2-(1,1-dimethylethyl)-6-[[3-(1,1-dimethylethyl)-2-hydroxy-5-methylphenyl]methyl]-4-methylphenyl acrylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb - 15 May 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted Sep 1998

Deviations:

not specified

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(1,1-dimethylethyl)-6-[[3-(1,1-dimethylethyl)-2-hydroxy-5-methylphenyl]methyl]-4-methylphenyl acrylate

EC Number:

262-634-6

EC Name:

2-(1,1-dimethylethyl)-6-[[3-(1,1-dimethylethyl)-2-hydroxy-5-methylphenyl]methyl]-4-methylphenyl acrylate

Cas Number:

61167-58-6

Molecular formula:

C26H34O3

IUPAC Name:

2-tert-butyl-6-[(3-tert-butyl-2-hydroxy-5-methylphenyl)methyl]-4-methylphenyl prop-2-enoate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The strain of rat was selected as a series of studies with the test substance was already performed in rats of this strain and compiled data were available at the testing facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: 132 - 173 g (females) and 173 - 216 g (males)
- Housing: 3 rats per cage were housed in plastic cages (41 x 26 x 20 cm) with hardwood chip bedding.
- Diet: Powdered diet CE-2 (CLEA Jpan, Inc., Tokyo, Japan), ad libitum
- Water: Drinking water (Ichinomiya City tap water), ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 60 ± 10
- Air changes (per hr): more than 15 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 Nov 1983 To: 22 Dec 1983

Administration / exposure

Route of administration:

oral: feed

Vehicle:

corn oil

Details on oral exposure:

DIET PREPARATION
The test material was incorporated at the required levels (without correction for the purity) into basal diet and mixed with a blender.
- Rate of preparation of diet (frequency): Weekly

VEHICLE
- Justification for use and choice of vehicle: Corn oil was added to all diets before mixing in order to prevent contamination with test material as dust.
- Concentration in vehicle: 2%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In order to analyze the content of the test material in the prepared diet, samples of the prepared diets were shipped to the sponsor. Analyses of stability and homogeneity of test material in the prepared diet had been performed prior to the commencement of the study. The homogeneity was satisfactory, and it was confirmed that test material in the diet was stable for 3 weeks in a freezer. The content of test material in the prepared diets was analyzed at every preparation. The prepared diet was started to use when the content values were confirmed to be within the acceptable range. Average contents of the test material were 2431, 4809, 9854 and 29431 ppm for the nominal concentrations of 2500, 5000, 10000 and 30000 ppm in the prepared diet, respectively.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 500 ppm

Remarks:

equivalent to 252.4 mg/kg bw/day for females and 175.1 mg/kg bw/day for males

Doses / concentrations 2

Dose / conc.:

5 000 ppm

Remarks:

equivalent to 506.9 mg/kg bw/day for females and 344.1 mg/kg bw/day for males

Doses / concentrations 3

Dose / conc.:

10 000 ppm

Remarks:

equivalent to 1019.3 mg/kg bw/day for females and 699.7 mg/kg bw/day for males

Doses / concentrations 4

Dose / conc.:

30 000 ppm

Remarks:

equivalent to 3281.3 mg/kg bw/day for females and 2193.3 mg/kg bw/day for males

No. of animals per sex per dose:

24 (12 animals per group were euthanized and necropsied after 6 weeks treatment with the test substance)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dietary levels in this study were set by considering the results of a previous 28 days repeated dose toxicity study entitled at the same testing facility. No treatment related changes were observed in the highest dietary level of 30000 ppm group, the dietary level of 30000 ppm was selected as the highest dose level for this study, then 10000, 5000, 2500 ppm were selected as higher middle, lower middle and low dietary levels, respectively.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, all animals were checked for general conditions and mortality
- Time schedule: Once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Two-day food consumption per cage was measured weekly, and mean daily food consumption values per rat were calculated from the number of animals housed in one cage and the number of days. Test material intake was calculated from the nominal dietary levels, group means of food consumption and body weight data.

FOOD EFFICIENCY: Yes
Food efficiency was calculated according to a standard method.

WATER CONSUMPTION: Yes
Two-day water consumption per cage was measured weekly, and mean daily water consumption values per rat were calculated from the number of animals housed in one cage and the number of days.

HAEMATOLOGY: Yes, blood samples were collected via the abdominal aorta under ether anesthesia at pretreatment and after experimental week 6 or 13. EDTA-2K was used as anticoagulant. Wright-Diemsa solution was used for the determination of differential white blood cell percentages
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight
- How many animals: All
- Parameters checked: red blood cell count (RBC), white blood cell count (WBC), haemoglobin (HB), haematocrit (HT), platelet count (PLT), differential white blood cell percentages and differential count of WBC (myelocytes, neutrophils (stab), neutrophils (segment), lymphocytes, monocytes, eosinophils, basophils)

CLINICAL CHEMISTRY: Yes
- Animals fasted: Yes, overnight
- How many animals: All
- Parameters checked: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total proteins, albumin/globulin ratio (A/G), blood urea nitrogen, sodium (Na), potassium (K), Na/K ratio (Na/K), glucose and albumin

URINALYSIS: Yes
- Time schedule for collection of urine: Fresh urine samples were collected from the animals for scheduled necropsy at pretreatment and week 6 or 13, and were subjected to the urinalysis.
- Parameters checked: pH, proteins, glucose, ketones, bilirubin, occult blood and urobilinogen

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals for scheduled necropsy were euthanized by exsanguination under ether anesthesia at pretreatment and after experimental week 6 or 13, and subjected to gross pathological examination for body surface and internal organs.
- Organs weights were recorded using an electric balance (1205MP, Sartorius K.K., Tokyo, Japan) after gross pathological examination: Brain, thymus, heart, lungs, liver, spleen, kidneys, adrenals, ovaries, testes and pituitary

HISTOPATHOLOGY: Yes, after fixation in 10% buffered formalin solution, the organs and tissues were routinely trimmed, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin (H&E), and then examined under light microscopically. In addition, bone tissues were decalcified by the rapid decalcification method, and processed for histopathological slides.
- Organs checked: Heart, lymph nodes (mandibular, mesenteric), spleen, bone and bone marrow (femur), thymus, adrenals, pituitary, lungs, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), pancreas, liver, kidneys, ovaries, testes, spinal cord, brain (cerebrum, cerebellum), and any gross lesions

Statistics:

Statistical comparisons between control and test material treated groups of numerical data for body weights, hematology data, clinical chemistry data and organ weights (absolute and relative) were assessed using the Student’s t-test. The significances of intergroup differences in incidences of findings of gross- and histopathology were analyzed by the Fisher’s exact probability test. The significance of differences for each parameters were analyzed and evaluated at 5 % (p <0.05) or 1 % (p <0.01).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males: Body weights in the 5000 ppm group were significantly lower than control values at weeks 7, 9 and 10.
Females: Body weights in the 10000 ppm group were significantly lower than control values at weeks 8 and 11, but within the control range at the end of treatment in week 13.
These effects were not considered to be related to the test material treatment, since these were transitory and not dose-dependent.

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):

effects observed, non-treatment-related

Description (incidence and severity):

Males: Decreased water consumptions were noted in the 30000 ppm group when compared to the control values from week 7 to week 13.
Females: Decreasing trend in water consumptions were observed in the 30000 ppm group when compared to the control values during the course of the study.
In addition, overall average water consumptions in both sexes of the 30000 ppm dose group were also reduced compared with the control group.

Since no adverse effects on general conditions, body weight, urinalysis or clinical chemistry were noted, the reduction of water consumption was not considered to be related to the test substance.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
A significantly decrease of haemoglobin values were observed in males of the 10000 ppm dose group after 6 weeks treatment with the test substance compared with the control group. After 13 weeks treatment with the test substance the hemoglobin values were within the range of the control animals.
Females:
In females of 5000, 10000 and 30000 ppm dose group a dose-dependent significantly reduced hemoglobin level after 6 weeks treatment with the test substance compared with the control group was determined.

However, the values were within the range of historical control data for rats of this strain according to data published by Charles River (reference: Charles River, Clinical Laboratory Parameters for Crl:CS (SD), March 2006). In addition, these changes were not considered to be treatment-related, since related parameters (RBC, WBC, HT and others) were not affected, and these alterations were reversible as the effects were not observed after 13-week treatment.
Please refer to Table 1 under "any other information on results incl. tables".

Reference: Charles River, Clinical Laboratory Parameters for Crl:CS (SD), March 2006; available online

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Compared to control animals significant higher values of potassium (lower value of NA/K ratio) were noted in females of 10000 and 30000 ppm dose groups after 6-week treatment. Significant increased potassium levels and significant decreased Na/K ratio were observed in females of the 5000, 10000 and 30000 ppm dose group after 13 weeks treatment with the test substance compared with control group. This effect was not considered to be related to the treatment, since the values were within acceptable range based on historical control data determined at the testing facility.
A dose-dependent significant reduction of total-cholesterol values was determined in animals exposed to 5000, 10000 and 30000 ppm compared to control animals. This effect was not considered to be related to the treatment, since the values were within acceptable range based on historical control data determined at the testing facility.
Significant higher values of alkaline phosphatase in both sexes of the 30000 ppm dose group, significant higher values of blood urea nitrogen in females of all treatment groups and males of the 30000 ppm dose group, significant higher values of albumin in females of the 30000 ppm dose group were observed after 6-weeks treatment. However, these changes were not considered to be related to the treatment, since they were not observed after 13-weeks treatment, and no related changes were found after histopathological examination.

Please refer to Table 2 and 3 under "any other information on results incl. tables".

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 13 weeks treatment with the test substance only traces of occult blood were observed in 1/12 females of the control group and the 5000 ppm dose group, respectively, and in 1/12 males of the control group, 2500 and 10000 ppm dose group, respectively.
However, it was considered that these changes were not related to the treatment, and they were incidental, since dose-dependence was lacking, and any related renal change was not determined after histopathological examination.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Significant lower absolute spleen weights (11%) were noted in males of the 5000 ppm dose group compared to controls. Significant higher relative kidney weights (8% and 9%, respectively) were observed in males of the 5000 and 30000 ppm dose group, respectively. In males of the 10000 ppm dose group a significant increase of the relative testis weight (11%) was determined.

Females:
Significant lower absolute thymus weights (17%), and significant higher relative brain weights (7%) and relative heart weights (9%) were noted in females of the 10000 ppm dose group compared to controls.

The increase of the relative kidney weight was considered not to be related to the test material treatment, since absolute kidney weight was comparable to control value, and any related renal change was not observed after histopathological examination. The effects on absolute organ weights observed in the study were not considered to be related to the test material treatment, since there were no changes in the relative organ weights and the effects occurred at individual doses without dose-dependence.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
Control: Single discolored spot (lung) in 7/12 males and small discolored testis in 1/12 males
2500 ppm: Single discolored spot (lung) in 3/12 males
5000 ppm: Crust cranium (skin/subcutis) in 1/12 males after 6-week treatment; single discolored spot (lung) in 1/12 males
10000 ppm: Single discolored spot (lung) in 2/12 males, small discolored testis, multiple discolored spots (lung) and discolored area (lung) each in 1/12 males
30000 ppm: Single discolored spot (lung) in 6/12 males, crust cranium (skin/subcutis) and reddened material perigenital (skin/subcutis) each in 1/12 males

Females:
Control: Single discolored spot (lung) in 4/12 females
2500 ppm: Single discolored spot (lung) in 4/12 females
5000 ppm: Single discolored spot (lung) in 2/12 females
10000 ppm: Single discolored spot (lung) in 3/12 females

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Heart, lymphocytic accumulation:
Control: 1/12 males (grade 1)
30000 ppm: 1/12 males (grade 1)

Pituitary, Cyst:
Control: 1/12 females (grade 1)
5000 ppm: 1/12 males (grade 1)
10000 ppm: 1/12 females (grade 1)

Lung, lymphocytic accumulation:
Control: 5/12 females (grade 1), 8/12 males (grade 1), 1/12 males (grade 2)
2500 ppm: 4/12 females (grade 1), 3/12 males (grade 1), 1/12 males (grade 2)
5000 ppm: 2/12 females (grade 2), 5/12 males (grade 1)
10000 ppm: 3/12 females (grade 1), 2/12 females (grade 2), 7/12 males (grade 1), 3/12 males (grade 2)
30000 ppm: 4/12 females (grade 1), 7/12 males (grade 1)

Lung, accumulation of foamy cells:
Control: 1/12 females (grade 1), 1/12 males (grade 1)
5000 ppm: 1/12 males (grade 1)
10000 ppm: 1/12 females (grade 1), 3/12 males (grade 1)

Lung, bronchopneumonia:
5000 ppm: 2/12 females (grade 1)

Lung, calcification arteria wall:
Control: 1/12 males (grade 1)
2500 ppm: 1/12 females (grade 1), 1/12 males (grade 1)
5000 ppm: 3/12 females (grade 1), 6/12 males (grade 1)
10000 ppm: 1/12 females (grade 1), 1/12 females (grade 2), 3/12 males (grade 1)
30000 ppm: 2/12 females (grade 1)

Large intestine, parasites in lumen:
Control: 1/12 females (grade 1), 3/12 males (grade 1)
2500 ppm: 3/12 females (grade 1), 3/12 males (grade 1)
5000 ppm: 1/12 females (grade 1), 3/12 males (grade 1)
10000 ppm: 2/12 females (grade 1)
30000 ppm: 1/12 females (grade 1), 3/12 males (grade 1)

Pancreas, atrophy of acinar cells:
5000 ppm: 1/12 females (grade 1)

Liver, fatty metamorphosis:
Control: 1/12 females (grade 1), 1/12 males (grade 1)
5000 ppm: 1/12 females (grade 1), 2/12 males (grade 1)

Adrenal, fatty metamorphosis:
Control: 1/12 males (grade 1)

Testis, atrophy:
Control: 1/12 males (grade 2)
1000 ppm: 1/12 males (grade 1)

Skin/subcutis, inflammation:
30000 ppm: 1/12 males (grade 1)

Preputial-clitoral gland, cyst:
30000 ppm: 1/12 males (grade 1)

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 30 000 ppm

Based on:

test mat.

Remarks:

equivalent to 3281.3 mg/kg bw/day for females and 2193.3 mg/kg bw/day for males

Sex:

male/female

Basis for effect level:

other: No adverse and treatment-related effects observed up to and including the highest tested dose level.

2

Key result

Dose descriptor:

NOAEL

Effect level:

3 281.3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse and treatment-related effects observed up to and including the highest tested dose level.

3

Key result

Dose descriptor:

NOAEL

Effect level:

2 193.3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse and treatment-related effects observed up to and including the highest tested dose level.

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Summay of hematological data

Parameter		Control males	1000 ppm males	3000 ppm males	10000 ppm males	30000 ppm males	Control females	1000 ppm females	3000 ppm females	10000 ppm females	30000 ppm females
	N	12	12	12	12	12	12	12	12	12	12
Hemoglobin (g/dL) at week 7	Mean ± SD	14.5± 0.4	14.2± 0.6	14.1± 0.7	14.0± 0.4 *	14.2± 0.6	14.8± 0.7	14.7± 0.8	14.1± 0.5 **	14.1± 0.4 **	13.9± 1.3 *
Hemoglobin (g/dL) at week 13	Mean ± SD	16.2± 0.9	15.7± 0.7	15.7± 0.5	15.7± 0.7	15.4± 1.0	15.8± 0.9	15.4± 0.5	15.5± 0.8	15.1± 0.5 *	15.1± 0.9

* Significantly different from control group at p < 0.05

** Significantly different from control group at p < 0.01

Table 2: Summary of clinical chemistry data

Parameter		Control males	1000 ppm males	3000 ppm males	10000 ppm males	30000 ppm males	Control females	1000 ppm females	3000 ppm females	10000 ppm females	30000 ppm females
	N	12	12	12	12	12	12	12	12	12	12
Aspartate aminotransferase (KU) at week 7	Mean ± SD	127 ± 32	140 ± 36	124 ± 28	130 ± 34	141 ± 28	118± 23	125 ± 25	124 ± 22	132 ± 29	139 ± 30
Aspartate aminotransferase (KU) at week 13	Mean ± SD	131 ± 39	121 ± 24	133 ± 35	140 ± 35	134 ± 49	119 ± 35	110 ± 24	118 ± 21	114 ± 19	115 ± 21
Alanine aminotransferase (KU) at week 7	Mean ± SD	26 ± 6	29 ± 7	32 ± 6 *	29 ± 6	30 ± 7	23 ± 6	27 ± 4	28 ± 4 *	29 ± 8 *	28 ± 10
Alanine aminotransferase (KU) at week 13	Mean ± SD	25 ± 8	25 ± 4	28 ± 9	29 ± 9	26 ± 7	31 ± 14	29 ± 4	25 ± 6	26 ± 5	27 ± 6
Total cholesterol (mg/dL) at week 7	Mean ± SD	54 ± 8	55 ± 8	52 ± 11	55 ± 9	52 ± 6	62 ± 13	62 ± 13	63 ± 9	61 ± 12	58 ± 12
Total cholesterol (mg/dL) at week 13	Mean ± SD	73 ± 16	67 ± 20	57 ± 9 **	53 ± 16 **	57 ± 13 *	71 ± 8	80 ± 13	71 ± 8	75 ± 10	73 ± 17
Potassium (meq/L) at week 7	Mean ± SD	4.9 ± 0.3	5.1 ± 0.4	4.9 ± 0.3	5.2 ± 0.4	5.4 ± 0.4 **	4.6 ± 0.2	4.7 ± 0.3	4.7 ± 0.4	4.9 ± 0.4 *	4.9 ± 0.3 *
Potassium (meq/L) at week 13	Mean ± SD	4.8 ± 0.3	4.7 ± 0.5	4.8 ± 0.4	4.9 ± 0.3	5.0 ± 0.4	4.0 ± 0.2	4.2 ± 0.3	4.3 ± 0.4 *	4.4 ± 0.5 *	4.5 ± 0.3 **

* Significantly different from control group at p < 0.05

** Significantly different from control group at p < 0.01

Table 3: Historical control data of CD(SD) rats at week 13 from 2 different examinations

	K (meq/dL)	Na (meq/dL)	Total cholesterol (mg/dL)
Female
N	19	19	19
Mean	4.4	142	72
S.D.	0.3	3	14
N	12	12	12
Mean	4.4	143	78
S.D.	0.4	1	13
Male
N	20	20	20
Mean	4.7	143	49
S.D.	0.6	1	7
N	12	12	12
Mean	4.7	144	68
S.D.	0.5	1	7