noted in the F1 females given 250 mg/kg/day compared with the control group.
This sporadic statistically significant change was not considered test item-related since the change was slight and showed no dose response.
ANDROLOGY
Summary Data: Table 42, Individual Data: Appendix 2, Table 37
No test item-related effects were observed on the spermatogenesis endpoints ( mean testicular and epididymal sperm numbers and sperm production rate, motility, and progressive motility) in F1 males at any dosage concentration. Differences from the control group were slight and were not statistically significant for these parameters. As per the protocol, assessments using the frozen right testis, as well as sperm morphology evaluations, were conducted initially on the control and high-dose groups only. As there were no test item-related effects in the group given 1000 mg/kg/day, evaluation of the lower dose groups was not conducted for these endpoints.
ANATOMIC PATHOLOGY – SCHEDULED DEATHS
MACROSCOPIC EXAMINATION
Summary Data: Tables 43, 61, 67, 68, Individual Data: Appendix 2, Tables 36, 38, 50, 58, 59, 60
There were no test-item related macroscopic findings at the F1 male scheduled necropsy. An equivocal macroscopic finding was that the left cranial lobe of the lungs from one F1 male given 1000 mg/kg/day was mottled. All macroscopic gross findings in F1 males were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
There were no test item-related macroscopic findings for the F1 females at the scheduled necropsies on LD (≡PND) 21. All macroscopic gross findings for F1 females at scheduled necropsy were noted with similar incidence among the groups, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
No test item-related effects were observed on the number of implantation sites. The number of females in proestrus, estrus, metestrus, and diestrus at demise were similar for all groups. The differences between the control and test item-treated groups were slight and not statistically significant.
ORGAN WEIGHTS
Summary Data: Tables 42, 67, Individual Data: Appendix 2, Tables 36, 58
There was a statically significant increase in relative lung weights (approximately 10.5% and 12%, respectively) in F1 males and females given 1000 mg/kg/day. Although not statistically significant when compared with the control group, absolute lung weights were also increased in the F1 males and females given 1000 mg/kg/day (approximately 8% and 9%, respectively). The increased lung weights correlated with the microscopic lung findings in rats given 1000 mg/kg/day. Other statistically significant changes in organ weights were not considered test item related as they showed no dose response.
MICROSCOPIC EXAMINATION
Summary Data: Tables 44, 67, 69, Individual Data: Appendix 3
Like the F0 males and females, chronic interstitial/alveolus inflammation was increased in incidence and severity in the lungs of F1 males and females given 1000 mg/kg/day compared with the controls, suggestive of aspiration of the dosing formulation. The increased lung weights correlated with the chronic interstitial/alveolus inflammation observed microscopically.
Also like the F0 animals, alveolar macrophage aggregation, alveolus was noted in F1 control and high-dose male and female lungs and was characterized by the presence of foamy appearing macrophages in alveolar spaces without any other significant changes associated with it. This non-adverse finding, although it can be seen as a spontaneous change in the lungs of rats, was most likely related to dosing with the corn oil vehicle and was also observed in the 90-day oral repeated dose toxicity study with the test material (see IUCLID section 7.5.1).
A test item-related slight increase in the incidence of renal tubule hyaline droplets was seen in the F1 males given 1000 mg/kg/day; however, kidneys from this study were only examined microscopically if a gross lesion was noted. In these males with gross kidney findings, lightly staining eosinophilic droplets were increased in proximal convoluted tubules of kidneys. The morphologic appearance of these droplets suggested hydrocarbon-induced α2μ-globulin male rat nephropathy, which is a common finding with hydrocarbons. In studies with closely related compounds, the finding of renal tubule hyaline droplets was confirmed to be α2μ-globulin. The severity of the hyaline droplets was subjectively graded as minimal. No degenerative/necrotic renal tubule changes were observed in association with the hyaline droplets. Control kidneys examined because of the presence of a gross lesion also had hyaline droplets as expected since male rats of this age contain α2μ-globulin; the number and distribution of the droplets in the control males were in the range considered normal for this age of male rat and, thus, not graded.
Hyaline droplets were not observed in the test item-treated females that had their kidneys examined. Hydrocarbon-induced α2μ-globulin male rat nephropathy was not unanticipated as this finding was also observed in studies with closely related compounds.
Other changes in the kidneys such as chronic progressive nephropathy and renal tubule regeneration were not increased in either incidence or in severity based on the small number of kidneys examined and were therefore not deemed test item-related.
The microscopic examination of reproductive tissues for low and mid dose animals suspected of reduced fertility did not have any finding related to test item administration.
A number of other histopathological findings were noted in a number of tissues which were typical of the spontaneous type of microscopic pathology that may be observed in this strain and age of rat.
Ovarian follicle counts were not statistically significantly different in the F1 females given 1000 mg/kg/day compared with the control group therefore, counts were not conducted on the lower dose groups.
F2 LITTER DATA
PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: Table 61, Individual Data: Appendix 2, Tables 50, 51
The mean number of pups delivered (live, dead, and total); number of live pups per litter on PND 0, 4, 7, 14, and 21 (for litters having at least 1 live pup); percentage of males per litter on PND 0, 4, 7, 14, and 21; lactational index; and postnatal survival indices (4-, 7-, 14-, and 21-day) were unaffected by the test item at all dosage concentrations.
Differences from the control group were slight and were not statistically significant.
GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: Table 63, Individual Data: Appendix 2, Tables 51, 54
The numbers of F2 pups found dead, euthanized, and/or missing, as well as the general physical condition of all F2 pups in this study were unaffected by test item administration. Pups that were found dead or euthanized prior to scheduled euthanasia on PND 21 numbered 10, 19, 11, and 16 in the control, 50, 250, and 1000 mg/kg/day groups, respectively. In addition, 1, 1, 9, and 2 pups in the same respective groups were missing (and presumed dead).
ANOGENITAL DISTANCE
Summary Data: Table 62, Individual Data: Appendix 2, Table 52
The anogenital distances (absolute and adjusted for body weight as the covariate) in the F2 male and female pups delivered from dams given 50, 250, and 1000 mg/kg/day were similar to the respective control group values. Differences from the control group were slight and not statistically significant.
OFFSPRING BODY WEIGHTS
Summary Data: Table 62, Individual Data: Appendix 2, Table 53
Mean male and female pup body weights and body weight changes in the 50, 250, and 1000 mg/kg/day groups were unaffected by parental test item administration throughout the postnatal period. No statistically significant differences from the control group were noted.
NECROPSIES OF PUPS STILLBORN, FOUND DEAD, OR EUTHANIZED
Summary Data: Tables 63, 64, Individual Data: Appendix 2, Tables 54, 55
The numbers of F2 pups (litters) stillborn, found dead, or euthanized from PND 0 through PND 21 numbered 9(8), 16(7), 17(4), and 16(9) in the control, 50, 250, and 1000 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration to the test item were noted at the necropsies of F2 pups that were stillborn, found dead, or euthanized.
Findings in the stillborn, found dead, or euthanized pups from test item-treated dams were similar to the findings in stillborn, found dead, or euthanized pups from vehicle-treated dams, culled PND 4 pups, and/or scheduled death PND 21 F2 pups.
NECROPSIES OF WEANLINGS - PND 21
MACROSCOPIC EXAMINATION
Summary Data: Table 66, Individual Data: Appendix 2, Table 57
At the PND 21 necropsy of F2 weanlings, there were no test item-related findings.
ORGAN WEIGHTS
Summary Data: Table 65, Individual Data: Appendix 2, Table 56
There were no adverse test item-related changes in F2 pup final body weights and organ weights compared with the control group. Although statistically significant slight decreases (approximately 3%) in absolute brain weights were observed for the F2 pups overall and for F2 male pups specifically in the group given 1000 mg/kg/day compared with the control group, a similar 3% decrease for the F2 female absolute brain weight and decreases in male and female relative brain weights were not statistically significant. The historical control ranges for PND 21 F2 male absolute and relative brain weights are 1.432 to 1.5422 g and 2.7095 to 3.0717 g, respectively, and the historical control ranges for PND 21 F2 female absolute and relative brain weights are 1.3803 to 1.4771 g and 2.6560 to 3.0876 g, respectively. Given the small magnitude of the change, the lack of statistical significance for most of the values, and since all of the weights except the male relative weight fall within the historical control data ranges, the decreased brain weights were considered equivocal and not adverse.
MICROSCOPIC EXAMINATION
Summary Data: Table 66, Individual Data: Appendix 3
There were no test item-related microscopic findings in the F2 PND 21 weanlings.
OBJECTIVE AND METHODS
The objective of this study was to characterize the effects of 'Distillates (Fischer - Tropsch), heavy, C18-50 – branched, cyclic and linear' on the integrity and performance of the male and female reproductive systems, including gonadal function, the estrus cycle, mating behavior, conception, gestation, parturition, lactation, and weaning of the F0 and F1generations, as well as the neonatal survival, growth, and development of the F1and F2offspring.
Three groups of male and female Crl:CD(SD) rats (25 or 26/sex/group for the F0generation and 25/sex/group for the F1generation) were administered the test item, daily by oral gavage for at least 70 consecutive days prior to mating. Dosage levels were 50, 250, and 1000 mg/kg/day for the F0and F1generations. A concurrent control group of 25 or 26/sex and 25/sex for the F0 and F1generations, respectively, received the vehicle, corn oil (CAS No. 8001-30-7), on a comparable regimen. F0animals were approximately 6 to 7 weeks (42 to 46 days) of age at the initiation of test item administration. The test item was administered to offspring selected to become the F1parental generation following weaning, beginning on postnatal day (PND) 22. The F0 and F1males continued to receive the test item throughout mating and through the day prior to euthanasia. The F0 and F1females continued to receive the test item throughout mating, gestation, and lactation, and through the day prior to euthanasia.
Animals were observed at least twice daily for mortality. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for males throughout the study and for females prior to mating and during gestation and lactation. Vaginal smears were performed daily for determination of estrous cycles beginning 21 days prior to pairing. All F0 and F1females were allowed to deliver and rear their pups until weaning on lactation day (LD≡PND) 21. Clinical observations, body weights, and sexes for F1and F2pups were recorded at appropriate intervals. For both generations (F1and F2), 10 pups per litter (5 per sex, when possible) were arbitrarily selected on PND 4 to reduce the variability among the litters.
Offspring (25/sex/group) from the pairing of the F0animals were selected on PND 21 to constitute the F1generation. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the selected F1rats. Nonselected F1pups were necropsied on PND 21, and F2pups were necropsied on PND 21. Selected organs (brain, spleen, and thymus) were weighed from 1 pup/sex/litter (when possible) from both F1and F2pups that were necropsied on PND 21. Each F0and F1parental animal received a complete detailed gross necropsy (the surviving female parental animals were necropsied on LD 21), and selected organs were weighed. Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F0and F1males as appropriate, and ovarian primordial follicle counts and the corpora lutea counts were recorded for all F1females in the control and high-dose groups. Designated tissues from the F0 and F1parental animals were examined microscopically.
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 750 mg/kg bw/day
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
There are no reproduction toxicity studies for Hydrocarbons, C18-C24, isoalkanes, <2% aromatics. However, there are studies available for GTL Gasoil (C8-C26) and GTL Base Oil Distillates (C18-C50). Thus, based on the similarity of constituents and carbon-range coverage, read-across is justified.
GTL Gasoil (C8 - C26, branched and linear)
A two-generation reproductive toxicity study (oral, gavage) has been carried out using GTL Gasoil (C8-C26, branched and linear, CAS 848301-67-7), following OECD Test Guideline 416 and conducted according to GLP (RTI, 2011a). The test material was administered by gavage to three groups of male and female Crl:CD (SD) rats, each of 25 and 28 animals of each sex for the F0 and F1 generations, respectively. Test concentrations were 0 (control), 50, 200 and 750 mg/kg bw/day for the F0 and F1 generations. Dosing was performed for 70 consecutive days prior to mating. Males continued to be dosed during the 14-day mating period and post-mating holding. Females were dosed during the mating period, gestation and lactation.
Administration of GTL Gasoil to male and female rats at dosages up to 750 mg/kg/day had no effect on reproductive performance or gestation length and parturition of both the F0 and F1 parental generations. In addition, there were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition/mortality, anogenital distance, and pup body weights. Vaginal patency of F1 females was unaffected by test item administration. There was a statistically significant, test article-related increase in the mean age and adjusted age of attainment of balanopreputial separation in F1 males given 750 mg/kg/day. However, this change was not considered adverse, as the age and adjusted age of attainment fell within the historical control data range for this parameter. Reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day, although slightly lower than the control group, were not statistically significantly different from the control group and also fell within the historical control data range. In addition, andrology parameters and sperm morphology data for the individual F1males in the high-dose group, with the longest delay in attainment of balanopreputial separation, were similar to the group mean data, further supporting the conclusion that the delay in preputial separation was non-adverse. Sperm morphology assessments of F1males showed a statistically significant increase in the percent of abnormal sperm in those males given 750 mg/kg/day; however, this change was not observed in F0 males and resulted primarily from a single male, and was therefore considered equivocal and non-adverse since the percent of abnormal sperm seen fell within the historical control data range for this parameter. Reproductive performance parameters (mating and fertility indices) for F1 males given 750 mg/kg/day were unaffected, and there were no microscopic findings in the testes. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, a dosage level of 750 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity.
Based on the available repeated dose oral studies to assess systemic, reproductive and developmental toxicity on GTL Gasoil (C8-C26) and considering the relative gut absorption discussed in Section 5.1 of the CSR, it is concluded that Hydrocarbons, C18-C24, isoalkanes, <2% aromatics (GS310) will have a similar toxicological profile after repeated oral exposure. Additional information on systemic toxicity of related GTL substances can be found in the literature (Boogaard et al., 2017).
Therefore, for repeated dose exposure, for GS310 the following is established NOAEL (reproduction) = 750 mg/kg body weight/day.
GTL Base Oil Distillates (C18-C50, branched, cyclic and linear)
In a two-generation reproductive toxicity study (RTI, 2011b),GTL Base Oil [0 (vehicle), 50, 250, or 1000 mg/kg/day)] was given orally to F0 and F1 parental male and female rats for at least 70 days prior to mating and throughout the 14-day mating, post mating holding (for males), and gestation and lactation (for females) periods. There was a test item-related, non-adverse decrease in Anogenital distance in F1 females born from dams given 250 and 1000 mg/kg/day. Test item-related histopathological lesions were identified in the lungs (chronic interstitial/alveolus inflammation) of both males and females of the F0 and F1 generations with corresponding macroscopic findings and/or increased lung weights and the kidneys (renal tubule hyaline droplets likely representing α2μ-globulin) of the F1 males only. The lung lesions were most likely secondary to aspiration of the test material and therefore not relevant for human risk assessment. The renal effects are a well-known male rat specific effect which is induced by hydrocarbons and has no relevance for humans. An equivocal, non-adverse slight decrease in F2 pup brain weights was noted. Based on the absence of adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, the absence of adverse findings directly attributable to the test item in non-reproductive tissues, and the absence of adverse effects on in-life parameters (such as body weight, feed consumption, and clinical observations), the dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive and systemic toxicity.
Based on the available weight of evidence for related substances in the relevant carbon number range it can be concluded that Hydrocarbons, C18-C24, isoalkanes, <2% aromatics is not reprotoxic at dose levels ≥750 mg/kg bw/day.
Effects on developmental toxicity
Description of key information
Pre-natal developmental toxicity (OECD 414) and two-generation reproductive toxicity studies (OECD 416) were conducted with GTL Gasoil and GTL Base Oil Distillates. In these oral gavage studies (Harlan, 2014a, Harlan, 2014b, RTI, 2011a and RTI, 2011b), there were no effects on any parameters examined in the F1 and F2 generations up to the highest doses tested, 750 mg/kg bw/day and 1000 mg/kg bw/day respectively.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted in compliance with agreed protocols, with no/minor deviations from standard test guidelines, which do not affect the quality of the results. Read-across is considered to be reliability 2.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Guidelines (Ministry of Agroculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- Number of Animals: 88 mated females, 22 mated females per group
Age (Day 0 Post Coitum): 11 weeks
Body Weight Range (Day 0 Post Coitum): 187 to 249 g
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Conditions:
Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually fol¬lowing room cleaning, which was considered not to have any influence on the study. These data were not reported but were retained in the raw data. There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation:
Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with pa-per enrichment (ISO-BLOX from Harlan Laborato-ries B.V., Netherlands).
Diet:
Pelleted standard Harlan Teklad 2018C (batch no. 56/12) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
Water:
Community tap-water from Füllinsdorf and Itingen was available ad libitum in water bottles. - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear) was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a solution was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
Dose formulations were stored in the refrigerator at 5 ± 3 °C in dark glass beakers.
Vehicle
Identification: Corn oil
Source: Carl Roth GmbH
Batch Number: 103197718
Expiry Date: 27-May-2014
Storage Conditions Room temperature (15 - 25 °C) - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- On the first dose formulation day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 1 g of each test item concentration were taken from the middle to confirm the stability (8 days in the refrigerator at 5 ± 3 °C protected from light). Towards the end of the study, samples were taken from the middle to confirm concentra-tion. The aliquots for analysis of dose formulations were delivered at ambient temperature to Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland and stored there at -20 ± 5 °C until analysis.
The samples were analyzed with a GC-system equipped with a FID. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.
Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Itingen / Switzerland. The samples were not discarded without written consent from the study director. - Details on mating procedure:
- After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day of mating was designated day 0 post coitum. Mating was performed in Füllinsdorf. After successful mating the animals were delivered on day 0 post coitum to Itingen to perform the rest of the study.
Male rats of the same source and strain were used only for mating. These male rats are in the possession of Harlan Laboratories and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored. - Duration of treatment / exposure:
- 15 days
- Frequency of treatment:
- Daily
- Duration of test:
- Day 6 - 20 post coitum
- No. of animals per sex per dose:
- 22 females per dose level
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The group identification and animal numbers assigned to treatment are stated in the following table:
Allocation and Group 1 Group 2 Group 3 Group 4
Dose Levels Control
mg/kg bw/day 0 50 200 1000
Females 1-22 23-44 45-66 67-88
- Maternal examinations:
- The following observations were recorded:
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).
Food Consumption: Recorded at 3-day intervals: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum.
Body Weights: Recorded daily from day 0 until day 21 post coitum.
Pathology
At the scheduled necropsy on day 21 post coitum, females were sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section. - Ovaries and uterine content:
- Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of fetuses in the uterus was performed and the data recorded. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites. - Fetal examinations:
- Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
Microdissection Technique
For Microdissection technique (sectioning/dissection technique) [see References (2)] at least one half of the fetuses from each litter were fixed in Bouin's fixative (one fetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This includes detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.
Skeletal Examination
The remaining fetuses were placed in a solution of potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The specimens were preserved individually in plastic vials. The foetuses were sent to the principal investigator and evaluated by Harlan Laboratories Ltd. The principal investigator was notified prior to shipment. After the evaluation the fetuses were send back to Harlan Laboratories Ltd, 4452 Itingen / Switzerland and will be archived there.
Investigations were performed by personnel appointed by the principal investigator and were conducted according to Harlan Laboratories Ltd., standard operating procedure 189.15 “Evaluation of Fetal Skeletons”. Fetuses were examined under a binocular microscope. Fetuses were examined in mixed group order. Each litter was examined in sequential order.
The assessment included, but not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles.
All findings were recorded on the appropriate proforma. These findings were collated and tabulated. These tables were audited by Harlan Laboratories Ltd., quality assurance department and dispatched together with the raw data to the study director.
Fetuses with abnormalities were photographed, when considered appropriate. - Statistics:
- The following statistical methods were used to analyze food consumption, body weights, macroscopical findings, reproduction and skeletal examination data:
• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test [see References (4)] (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test [see References (5)] (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test [see References (6)] was applied if the variables could be dichotomized without loss of information. - Details on maternal toxic effects:
- Maternal toxic effects:no effects
Details on maternal toxic effects:
Viability / Mortality
All females survived until the scheduled necropsy.
Clinical Signs
No test item-related clinical symptoms or signs were observed at any dose during the study.
Incidentally towards the end of the study, one female at 200 mg/kg bw/day showed a cloudy inclusion in the right eye.
Food Consumption
No test item-related changes were observed in the food consumption during the study.
Body Weights
Mean absolute body weight and body weight gain were not affected by treatment with the test item.
Corrected body weight gain (corrected for the gravid uterus weight) was not affected by treat-ment with the test item (+10.9%, +10.3%, +11.3%, +11.5% in order of ascending dose level).
Reproduction Data
The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the test item.
Pathology (Maternal Data)
Macroscopic Findings
No findings were observed at any dose level during macroscopical examination. - Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
Observations (Fetal Data)
External Abnormalities and Variations
No test item-related findings were observed during external examination of the fetuses in any group.
At 200 mg/kg bw/day, one pup had incidentally a short lower jaw (confirmed by fetal pathology).
Sex Ratios
No test item-related effects on the sex ratio of the fetuses were noted in any group.
The proportion of male fetuses was 50.0%, 45.8%, 50.7% and 52.1% in order of ascending dose level.
Body Weights
No test item-related effects on fetal body weights were noted.
Mean body weights of live fetuses calculated on litter and individual basis were statistically significantly increased at 1000 mg/kg bw/day (+6% when calculated on a litter basis for male and female fetuses).
Visceral Abnormalities and Variations
No test item-related findings were noted.
At 50 mg/kg bw/day, one fetus had a severely dilated renal pelvis. At 200 mg/kg bw/day, one fetus had a malpositioned subclavian artery and at 1000 mg/kg bw/day, one fetus showed a situs inversus. All these isolated abnormalities were considered to be incidental.
The type and frequency of commonly noted variations were similar in nature for the groups treated with the test item and the control group and did not indicate any dose-dependency. They were therefore considered not to be test item-related.
Bone Abnormalities and Variations
No test item-related findings were noted.
At 50 mg/kg bw/day and 200 mg/kg bw/day, there were two fetuses that showed similar find¬ings. One fetus had fused mandibles and lower incisors, misshapen exoccipital/basisphenoid and absent/reduced in size cervical vertebral arches. Another fetus showed a short lower jaw, fused mandibles, lower incisors and misshapen basisphenoid. However, in isolation and in the absence of any dose treatment relationship, this is not considered to be an adverse effect of treatment on fetal development.
The type and frequency of commonly noted variations were similar in nature for the groups treated with the test item and the control group and did not indicate any dose-dependency. They were therefore considered not to be test item-related. - Dose descriptor:
- NOAEL
- Effect level:
- 750 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: developmental toxicity
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Conclusions:
- This study is an investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear) to rats. The test item was admin¬istered in corn oil as vehicle at dose levels of 50, 200 and 1000 mg/kg body weight/day.
No maternal toxicity was observed at any dose level.
The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the test item and also no effects on the development of fetuses were observed at any dose level. However, the fetal body weight was slightly statistically significantly increased at 1000 mg/kg bw/day (5.1 ± 0.2 g for the high dose-group versus 4.9 ± 0.2 g for the controls and 5.0 ± 0.2 g and 4.9 ± 0.3 g for the low and mid dose-groups, respectively). A test item-related effect cannot be excluded, most probably due to the higher nutri¬tional value that the test item provides.
Based on this result a maternal NOEL (No Observed Effect Level) for GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear), was considered to be 1000 mg/kg body weight/day and a fetal NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/ day.
Under the conditions described for this study, GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear) did not reveal teratogenic potential up to and including a dose of 1000 mg/kg body weight/day. - Executive summary:
Purpose
The purpose of this study was to detect effects on the pregnant rat and development of the embryo and fetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section).
Rationale for Choice of Species, Route of Administration and Dose Levels
The rat is a suitable species for repeated dose and reproduction/developmental toxicity studies required by regulatory authorities. The oral route is one possible route for human exposure.
Based on the results of a OECD408 study (Harlan Study Number2041/0061) the dose levels of 0, 50, 200, and 1000 mg/kg bw per day were selected for the present study
Guidelines / Regulations
This study was performed in accordance with the procedures indicated by the following internationally accepted guidelines and recommendations:
· OECD guidelines (OECD guideline for testing of chemicals proposal for updating guideline 414, prenatal developmental toxicity study. Adopted: 22nd January 2001) and EEC guidelines (EC 2004/73 B31, dated April 29, 2004).
· US-EPA guidelines (Health Effects Test Guidelines, OPPTS 870.3700 ‘Prenatal Development Toxicity Study’, EPA 712-C-98 -207, dated August 1998).
· Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000).
Maternal Data
Mortality / Viability
All females survived until the scheduled necropsy.
Clinical Signs
No test item-related clinical symptoms or signs were observed at any dose during the study.
Food Consumption
No test item-related changes were observed in the food consumption during the study.
Body Weights
Mean absolute body weight, body weight gain and corrected body weight gain were not affected by treatment with the test item.
Reproduction Data
The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the test item.
Macroscopic Findings
No findings were observed at any dose level during macroscopical examination.
Fetal Data
External Abnormalities and Variations
During the external examination of the fetuses, no test item-related findings were observed.
Sex Ratios
No test item-related effects on the sex ratio of the fetuses were noted in any group.
Body Weights
Mean body weights of live fetuses were slightly increased at 1000 mg/kg bw/day. The minor increase was not in the range of the historical control data and an effect of the test item cannot be excluded.
Visceral Abnormalities and Variations
No test item-related findings were noted during external and fresh visceral examinations of fetuses.
Bone Abnormalities and Variations
No test item-related findings were noted during skeletal examination of the fetuses. No delay in the stage of ossification was observed.
The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the test item and also no effects on the development of fetuses were observed at any dose level. However, the fetal body weight was slightly statistically significantly increased at 1000 mg/kg bw/day (5.1±0.2 g for the high dose-group versus 4.9±0.2 g for the controls and 5.0±0.2 g and 4.9±0.3 g for the low and mid dose-groups, respectively). A test item-related effect cannot be excluded, most probably due to the higher nutritional value that the test item provides.
Based on this result a maternal NOEL (No Observed Effect Level) for GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear), was considered to be 1000 mg/kg body weight/day and a fetal NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/ day.
Under the conditions described for this study, GTL base oil distillate (Distillates (Fischer-Tropsch), heavy, C18-50-branched, cyclic and linear) did not reveal teratogenic potential up to and including a dose of 1000 mg/kg body weight/day.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2nd September 2013 (animal preparation and dosing) to 16th September 2012 (necropsy)
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study conducted in compliance with agreed protocols, with no/minor deviations from standard test guidelines, which do not affect the quality of the results. Read-across is considered to be reliability 2.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 191 to 272g.
The animals were housed individually in solid-floor polypropylene cages with stainless steel lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly mean temperatures and humidity were included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively; Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan descried below in "Any other information on method".
The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories. - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Corn Oil. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK Analytical Services. Results show the formulations to be stable for at least twenty-five days. Formulations were therefore prepared once and stored at approximately +4 °C in the dark.
Samples were taken of the test item formulations and were analyzed for concentration of GTL diesel (Distillates (Fischer-Tropsch), C8-26 branched and linear) at Harlan Analytical Laboratory, Shardlow. The method used for analysis of formulations and the results obtained are given in the attached Appendix 12. The results indicate that the prepared formulations were within ± 2% of the nominal concentration. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Introduction
The test item concentration in the test samples was determined by gas chromtaography (GC) using an external standard technique. The test item have a chromatograohic profile consisting of multiple peaks.
Preparation of standard solutions
Syock solutions of test item in tetrahydrofuran were prepared for external standard calibration. An aliquot of test item was accurately weighed into a volumetric flask and brought to volume with tetrahydrofuran to yield a solutin with a concentration of 1 mg/ml. The standard solutions contained the equivalent amount og vehicle to that of the relevant samples.
Analysis of samples
The formulations received were diluted with tetrahydrofuran. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with tetrahydrofuran and then shaken to dissolve. Where necessary, sample solutions were further diluted with tetrahydrofuran to achieve the working concentration.
Preparation of accuracy samples
Samples of corn oil were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as per the test samples referred to in the previous paragraph.
Preparation of linearity standards
A range of standard solutions were prepared in tetrahydrofuran from a stock solution of 2.02 mg/ml by serial dilution covering the concentration range 0 to 2.02 mg/ml.
Instrumental Setup
GC system: Agilent Technologues 5890, incorporating autosampler and workstation
Column: ZB 1 HT (30 m x 0.25 mm id x 1 um film)
Oven temperature program: Ocen: 30°C for 0 minutes with 10°C/minute to 300°C for 5 minutes
Injection temperature: 250°C
Flame ionisation detector temperature: 250°C
Injectionn volume: 2 micor-l
Retention time: Profile of peaks from 5 to 25 minutes - Details on mating procedure:
- Not described in study
- Duration of treatment / exposure:
- Day 5 to Day 19 of gestation,
- Frequency of treatment:
- Daily
- Duration of test:
- 20 days - animals sacrificed on Day 20 after gestation
- No. of animals per sex per dose:
- 24 females per dose
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
- Maternal examinations:
- Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.
Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).
Food Consumption
Food consumption was recorded for each surviving individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.
Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes. - Ovaries and uterine content:
- Post Mortem
All surviving animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):
Left Horn Cervix Right Horn
L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16
V = viable fetus - Fetal examinations:
- The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed in 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
- Statistics:
- The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis non-parametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant) - Indices:
- All data was summarized in tabular form, including reproductive indices. Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation. Values given in appendices may represent rounded values for presentation purposes. Group mean values were generally calculated using unrounded valves therefore it is not always possible to calculate the exact group mean values from values presented in the appendices.
As the litter was the standard unit of assessment, values were first calculated within the litter and group mean values represent the mean of these individual litter values.
Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:
((Number of corpora lutea - number of implantations) / number of corpora lutea) x 100
Percentage post-implantation loss was calculated as:
((Number if implantations - number of live foetuses) / number of implantations) x 100
Sex Ratio
Sex ratio was calculated as:
% male fetuses (sex ratio) = (Number of male foetuses / number of females foetuses) x 100 - Details on maternal toxic effects:
- Maternal toxic effects:no effects
- Dose descriptor:
- NOAEL
- Effect level:
- 750 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Abnormalities:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
- Dose descriptor:
- NOAEL
- Effect level:
- 750 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 750 mg/kg bw/day.
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Conclusions:
- The oral administration of GTL diesel (distillates (Fischer-Tropsch), C8-26 branched and linear) to pregnant rats by oral gavage during gestation at dose levels of 50, 200 and 750 mg/kg bw/day did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 750 mg/kg bw/day.
No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 750 mg/kg bw/day. - Executive summary:
Introduction
The study was performed according to the study plan presented in Appendix13and was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.
The study was designed to comply with the following guidelines:
· US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)
· Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)
· OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)
· Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH)
Method
The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 50, 200, and 750 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (corn oil) to serve as a control.
Clinical signs, body weight change, food and water consumptions were monitored during the study.
All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.
Results…….
Mortality
There were no treatment-related deaths.
One control female was killed in extremison Day 15 due to a mal-dose.
Clinical Observations
No clinically observable signs of toxicity were evident in treated females.
Body Weight
No toxicologically significant effects in body weight development were detected.
Food Consumption
No treatment related effects were detected in food consumption.
Water Consumption
No adverse effect on water consumption was detected.
Post Mortem Studies
No treatment related macroscopic abnormalities were detected.
Litter Data and Litter Placental and Fetal Weights
No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in growth and development.
Fetal Examination
No treatment-related effects were detected on external development or in the type and incidence of skeletal or visceral findings.
Conclusion
The oral administration ofGTL diesel (distillates (Fischer-Tropsch), C8-26 branched and linear)to pregnant rats by oral gavage during gestation at dose levels of 50, 200 and 750 mg/kg bw/day did not result in any toxicologically significant effects in parental females. The ‘No Observed Adverse Effect Level’ (NOAEL) was therefore, considered to be 750 mg/kg bw/day.
No toxicological significant changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity was therefore considered to be 750 mg/kg bw/day.
Referenceopen allclose all
Analysis of Dose Formulations
The GTL Base oil distillate peak was assigned in sample chromatograms by comparison to that of calibration solutions. In blank sample chromatograms no significant peak appeared at the retention time of GTL Base oil distillate and, therefore, the absence of the test item in the vehicle control samples (corn oil) was confirmed.
The GTL Base oil distillate concentrations in the dose formulations ranged from 82.6% to 119.5% with reference to the nominaland were within the accepted range of±20%,with exception of sample of group 2-top prepared on 17-Aug-2013 (71.1%).However, the result was accepted as homogeneity was in range.
The homogeneous distribution of GTL Base oil distillate in the preparations was approved because single results found did not deviate more than 13.6% from the corresponding mean and met the specified acceptance criterion of ≤15%.
In addition, the test item was found to be stable in application formulations when kept eight days in the refrigerator (5 ± 3 °C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.
Adult Response
Mortality
There were no treatment-related deaths.
One control female was killedin extremison Day 15. Macroscopic observations revealed evidence of a physical trauma following the dosing procedure therefore this death was considered unrelated to test item toxicity.
Clinical Observations
No toxicologically significant clinical signs were evident in treated females.
One female treated with 750 mg/kg bw/day showed an isolated incident of increased salivation on Day 6 and a further female from this treatment group had a decreased respiratory rate on Day 16. No such effects were detected in females treated with 200 or 50 mg/kg bw/day. Observations of this nature are commonly observed following the oral administration of an unpalatable test item formulation and in isolation are considered not to be of toxicological significance.
One female treated with 200 mg/kg bw/day and one female treated with 50 mg/kg bw/day showed generalized fur loss between Days 6 and 7 and Days 16 and 20 (respectively). Observations of this nature are commonly observed in this type of study and in isolation are not considered to be related to test item toxicity. One female treated with 200 mg/kg bw/day had a damaged tail between days 13 and 20. This was a physical injury and unrelated to test item.
The female that was killedin extremison Day 15 showed red/brown staining around the snout, eyes and mouth, hunched posture, pilo-erection and respiratory pattern changes. One female treated with 750 mg/kg bw/day had a mass under the left forelimb. At necropsy, macroscopic observations for both females revealed evidence of a physical trauma following the dosing procedure therefore the clinical observations were considered unrelated to test item toxicity.
Body Weight
No toxicologically significant effects in body weight development were detected.
Females treated with 750 mg/kg bw/day showed a statistically significant reduction (p<0.05) in body weight gain between Days 5 and 6. The statistical significance was minimal and recovery was evident thereafter. Therefore in isolation and in the absence of an overall effect on body weight development the intergroup difference was considered of no toxicological significance.
Food Consumption
No adverse effect on dietary intake was detected.
Statistical analysis of the data did not reveal any significant intergroup differences.
Water Consumption
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
Post Mortem Studies
No treatment-related macroscopic abnormalities were detected in treated females at terminal kill.
The female that was killed in extremis had a fluid filled thoracic cavity, a damaged trachea and an oil like substance in the stomach. One terminal kill female treated with 750 mg/kg bw/day had a mass under the left forelimb which was filled with a food like substance and a small hole in the trachea. These macroscopic observations were considered to be the result of a physical trauma following the dosing procedure and therefore unrelated to test item toxicity.
Litter Responses
Litter Data and Litter Placental and Fetal Weights
There was no treatment related effects on in utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and post-implantation losses. There were also no toxicologically significant effects on pre-implantation losses or in sex ratio.
Females treated with 750 and 200 mg/kg bw/day showed a statistically significant reduction in pre-implantation loss. A true dose related response was not evident and a reduction in this parameter is considered not to represent an effect of treatment due to implantation occurring prior to the administration of the test item.
Fetal Examination
For all dose groups, there were no significant treatment-related trends in the proportion of fetuses (or litters) with evidence of external, visceral or skeletal anomalies. The type of external, visceral and skeletal anomalies, were those commonly observed for this type of study. There were no findings that were considered to represent any known malformations.
During visceral assessment, litters from females treated with 50 mg/kg bw/day showed a statistically significant increase in the total percent of affected offspring. No significant increases were evident in any of the individual parameters examined and in the absence of a true dose related response the intergroup difference was considered not to be of toxicological significance.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 750 mg/kg bw/day
Additional information
No specific developmental toxicity studies are currently available for Hydrocarbons, C18-C24, isoalkanes, <2% aromatics or related Fischer-Tropsch-derived substances. However, evidence from pre-natal developmental studies (OECD 414) and two-generation reproductive toxicity studies (OECD 416) conducted with GTL Gasoil (C8-C26) and GTL Base Oil Distillates (C18-C50) indicates that the substances are not developmental toxins at dose levels ≥750 and 1000 mg/kg bw/day.
GTL Gasoil (C8-C26, branched and linear)
A pre-natal developmental toxicity study in Sprague-Dawley rats was performed with GTL Gasoil (C8-C26) in accordance with OECD Test Guideline 414 and in compliance with GLP (Harlan, 2014a). Four groups of 24 mated females were dosed once daily by gavage at dose levels of 0, 50, 200 and 750 mg/kg bw/day from Day 5 until Day 19 of gestation. There were no mortalities or test substance-related clinical findings at any dose level. There were no test substance-related changes in food consumption, water consumption or body weight. No treatment-related macroscopic abnormalities were detected at post mortem. There were no treatment-related effects on the uterine parameters examined, in foetal viability or in growth and development. No treatment-related effects were detected on external development or in the type and incidence of skeletal or visceral findings. The No Observed Adverse Effect Level for both parental females and for development was therefore considered to be 750 mg/kg bw/day.
In a two-generation reproductive toxicity study (OECD 416) with GTL Gasoil (RTI, 2011a), there were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition, mortality, pup body weights and anogenital distance. Equivocal, non-adverse decreases (not statistically significant) in absolute and relative spleen weights were observed in the F1 and F2 male and female PND 21 pups in the group given 750 mg/kg/day, compared with the control group. Macroscopic and microscopic findings in PND 21 pups were not test item related. Further details are presented in Sections 5.6 and 5.9.1. Based on this study, NOAEL for developmental effects can therefore be considered to be at least 750 mg/kg bw/day (the highest dose tested).
Based on the available repeated dose oral studies to assess systemic, reproductive and developmental toxicity on GTL Gasoil (C8-C26) and considering the relative gut absorption discussed in Section 5.1 of the CSR, it is concluded that Hydrocarbons, C18-C24, isoalkanes, <2% aromatics (GS310) will have a similar toxicological profile after repeated oral exposure. Additional information on systemic toxicity of related GTL substances can be found in the literature (Boogaard et al., 2017).
Therefore, for repeated dose exposure, for GS310 the following is established NOAEL (developmental) = 750 mg/kg body weight/day.
GTL Base Oil Distillates (C18-C50, branched, cyclic and linear)
A pre-natal developmental toxicity study in Han Wistar rats was performed with GTL Base Oil Distillates (C18-C50) in accordance with OECD Test Guideline 414 and in compliance with GLP (Harlan, 2014b). Four groups of 22 mated females were dosed once daily by gavage at dose levels of 0, 50, 200 and 1000 mg/kg bw/day from Day 6 to Day 20 post coitum. There were no mortalities or test substance-related clinical findings at any dose level. There were no test substance-related changes in food consumption, mean absolute body weight, body weight gain or corrected body weight gain. The number of post-implantation losses and number of foetuses per dam were not affected by treatment. There were no findings at any dose level during macroscopic examination. There were no test-substance related findings during external examination of the foetuses, and no effect on the sex ratio of the foetuses in any group. Mean body weights of live foetuses were slightly increased at 1000 mg/kg bw/day. This increase was not in the range of the historical control data and a treatment-related effect therefore could not be excluded. There were no test substance-related findings noted during external and fresh visceral examination or skeletal examination of the foetuses and no delay in the stage of ossification was observed. The study author concluded that there were no adverse effects on skeletal development up to and including 1000 mg/kg bw/day. In isolation, the increased mean body weight of live foetuses is not considered to be adverse and therefore the No Observed Adverse Effect Level is 1000 mg/kg bw/day.
Administration of GTL Base Oil Distillates in a two- generation reproductive toxicity study in male and female rats at dosages up to 1000 mg/kg/day had no effect on reproductive performance or gestation length and parturition of both the F0 and F1 parental generations (RTI, 2011b). Andrology parameters for F0and F1males were unaffected by test item administration. In addition, there were no test item-related effects on F1 and F2 litter parameters, postnatal survival, physical condition/mortality, and pup body weights. Vaginal patency of F1 females and balanopreputial separation in F1 males were also unaffected by test item administration. Based on the absence of adverse, test item related findings a dosage level of 1000 mg/kg/day for developmental effects was considered to be the NOAEL.
Justification for classification or non-classification
Based on the available data Hydrocarbons, C18-C24, isoalkanes, <2% aromatics is not classified for reproductive or developmental toxicity according to Regulation (EC) No 1272/2008.
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