reaction mass of isomers of: C7-9-alkyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

between 5 April 2002 and 15 July 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

other: Body responsible for the test

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): OS162170C
- Molecular formula (if other than submission substance): C21H34O3
- Molecular weight (if other than submission substance): 334.491
- Smiles notation (if other than submission substance): Oc1c(C(C)(C)C)cc(cc1C(C)(C)C)CCC(=O)OCCCC
- InChl (if other than submission substance): 1S/C21H34O3/c1-8-9-12-24-18(22)11-10-15-13-16(20(2,3)4)19(23)17(14-15)21(5,6)7/h13-14,23H,8-12H2,1-7H3
- Structural formula attached as image file (if other than submission substance): see Fig.1
- Substance type: alkylated phenol
- Physical state: light yellow liquid
- Analytical purity: 98%
- Purity test date: responsibility of the sponsor
- Lot/batch No.: none provided
- Expiration date of the lot/batch: 2003-11-15
- Stability under test conditions: responsibility of the sponsor
- Storage condition of test material: A five-gram retention sample of the test article was taken and stored at room temperature at SLI. However, the bulk test article was subsequently observed to have crystallized following receipt. Therefore, the bulk test article was stored in an -40°C oven at the suggestion of the Sponsor. However, -40°C storage did not maintain the test article in liquid form so the storage temperature was increased to -50°C for the remainder of the study.
- Other:
- Receipt date of the sample: 2002-04-05
- The test article was dosed as received from the Sponsor, therefore, no analytical analysis of the dose material was performed at SLI. A 5 mL sample of the bulk test article was collected, in an amber glass vial, prior to study initiation and following study completion, maintained at ambient room temperature and shipped to the Sponsor by overnight courier at ambient temperature for possible analysis by the Sponsor.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan.
- Age at study initiation: nine weeks
- Weight at study initiation: Individual body weights were recorded on the day following receipt and prior to randomization on day -1 (260 to
306 grams for males and 194 to 230 grams for females).
- Fasting period before study: no
- Housing: individually in suspended stainless steel cages.
- Diet (e.g. ad libitum): ad libitum, except when feed was withheld overnight prior to blood collection for clinical pathology determinations.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days for the males and 13 days for the females prior to randomization.
On study days -7 to -2, the animals were acclimated to the dermal wrapping procedure. Each animal was wrapped with an elastic wrap (Coflex) for
approximately two hours on days -7 and -6, approximately four hours on days -5 and -4 and approximately six hours on days -3 and -2.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: June 5, 2002 (day 0 of test material administration),To: July 15, 2002 (day 39, final terminal euthanasia ).

Administration / exposure

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST SITE
- Area of exposure: dorsal skin area: the clipped area included the area starting at the scapulae to the hipbone and halfway down the flank on each side.
- % coverage: 10
- Type of wrap if used: The test site was then covered with a 4-ply gauze pad, the torso was wrapped with an elastic wrap (Coflex) and secured with adhesive tape.
- Time intervals for shavings or clipplings: prior the first treatment on day 1 and then the application area was re-clipped as necessary throughout the study.
- The test article and control dosing containers were placed in an -50°C water bath during dosing. In addition, the dosing syringeheedles were placed in the -50°C water bath (inside a plastic bag to prevent contamination of the equipment with the water) between doses to insure the residual test article in the gavage needle syringe did not crystallize.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 101, 253, 505 and 1010 µL/kg bw (based on density of 990 mg/mL)
- Concentration (if solution): pure substance
- Constant volume or concentration used: no


VEHICLE
no vehicle was used. Pure test material was applied to skin of animals.

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The test article and control material were administered as received. Dose volumes (mL/kg) for each test group were determined using the Sponsor-supplied dose (mg/kg) and the Sponsor-supplied density (990 mg/mL). The control material was administered at the same dose volume as the high-dose level. The bulk test article and control material containers were maintained in a heated oven. Dosing materials were dispensed fresh daily following inversion of the bulk test article container several times. The daily dosing containers were then placed in a heated water bath to insure that the test article did not crystallize during the dosing procedure.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Duration of exposure per day: 6 hours
Dosing regime: 5 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 250, 500 and 1000 mg/kg bw/day
Basis:
nominal per unit area

No. of animals per sex per dose:

10;
5 (recovery control and the highest dose level )

Control animals:

other: mineral oil (at the same dose volume as the high-dose level (1010 µL/kg))

Details on study design:

- Dose selection rationale: defined in a 14-day range-finding study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: to study reversibility of effects
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): random

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily in the morning and afternoon. For each animal between one-half and two hours following dosing. In addition, cage-side observations were performed once daily on days the animals were not dosed and daily during the recovery period, except on the days of
detailed observations.
- Cage side observations checked: general heaIth/mortality and moribundity checks.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On day -1, prior to dosing on days 7, 14, 21 and 25 (main phase), and on days 32 and 38 (recovery phase), the animals received a detailed clinical observation.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: prior to dosing on days 7, 14, 21 and 25 (main and recovery animals) and days 32 and 38
(recovery animals).

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed prior to the initiation of treatment on day -1. During the study period, individual body weights were recorded on days 7, 14, 21 and 25 (main and recovery animals) and days 32 and 38 (recovery animals). In addition, a terminal body weight was recorded on the day of scheduled euthanasia (day 26 or 39) for calculation of relative organ weight data.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Individual food consumption was measured on days -1, 7, 14, 21 and 25 (during the treatment phase) and days 32 and 38 (during the recovery phase).

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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled euthanasia at the end of the main (day 26) and recovery (day 39) phases
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled euthanasia at the end of the main (day 26) and recovery (day 39) phases
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
- BIOCHEMISTRY PARAMETERS:
Prior to dosing on days 0 and 14, the animals were lightly anesthetized by isoflurane inhalation prior to blood collection via the orbital plexus. All blood samples were obtained in the morning with the time of collection recorded for each animal. In addition, blood samples were collected sequentially by group (Le., one group 1 animal, one group 2 animal, one group 3 animal, one group 4 animal, one group 5 animal, one group 1 animal, etc.). The biochemistry determinations are summarized in the table 2.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3).
All animals were subjected to a gross necropsy at the time of scheduled euthanasia (day 26 or 39). The necropsy examination included evaluation of the external surfaces of the body and all viscera.
Fresh organ weights were obtained at scheduled euthanasia for the adrenal glands, brain, heart, kidneys, liver, ovaries, prostate, spleen, testes with epididymides, thymus, uterus (weighed with intact cervix) and vagina. The thyroids were fixed in 10% neutral buffered formalin (NBF) and then trimmed under a microscope prior to weighing. Paired organs were weighed together. The organs/tissues preserved in 10% NBF are listed in the table 3.

HISTOPATHOLOGY: Yes (see table 3)
All tissues and organs collected at necropsy (days 26 and 39) from all animals in the control and high-dose groups, as well as the adrenal glands, brain, heart, kidneys, liver, ovaries, prostate, spleen, testes with epididymides, thymus, thyroids, uterus and vagina of the Groups 2, 3 and 4 animals (100, 250 and 500 mg/kg bw/day respectively), were processed for histopathological examination. The tissues were trimmed, embedded in paraffin, sectioned and slides stained with hematoxylin and eosin. Histology was performed by HistoTechniques, Powell, Ohio, and the tissues were examined microscopically by a board-certified veterinary pathologist.

Other examinations:

No

Statistics:

Inferential statistical analyses were performed on the SLI Compaq Alpha DS-10 GenTox Computer System. Body weights, body weight gain and food consumption were analyzed by One-way Analysis of Variance (ANOVA). If significance was detected with ANOVA (p<0.05), pair-wise group comparisons proceeded using the Tu key-Kramer test. Absolute and relative organ weights, and clinical pathology data were analyzed for homogeneity of variance using Levene’s test. If significance was detected with Levene’s test (p < 0.01), multiple group comparisons proceeded using the Kruskal-Wallis non-parametric ANOVA, followed by Dunn’s test, when p < 0.05 .If significance was not detected with Levene’s test, parametric procedures were used to analyze the data, i.e., ANOVA followed by Tukey-Kramer test when p < 0.05.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

decreased in the 100 and 500 mg/kg bw (no clear dose-response relationship)

Food consumption and compound intake (if feeding study):

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In 1000 mg/kg bw males only: statistically increased haematocrit and MCV, decreased MCHC (at study day 26), increased platelets (at study day 39).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In 1000 mg/kg bw males and females, increased bilirubin (at study day 26); In 1000 mg/kg bw males, decreased urea nitrogen (at study day 39). In 1000 mg/kg bw females, increased creatinine (at study day 26). In females, decreased enzyme levels.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased liver and thyroid weight; decreased adrenal and kidney weights,

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the high dose female animals, minimal thyroid follicular cell hypertrophy etc.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:
No mortality was observed in males or females during the main and recovery phases of this study. In addition, there were no test article-related clinical abnormalities noted during this project. All clinical findings were typical of this age and strain of rat the dermal dosing procedure utilized on this project.
There were no test article-related dermal findings observed during this study. Sporadic instances of mild irritation were observed in all groups during the main phase of this study, but were more prominent in the males. These findings generally consisted of minimal erythema, focal/pinpoint eschar and/or desquamation. Minimal erythema and desquamation was observed sporadically in the females. These changes were not considered to be toxicologically significant since they generally occurred at similar or greater frequencies in the control animals. In addition, these types of dermal changes are not uncommon in this species with oil-based vehicles and a semi-occlusive dermal exposure. No dermal irritation was observed in either
males or females during the recovery phase of this study.

BODY WEIGHT AND WEIGHT GAIN
There were not significant changes in mean body weight or body weight gain during the main or recovery phases of the study. Body weight gain was statistically decreased in the 100 and 500 mg/kg/day females during the day -1 to 7 interval. However, this was not considered significant as
there was no clear dose-response relationship. In additional, weight gain for the control females was higher than is typically observed.

FOOD CONSUMPTION
There were no statistically or toxicologically meaningful changes in the amount of food consumed during the main or recovery phases of this study. Food consumption was comparable between the test article-treated groups and the controls.

HAEMATOLOGY
In males, statistically increased haematocrit (1000 mg/kg/day males) and MCV (100 and 1000 mg/kg/day males) and statistically decreased MCHC
(1000 mg/kg/day males) was noted on study day 26. By the end of the recovery phase on day 39, the above haematology parameters were comparable to controls. Statistically increased platelets were also observed in the 1000 mg/kg/day males on day 39. However, this finding was not considered to be meaningful since this change occurred at the end of the recovery phase and was not present following four weeks of treatment on day 26.
In females, there were no statistically or toxicologically meaningful changes in the haematology parameters examined during the main or recovery phases of this study.

CLINICAL CHEMISTRY
In males, statistically increased bilirubin was observed in the 1000 mg/kg males on study day 26. By the end of the recovery phase on day 39, serum bilirubin concentrations in the 1000 mg/kg/day males were comparable to controls. Statistically decreased urea nitrogen was also observed in the 1000 mg/kg males on study day 39. However, this finding was not considered to be meaningful since this change occurred at the end of the recovery phase and was not present following four weeks of treatment on day 26. In addition, there were no associated histopathological changes in the kidneys.
In females, statistically increased bilirubin was observed in the 1000 mg/kg/day group on study day 26. By the end of the recovery phase on day 39, serum bilirubin concentrations in the 1000 mg/kg/day females were comparable to controls. In addition, statistically increased creatinine was observed in the 1000 mg/kg/day group on study day 26. The elevated creatinine was not thought to be toxicological significant since there were no microscopic abnormalities in the kidneys and the other biochemical indicators of renal disease or altered glomular filtration were not affected. By the end of the recovery phase on day 39, serum creatinine concentrations in the 1000 mg/kg/day females were comparable to controls.
Statistically decreased alkaline phosphatase (1 00 mg/kg/day group), glucose (500 mg/kg/day group) and GGT (250 mg/kg/day group) were also observed in the females. However, these changes were not considered toxicologically meaningful since there was no dose-response relationship.

ORGAN WEIGHTS
In males, there were no toxicologically meaningful changes in organ weight data. Relative liver weights were statistically increased in the 1000 mg/kg/day animals on study day 26. However, this change did not appear meaningful since the absolute liver weight for this group was not affected, hepatic biochemistry parameters were normal and there was no abnormal pathology observed in the liver following microscopic evaluation. By the end of the recovery phase on day 39, relative liver weights in the 1000 mg/kg/day males were comparable to controls.

In females, a statistically significant increase in absolute and relative thyroid weights (1000 mg/kg/day group) was observed on study day 26 with a notable, but not statistically significant, increase persisting to the end of the recovery phase. The increased thyroid weights appeared to be a secondary result of the follicular cell hypertrophy observed in this group following histopathological evaluation. Statistically decreased absolute adrenal weights (500 and 1000 mg/kg/day groups) and decreased absolute kidney weights (500 mg/kg/day group) were also observed on study day 26. The significance of the decreased absolute adrenal weight is questionable since the relative weight ratio was not affected and there was no microscopic pathology observed in the adrenal glands. The significance of the decreased absolute kidney is also of questionable significance since there was no dose-response relationship, no abnormal renal biochemistry in this group and no microscopic pathology observed in the kidneys.

GROSS PATHOLOGY
There were no apparent test article-related changes observed at gross necropsy at the end of the main and recovery phases of this study. All internal findings were typical of this age and strain of rat.

HISTOPATHOLOGY: NON-NEOPLASTIC
Minimal thyroid follicular cell hypertrophy was observed in the 1000 mg/kg/day females rats on day 26 (7110) and at the end of the recovery period on day 39 (315). No follicular cell hypertrophy was observed in the 100, 250 or 500 mg/kg/day females or in the 100, 250, 500 and 1000 mg/kg/day male groups. A few commonly observed spontaneous lesions were diagnosed and generally included minimal focal mixed cell inflammatory infiltrate in the liver, minimal haemorrhage in an occasional thymus and lymph node and a small focal lymphocytic infiltrate in an occasional organ/tissue. Most of these findings were minimal in degree of severity and the incidence was similar in all groups, including controls.


OTHER FINDINGS
THYROID HORMONE ANALYSIS

In males, there were no statistically significant changes in any of the thyroid hormone levels evaluated during the main or recovery phases of this study.
In females, RT3 was statistically increased in the 1000 mg/kg/day group on study day 26. By the end of the recovery period on day 36, serum RT3 concentrations in the 1000 mg/kg/day females were comparable to controls.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Findings were considered to be of no toxicologically significance

2

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Minimal thyroid follicular cell hypertrophy in the highest dose group. However, the change did not have a notable effect on the thyroid serum hormones levels and had no apparent effect on the overall health on the animals.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Dermal administration of test material for four consecutive weeks (5 days/week) at up to 1000 mg/kg/day did not produce any mortality, significant clinical abnormalities or notable dermal findings. Minimal thyroid follicular cell hypertrophy was observed in the 1000 mg/kg/day females at the end of the treatment and recovery periods. However, the above change did not have a notable effect on the thyroid serum hormones levels and had no apparent effect on the overall health on the animals. Therefore, the no-observed-adverse-effect level (NOAEL) for this study was considered to be 1000 mg/kg/day in the males and 500 mg/kg/day in the females.

Executive summary:

The present study was conducted in accordance with US-EPA Health Effects Test Guidelines OPPTS 870.3200 (August 1998) and was focusing more specifically on the potential target organ effects. The study is considered to be of the highest quality and therefore is reliable without restrictions (Klimisch 1). The purpose of this study was to evaluate the potential toxicity of OS162170C when administered to rats by the dermal route for four consecutive weeks (five days/week) followed by a two-week recovery phase. The study design consisted of a control group and four treatment groups with fifteen animals per sex in the control and high-dose groups and ten animals per sex in the low- and mid-dose groups. The test article was administered, in the morning, as a single daily dose at dosage levels of 100, 250, 500 and 1000 mg/kg/day for five days/week for four weeks. Control animals received the control material (mineral oil) at the same dose volume as the high-dose level under the same experimental conditions. Detailed clinical observations were performed on day -1, prior to dosing on days 7, 14, 21 and 25 and on days 32 and 38 during the recovery phase. Cage-side observations were performed between approximately one-half to two hours following dosing on days 0-4, 7-11, 14-18, and 21-25. Cage-side observations were also performed once daily on days the animals were not dosed and daily during the recovery period, except on the days of detailed observations. Dermal irritation examinations were conducted, using a dermal scoring system, prior to dosing on days 7, 14, 21 and 25 and on days 32 and 38 during the recovery phase. Food consumption and individual body weights were recorded on days -1, 7, 4, 21, 25, 32 and 38. A final body weight was obtained from each animal prior to scheduled euthanasia (day 26 or 39). Blood samples were obtained in the morning from all animals, sequentially by group, prior to dosing on days 0 and 14 for triiodothyronine (T3), reverse triiodothyronine (RT3), thyroxine (T4) and thyroid stimulating hormone (TSH) analyses. Blood samples were also collected on the day of scheduled euthanasia (day 26 or 39) for evaluation of selected clinical pathology parameters, including T3, RT3, T4 and TSH analyses. Following collection of blood samples, serum was obtained and all serum samples designated for T3, RT3, T4 and TSH analyses were frozen at approximately -70°C and shipped following study completion to Ani Lytics Inc., Gaithersburg, Maryland. Each rat was subjected to a complete gross necropsy examination at scheduled euthanasia (day 26 or 39). Ten males and ten females per group were euthanized at the end of the main phase (day 26). The remaining males and females in the control and high-dose groups were euthanized at the end of the recovery phase (day 39). Fresh organ weights were obtained for all animals at scheduled euthanasia, with the exception of the thyroid glands, which were fixed and trimmed under a microscope prior to weighing. Selected tissues, including treated skin, were preserved from all rats. All tissues and organs collected at necropsy from animals in the control and high-dose groups were examined microscopically, as well as the adrenal glands, brain, heart, kidneys, liver, ovaries, prostate, spleen, testes with epididymides, thymus, thyroids, uterus and vagina from the low- and mid-dose groups.

Results: No mortality, test article-related clinical abnormalities or notable dermal findings were observed during the main and recovery phases of this study. In addition, there were no toxicologically meaningful changes in body weights, food consumption or gross necropsy observations during this project. In the 1000 mg/kg/day males, a slight increase in haematocrit, MCV and bilirubin and a slight decrease in MCHC was observed on day 26. These changes could be interpreted as slight hemolytic anemia, however, the absence of microscopic changes in the bone marrow, spleen or liver along with the lack of increases in the number of reticulocytes or polychromatic cells suggests that, if anaemia was present, it was minimal and had little impact on the overall health of the animal. In the 1000 mg/kg/day females, statistically elevated RT3 was observed at the end of the treatment period on day 26. In addition, statistically increased absolute/relative thyroid weights were noted in these animals on day 26 and follicular cell hypertrophy was observed microscopically at both the day 26 and 39 intervals. These findings appear to be associated with test article treatment. However, the absence of correlative changes in the other key thyroid hormones (ex., increases in serum TSH or decreases in T3 or T4) suggests that the above changes, if real, had minimal physiological effect on the thyroid and on the overall health of the animal.

Conclusion: Based on the results of this study, dermal administration of OS1 621 70C for four consecutive weeks (5 days/week) at up to 1000 mg/kg/day did not produce any mortality, significant clinical abnormalities or notable dermal findings. Minimal thyroid follicular cell hypertrophy was observed in the 1000 mg/kg/day females at the end of the treatment and recovery periods. However, the above change did not have a notable effect on the thyroid serum hormones levels and had no apparent effect on the overall health on the animals. Therefore, the no-observed-adverse-effect level (NOAEL) for this study was considered to be 1000 mg/kg/day in the males and 500 mg/kg /day in females.