Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral
Remarks:
other: In utero exposure followed by subchronic dosing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
In life phase, from 1 JUN 1993 to 30 NOV 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented study report which meets basic scientific principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: FDA IV.C.8. In utero exposure phase for addition to carcinogenicity studies or chronic toxicity studies with rodents.
Deviations:
yes
Remarks:
exposure limited to subchronic
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Assigned SLS ID: S92.003.3196 and S93.001.3196
- Substance type: Polyalphaolefins
- Physical state: Clear colorless liquid
- Lot/batch No.: 200-135 and 200-185
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Sprague-Dawley Crl:CD BR VAF/Plus rats
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA.
- Age at study initiation: 10 weeks
- Weight at study initiation: Males 319 to 422g; females 202 to 274g
- Fasting period before study: no
- Housing: Individually during acclimation and while on study (except during cohabitation) in suspended stainless steel cages. Females scheduled to deliver were transferred to individual plastic nesting boxes with corncob bedding.
- Diet (e.g. ad libitum): Purina Certified Rodent Meal #5002. Ad libitum except prior to blood collection when feed was withheld overnight.
- Water (e.g. ad libitum): Tap water purified by reverse osmosis and supplied by automatic watering system or from water bottles.
- Acclimation period: Minimum 17 days prior to randomization.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24.5
- Humidity (%): 40-70
- Air changes (per hr): 10-12
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 1 JUN 1993 To: 30 NOV 1993

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
A specified amount of EthylFlo 166 for each dose group was weighed into a precalibrated beaker. An appropriate amount of PEG 400 was added to each beaker and the mixtures were stirred manually. A sufficient quantity of PEG400 was then added to each beaker to achieve the desired concentration. The dosing mixture were prepared a minimum of once every two weeks and stored refrigerated.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: 0, 20, 100, 200 mg/ml
- Amount of vehicle (if gavage): 5ml/kg
- Lot/batch no. (if required): 920897 and 933384 (Source: Fisher Scientific, Cincinnati, OH, USA)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All dosing mixtures were analyzed for verification of test article concentration. In addition, at initiation, midway through and termination of the study a 5ml sample of neat EthyFlo 166 was analyzed for purity and stability.
Duration of treatment / exposure:
F0 generation: minimum 4 weeks premating+ 21 days during gestation (all rats) +21 days during lactation( all rats)
F1 generation: 21 days during gestation+21 days during lactation+ 91 days post lactation
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 500, 1000 mg/kg/day
Basis:
other: gavage
No. of animals per sex per dose:
F0 generation: See table 1
F1 generation: See table 2
Control animals:
yes, concurrent vehicle
Details on study design:
Following treatment for males and females, each female was cohabitated with a single male from the same treatment group. Each mating pair was observed daily for evidence of copulation determined by the presence of a copulatory plug in the vagina or a sperm positive vaginal smear and this was designated as day 0 of gestation. Female was then separated from the male and housed individually.

The first day of dosing was defined as study day 1. F0 males were dosed for a minimum of four weeks prior to mating, continuing through mating and until euthanasia. F0 females were dosed for a minimum of four weeks prior to mating, continuing through gestation and through lactation day 20. Treatment continued until euthanasia for females without evidence of mating and/or failure to deliver.

On lactation day 4, litters were randomly culled to a maximum of 8 pups, 4 per sex, when possible. Pup viability was determined daily throughout lactation. A detailed external examination of the pups was performed on lactation days 0, 4, 7, 14 and 21. On day 21 pups were randomly selected for the 91-day toxicity phase from the twenty oldest litters in each group. Only healthy animals (without ocular abnormalities) were acceptable for selection. All F1 selected pups were gang-housed by sex (2 or 3/cage/group) for 3 days beginning on postpartum day 21 to allow animals to become accustomed to the automatic watering system and then single-housed for dosing period.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Minimum once daily, except on the day of detailed clinical observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly, except during gestation and lactation when they were performed daily. In addition, the animals were observed for toxic effects between one-half hour and two hours following dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were measured:
F0 Males: weekly throughout study; F0 females: weekly before mating and on gestation days 0, 7, 14 and 20; and lactation days 1, 7, 14 and 21.
F1 Males and females: Weekly


OPHTHALMOSCOPIC EXAMINATION: Yes (F1)
- Time schedule for examinations: Prior to study initiation (day -7) and day 92
- Dose groups that were examined: All


HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes (F1)
- Time schedule for collection of blood: On the day of scheduled euthanasia (days 96 to 99)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked in table [No.3] were examined.


Sacrifice and pathology:
GROSS PATHOLOGY AND HISTOPATHOLOGY: Yes;
For F1 see table 4 for organs and tissues preserved in 10% neutral buffered formalin. All tissues collected at necropsy from control animals, high dose animals and animals found dead were processed for examination. Fresh organ weights were obtained for the liver, kidneys, thyroid/parathyroid, adrenal glands, gonads and brain of surviving animals.

For F0, the thoracic, abdominal and pelvic cavities were opened and the viscera examined. The number of implantation scars were recorded, if noted. Uteri with no macroscopic evidence of implants were placed in a 10% aqueous ammonium sulphide solution for detection of early embryolethality.
Abnormalities were recorded and representative samples preserved in 10% neutral buffered formalin for possible histological examination.
Statistics:
Continuous data, including body weights, weight gain, food consumption, preimplantation loss, gestation length, mean live litter size, implantation scar counts, clinical pathology and organ weights were analyzed by ANOVA.
When significance was observed with ANOVA, group by group comparisons were performed using Dunnett's Test or its modified version.
Count data were analyzed using Chi-Square test for copulation, fertility and pup sex ratios, the number of live and dead pups per group on lactation day 0, and pup survival after lactation day 0.
Mann-Whitney U Test was utilized for resorptions.
All tests were two tailed with a minimum significance level of 5% comparing the control group to each treatment group.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
F0: 1 female (100mg/kg/day) had an accidental death and another in the 0 mg/kg/day group was euthanized moribund during an incomplete delivery. Some vehicle related findings were observed.

F1: All except 3 rats that were found dead survived to scheduled euthanasia (one male each in the 500 and 1000mg/kg/day groups, and one female in the 100 mg/kg/day group). There were some vehicle-related minor gastrointestinal disturbances and some incidental findings reported but no clear test-substance related clinical findings.

BODY WEIGHT AND WEIGHT GAIN
F0: A significant decrease in mean body weight observed in the 1000mg/kg/day group over week 4 to 5 which was transient and considered not to be biologically meaningful.

F1: In the 1000mg/kg/day group, statistically significant increases in body weight gain were noted in males over weeks 11 to 12 and in females over weeks 3 to 4. These changes were not believed to be test article-related because of their sporadic occurrence.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
F0: Significant decreases during lactation (g/animal/day) were seen in the 1000mg/kg/day group females over days 1-7 and 7-14. However, these were not considered significant when calculated as g/kg/day and thus do not appear to be biologically meaningful.

F1: In females, statistically significant decreases were noted in the 500mg/kg/day group (weeks 6 to 7), in the 100, 500 and 1000mg/kg/day groups (weeks 12 to 13), and in the 100 and 500 mg/kg/day (weeks 13 to 14). They were not considered to be biologically meaningful because of lack of dose response or an abnormally increased control food consumption value.

OPHTHALMOSCOPIC EXAMINATION
F1: No abnormal effects noted.

HAEMATOLOGY
F1: A statistically significant increase in prothrombin time was seen in the males of the 1000mg/kg/day group. In the females of the 500mg/kg/day group, statistically significant increases in erythrocytes and hematocrit and statistically significant decreases in MCHC and prothrombin time were noted. Because of a lack of dose response, these changes were not considered to be test article related.

CLINICAL CHEMISTRY
F1: No apparent test article-related effects noted.

ORGAN WEIGHTS
F1: A statistically significant decrease in liver weight relative to brain weight was observed in females of the 100mg/kg/day group, while liver weight relative to final body weight was statistically decreased in the females of the 500 and 1000mg/kg/day groups. They were not considered to be biologically meaningful because of the lack of dose response.

GROSS PATHOLOGY
F0: Female at 0mg/kg/day that was found moribund, findings indicated reason to be parturition difficulty and not vehicle treatment. There appear to be no test article-related gross findings in any animals that were euthanized at post-breeding day 25 or scheduled euthanasia.

F1: One male each in the 500 and 1000mg/kg/day groups and one female in the 100mg/kg/day group were found dead apparently due to intubation errors.

HISTOPATHOLOGY:
There were no apparent test article-related changes in any of the groups. Lesions commonly seen in rats of the same age and species were noted, but did not interfere with the interpretation of the study results.

OTHER FINDINGS
There did not appear to be any test article-related effects on fertility, the length of gestation, pregnancy status, parturition, lactation, or in any of the litter parameter data.

Effect levels

Dose descriptor:
NOEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no apparent gross necropsy observations or histopathologic lesions that could be related to EthylFlo166 treatment. Because there were no apparent toxic effects on the numerous parameters measured, the NOEL for EthylFlo 166 was judged to be 1000mg/kg/day.
Executive summary:

The potential toxicity of a C30 -C60 hydrogenated polyalphaolefin was evaluated in a study following single daily oral doses by gavage for a minimum of 91 days. The animals utilized were the offspring of parental animals administered the test article. The in utero phase consisted of a control vehicle group and three doses of the test article suspended in polyethylene glycol 400 and administered at dosage levels of 100, 500 and 1000mg/kg/day. For the in utero phase, the F0 males were treated for a minimum of four weeks prior to mating. Treatment of the F0 females was initiated four weeks prior to mating and continued through lactation day 20. F0 females were allowed to deliver and rear their offspring. F1 pups were treated beginning at 22 days and continuing for the 91-day toxicity phase.

No apparent toxicity was observed in F0 male and female rats including no effects on their fertility. In addition, F1 pups did not demonstrate any test article-related toxicity during the parturition and lactation phases. In the F1 rats during the 91 -day toxicity phase, minor gastrointestinal disturbances were seen in all groups, judged to be vehicle-related. No apparent test article-related clinical observations were noted. There were transient changes in body weights, weight gain, food consumption, hematology parameters and organ weights at a few intervals, but were not considered to be biologically meaningful. A statistically significant increase in prothrombin time was seen in the males of the 1000mg/kg/day group, however, this change did not correlate with a decrease in platelets, gross necropsy findings or any lesions noted histopathologically. Therefore, this increase in prothrombin time was not considered to be biologically meaningful. There were no apparent gross necropsy observations or histopathologic lesions that could be related to EthylFlo 166 treatment and no apparent toxic effects on the numerous parameters measured. Therefore, the NOEL was judged to be 1000mg/kg/day. This result can be reliably read across to the C28 -C80 polyalphaolefin "Pentadecane, 7-methylene mixed with 1-tetradecene, dimers and trimers, hydrogenated".