Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Between 20 April 2010 and 06 January 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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

Administration / exposure

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.

Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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 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).

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

LITTER DATA F1

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.

Effect levels (F1)

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'

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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. 

Applicant's summary and conclusion

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.