Hexadecyl 3,5-bis-tert-butyl-4-hydroxybenzoate

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 10, 1980 through December 9, 1980

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptable, well-documented study report which meets basic scientific principles, basic data given, and comparable to guideline study. In general, this study was performed according to the SOPs, which are in conformity with the FDA GLP Regulations. Deviations were limited, listed below, and are believed not to impact the reliability of the test result.

Qualifier:

equivalent or similar to guideline

Guideline:

other: 91-DAY FEEDING STUDY IN THE RAT Protocol 406

Deviations:

not specified

Principles of method if other than guideline:

The following were deviations from GLP.

a. The first amendment was made to comply with the Sponsor's request to collect blood from rats at various stages of the study and to perform analysis on the quantity of compound absorbed.

b. The second amendment was made to specify the zero day for the second phase of the study and to specify the dose levels employed,

c. The third amendment was made to specify the final report date,

d. The fourth amendment was made to modifv the final report date, and

e. The fifth amendment was made to document that feed assays were performed by the Sponsor.

GLP compliance:

yes

Remarks:

See above

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington Mass.
- Age at study initiation: 21 Day Old Weanlings
- Housing: The animals in both phases of the study were individually housed in wire mesh suspended cages, except during breeding and weaning, when the dams and their offspring were kept in plastic cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:Quarantinen for 14 day upon arrival.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 73 + or - 2
- Humidity (%): 45% to 70% except during weekly hosing of tile floor for cleaning.
- Air changes (per hr): 10 times

Route of administration:

oral: feed

Vehicle:

other: First 20 days of Phase I of the study: Certified Rodent Chow, No. 5002 from Purina Company, St. Louis, Missouri.; May 27, 1980 until the end of the study: Charles River R-M-H 3200 Certified Meal, manufactured by Agway of Syracuse, N.Y.

Details on oral exposure:

The diets were prepared weekly by mixing the appropriate amount of UV-2908 with the appropriate amount of the basic diet with a Patterson-Kelley
2 cubic feet V-shaped mixer. Fresh diets were prepared weekly bassed on the feed consumption data obtained two weeks before. Feed consumption for each animal was measured weekly during the quarantine and for the next ten weeks on test during Phase 1., and also for the 13 weeks on test in Phase 2. Feed and water was provided ad libitum.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

The dose levels were set in advance by the Sponsor. Mixing Analyses: These analyses were performed by the Sponsor. Results sent to the lab indicated that the test article was properly mixed in the feed.

Duration of treatment / exposure:

91 Days

Frequency of treatment:

Daily thoroughly mixed in the feed.

Dose / conc.:

5 000 ppm

Remarks:

The low dose group received daily, thoroughly mixed in the feed, 5000 parts of UV-2908 per million parts of feed PPM corresponding to 0.5%.

Dose / conc.:

10 000 ppm

Remarks:

The mid-dose group received daily, thoroughly mixed in the feed, 10000 parts of UV-2908 per million parts of feed PPM corresponding to 1.0%.

Dose / conc.:

50 000 ppm

Remarks:

The high-dose group received daily, thoroughly mixed in the feed, 50000 parts of UV-2908 per million parts of feed PPM corresponding to 5%.

No. of animals per sex per dose:

High Dose: 25 Female and 25 Male
Mid Dose: 25 Female and 25 Male
Low Dose: 25 Female and 25 Male
Control: 25 Female and 25 Male

Control animals:

yes, plain diet

Details on study design:

The first phase (Breeding/Reproductive) started with 20 Sprague-Dawley CD weanling rats per sex per group and lasted for 10 weeks. Beginning with the first dav of the 11th week., the rats were pair-mated within their respective groups. The animals were allowed to pair-mate for exactly six davs, for three successive breedings, after which the males were removed on the 18th day of breeding, euthanized and teste saved in 10% buffered formalin. The dams were allowed to deliver their young. All female rats, except one, produced liveborn Iitters with litter size ranginq from 7 to 17. The Pups in each litter were sexed and culled to 10 at day 4, and were again resexed and culled to 2 per sex at day 21.

Weanlings for the second phase (91-Day Phase) of the study were chosen by biasing randomization in favor of body weight. Twenty-five rats per sex per group were chosen for the second phase of the study that lasted for 13 consecutive weeks.

Positive control:

The control group of animals received the basic ration of feed for the duration of the study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Observed daily by animal caretaker and/or Study Coordinator (Junior Toxicologist); Study Coordinator documented the observed weekly; Study
Director and the Veterinarian also observed these animals according to the SOPs.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; Terminal Sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Phase 2: 45 + or - 5 Days and Termination
- Animals fasted: Yes
- How many animals: Same animals as Clinical Chemistry (5 Males and 5 Females)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Phase 2: 45 + or - 5 Days and Termination
- Animals fasted: Yes
- How many animals: 5 Males and 5 Females

URINALYSIS: Yes
- Time schedule for collection of urine: Phase 2: 45 + or - 5 Days and Termination
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes

NECROPSY EXAMINATION: Yes
- Time schedule for examinations: 45 Days and Termination
- Dose groups that were examined: All
- Battery of functions tested: other: Complete Gross Necropsy

ORGAN WEIGHT: Yes
- Organ weighing and tissue blocking were done after fixation in 10% buffered formalin for at least 72 hours.
- Organ weights were measured from 10 male and 10 female rats per group at terminal sacrifice.
- Organ-to-body weight ratios were calculated.

HISTOPATHOLOGICAL EXAMINATION: Yes
- Phase 1: Reproductive Organs were preserved in case of abnormalities were detected in the reproductive organs of the offspring and the need arose to go back and examine them histopathologically.
- Phase 2: All animals received complete histopathology, that is microscopical examination of all tissues appearing on our lADRs.

Sacrifice and pathology:

GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes

Statistics:

The following determinations were statistically evaluated by PRL using the independent, two sided Students t-test at the 9S% confidence level:
a. Feed consumption data
b. Body weight data
c. Clinical chemistry data
d. Hematology data
e. Organ weight data

Scheffe, H.: Analysis of Variance, Wiley &: Sons, New York, N.Y., 1959.

Snedecor, G.W. an. Cochran, W.G.: Statistical Analysis, 6th ed., Iowa State Univ. press, Ames, La, 1967.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Details On Results

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See Details On Results

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See Details On Results

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

One or more tissues were founn to he abnormal, thus rendering a specific diagnosis.See Details On Results

Histopathological findings: neoplastic:

not examined

Details on results:

BODY WEIGHT AND WEIGHT GAIN
Phase 2 Males showed the following significance as compared to the control group): (a) the low-dose group showed a sporadic decrease at weeks 2 and 5, (b) the min-dose group showed a sporadic decrease at week 12, and (c) the high-dose group showed a persistent decrease from day 0 until the end of the study (week 13). We believe that this persistent statistical decrease in the bodv weight of the high-dose group in the males is pertinent, while the fluctuations in the low and mid-dose groups are not biologically meaningful. In the female rats belonging to the second phase of the study the following significance was observed: (a) the mid-dose group showed a statistical decrease at weeks 12 and 13, and (b) the high-dose group showed a statistical decrease from dav 0 unti the end of the study (week: 13). We believe that this persistent statistical decrease in the mean body weight of the high-dose level in the female rats is pertinent while the decrease in the mid-dose female group is a borderline effect.

It should be noted that the mean body weight values of the high-dose group in both sexes were much less than the corresponding controls at day zero and remained so throughout the study though the rates of body weight gains were comparable to those of the controls. This would suggest that the compound employed at the high-dose level leads to a pertinent effect on the offsping that manifests itself in a generalized decrease of body weight.

ORGAN WEIGHTS
Liver: There were statistically significant decreases in the mean hepatic weight between all test groups and the control group in the males, but not in the females. We do not believe that this decrease is biologically meaningful since there was no histopathological correlation, particularly in the males. Moreover, historical control data indicate that the decrease is a reflection of a higher mean liver weight for the corresponding control males than a biologically meaningful decrease.

Adrenals: There were statistically significant increases in the mean adrenal weights between the high-dose group and control groups in both sexes. We do not believe that these increases are biologically meaningful since there were no histopathological correlations. The changes observed were minimal and lacked a clear-cut dose response reIationship.

Most of the organ-to-body weiqht ratios in either sex did not show remarkahle
differences as compared to the control groups, except as follows: (a) the low and mid-dose groups in the males showed a remarkable decrease in liver-to-body weight ratio, and (b) the mid and high-dose groups in the females showed a remarkable increase in liver-to-body weight ratio. The changes observed in the male liver-to-body weight ratios were minimal, without histopathological correlates, and lacked a clear-cut dose response
relationship whereas the changes in the female liver-to-body weight ratios were biologically meaningful because of the presence of correlated histopathology in some of the female rats as explained below.

GROSS PATHOLOGY
No abnormal gross pathology findings were observed during the first phase of the study, during the second phase, the following abnormal gross pathology findings were observed: (a) Control male rat #3515 exhibited slightly congested tissue beneath the skin over the mid dorsal aspect of the body measurln 2.S x 2.0 x 0.5 cm, with several small (1-2 mm) clear cysts on its surface; (b) Control male rat #3517 showed the right anterior lobe of the liver to be herniated into the pleural cavity; (c) Control male rate #3535 showed the right anterior lobe of the liver to be herniated into the pleural cavity; (d) High-dose male rat #3685 had an overgrown lower incisor which had ulcerated the palate; (e) Mid-dose female rat #3632 showed two capsular, white circumscribed lesions present on the underside of the anterior gastric lobe of the liver, each measuring 3 mm in diameter; (f) Mid-dose female rat #3648 showed several capsular lesions on the left ventral lobe which were pale and slightly depressed. They extenden into the liver parenchyma for approximately 3 mm and all measured approximately 3 mm in diameter; (g) high-dose female rat #3658 showed the capsular surface and parenchyma of the right ventral lobe of the liver to be pale with a lesion approximately 1 cm in diameter; (h) High-dose female rat #3698 showed the capsular surface of the right anterior lobe of the liver to have a pale white lesion that extended into the parenchyma, measuring approximately 8 mm in diameter.

We do not believe that the first four (a-d) gross pathology findings are compound-related, whereas we consicler the last four (e-h) to be pertinent (compound-related).

HISTOPATHOLOGY: NON-NEOPLASTIC
High-dose female rat #3666 sacrificed at 91 days had a low SGOT value of 11 IU/l. Histopathologically this animal revealed a moderate nonzonal hepatic fatty infiltration, which may have accounted for this low SGOT value.

OTHER RESULTS:
Gross Pathology (#3632, :#:3648, #3658 and #3698) as well as in high-dose female rats #3666 and #3996. All six animals had similar type hepatic
lesions present which differed slightly in location ann degree of severity. Fatty infiltration, when observed, was present multifocallv although it was
predominantly periportal. Necrosis of an "acute" nature occurred in these foci in varying severity. A subacute to chronic predominantlv periportal inflammatory response accompanied this necrosis. Bile duct hyperplasia was observed in chronic, periportal areas of inflammation. An acute to chronic multifocal parenchymal hepatitis of an inflammatory nature was observed in the following female rats: control rat #3508; low-dose rats :#:3554 and #3580; mid-dose rats #3600 , #3604, #3606, #3618, #3624 and #3634; high-dose rats #3652, #3654, #3674 ann #3676 and in the following male rats: min-dose rat #3621; high-nose rats #3667, #3669 anri #3685. This lesion was characterized by multifocal discrete foci of inflammatory cells. Fatty infiltration and necrosis were not observed in these animals. This lesion appeared to be of an unrelated pathogenesis compared to the other six animals previously discussed and most likely of an endemic origin; the increase of affected animals in the mid and high-dose levels is most likely the result of the fact that these animals were stressed by the compound, allowing greater numbers of inflammatory type lesions to appear. Additional histopathology observed was limited to lesions also known to be endemic in this strain of rat, and is not considered to be compound-related.

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

gross pathology

histopathology: non-neoplastic

Critical effects observed:

not specified

Conclusions:

From the data presented we conclude that UV-2908, when fed to rats under the conditions of this study, caused a statistically and biologically meaningful
decrease in the body weight of the high-dose group in the F1a female rats and a borderline decrease in the mid-dose female rats. Histopathologically, a liver
lesion was present in six female rats (4 high-dose and 2 mid-dose) that appeared to begin as a fatty infiltration of the hepatic cells, periportally as well as nonzonally distributed. These cells progressed through degeneration to franknecrosis accompanied by a subacute to chronic inflammatory response. Although
in severe stages central areas were involved, it appeared that the main and original lesion was more periportal in distribution. This lesion, although noted at times to be multifocal, han a strong tendency to remain focal in moderate to severe cases. This is considered to be unusual in toxic liver injury where damage usually is widespread and affects most lobules to some degree. Another interesting feature of this lesion is that all six animals were female which suggests a difference in metabolism or hormonal influence in the development of said lesion, The low-dose group in both sexes was without any biologically meaningful
significance.

Executive summary:

From the data presented we conclude that UV-2908, when fed to rats under the conditions of this study, caused a statistically and biologically meaningful decrease in the body weight of the high-dose group in the F1a female rats and a borderline decrease in the min-dose female rats. Histopathologically, a liver lesion was present in six female rats (4 high-dose and 2 mid-dose) that appeared to begin as a fatty infiltration of the hepatic cells, periportally as well as nonzonally distributed. The study followed GLP and Protocol 406. The test substance doses used in this study were as follows: low dose group received daily, thoroughly mixed in the feed, 5000 parts of UV-2908 per million parts of feed PPM 0.5%; mid-dose group received daily, thoroughly mixed in the feed, 10000 parts of UV-2908 per million parts of feed PPM 1.0%; high-dose group received daily, thoroughly mixed in the feed, 50000 parts of UV-2908 per million parts of feed PPM 5%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

375 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available