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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 July 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(2-hydroxyphenyl)methyl]phenol; 2-[(4-hydroxyphenyl)methyl]phenol; 4-[(4-hydroxyphenyl)methyl]phenol
EC Number:
908-912-9
Cas Number:
1333-16-0
Molecular formula:
C39H36O6
IUPAC Name:
2-[(2-hydroxyphenyl)methyl]phenol; 2-[(4-hydroxyphenyl)methyl]phenol; 4-[(4-hydroxyphenyl)methyl]phenol
Test material form:
solid

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Crj:CD (SD) rats were purchased from Charles River Japan, Inc (Shiga, Japan). Animals were weighed, weight ranked, and randomly assigned to each of the treatment groups and control group before administration, and then housed individually in stainless steel, wire-mesh cages throughout the study. Rats were provided with water automatically and with a commercial diet (MF, Oriental Yeast
Co., Tokyo, Japan) ad libitum. The animal room was maintained at a temperature of 23 ± 2°C and a relative humidity of 55 ± 15%, and it was artiWcially illuminated with Xuorescent light on a 12- h light/dark cycle (07:00–19:00 h).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
The volume of the olive oil solution containing the chemical for gavage was 10 ml/kg
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Each group consisted of 10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
In the preliminary test, rats were orally gavaged with 0, 50, 250, 500 and 1,000 mg/kg per day for 14 days and resulted that decreased body weights, hematological and biochemical
abnormalities and organ weight changes were detected in 500 and 1,000 mg/kg groups, so we selected these doses in this study. A vehicle control group was gavaged with olive oil alone.

The concentration and stability of the test chemical were confirmed.

Animals were killed by exsanguination under ether anesthesia, and blood samples were obtained from the abdominal aorta and examined for hematological, clinical biochemistry and hormonal parameters.

Examinations

Observations and examinations performed and frequency:
General observations
Clinical signs were recorded daily. Once before the first dose and once a week thereafter, detailed clinical observations of all animals were made outside the home cage. The signs for which the animals were examined included changes in skin, fur, eyes, and mucous membranes, frequency of urine and feces, and autonomic activity (e.g.,lacrimation, piloerection, pupil size, respiratory pattern). Changes in gait, posture, response to handling, the occurrence of clonic or tonic movements, stereotypes (e.g., excessive grooming, circling), or bizarre behavior (e.g., self-mutilation, walking backwards), were also recorded. In the 4th week, a functional observation battery (FOB) that tested sensory reactivity to stimuli of diVerent types (e.g., auditory, visual, and proprioceptive), assessed grip strength, and assessed motor activity, was also conducted.

Body weight and food consumption
Individual body weight was recorded twice weekly and immediately before necropsy. Food consumption was measured weekly

Hematology
The following were examined in the hematology examinations: red blood cell count, white blood cell count, hemoglobin concentration, hematocrit value, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, prothrombin time, activated partial thromboplastin time, and diVerential leukocyte count.

Clinical biochemistry
Serum levels of the following were measured in the clinical biochemistry examination: glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, alkaline phosphatase,cholinesterase, γ-glutamyl transpeptidase, total cholesterol, triglyceride, glucose, total protein, albumin, blood urea nitrogen, creatinine, total bilirubin, calcium, inorganic phosphorus, sodium, potassium and chlorine, and the albumin–globulin ratio was calculated.

Hormone analysis
The serum concentrations of the following hormones were measured at the end of the test period: thyroid-stimulating hormone (TSH), thyroxin (T4) and triiodothyronine (T3). T3 and T4 were determined with an automatic immunoassay system (IMX, Abott laboratories), and TSH was measured with a microplate reader (FLUOSTAR OPTIMA, BMG Labtechnologies).

Spermatology
Sperm morphology (200 formalin-Wxed, Giemza-stained spermatozoa) and (heated to 70°C) the sperm count were determined by examining by using specimens obtained from the right epididymis. The number of homogenization- resistant (0.9% NaCl solution plus Triton-X100) sperm was determined on specimens from the right testis.

Estrous cycling
The estrous cycle of all females was assessed daily from day 22 until the day of sacriWce by examining vaginal smears stained with Giemsa stain.
Sacrifice and pathology:
Necropsy
Males were necropsied on day 29. Females were necropsied after having been dosed for at least 29 days and sacrificed on days 30–34 to allow them to be sacriWced in the diestrous stage.

Organ weight
The following organs were weighed after necropsy: testes, epididymes, ventral prostate, dorsolateral prostate, seminal vesicles, ovaries, uterus, adrenals, liver, spleen, kidneys, heart, brain, and thymus, as fresh organs, and the thyroid and pituitary gland, after organ Wxation.

Histopathology
The following organs were Wxed in 10% neutral buffered formalin and examined: prostate, including ventral prostate and dorsolateral prostate, seminal vesicles, ovaries, uterus, vagina, mammary gland, brain, thyroid, adrenals, liver, spleen, kidneys, stomach, intestine, pancreas, thymus, axillar and mesenteric lymph nodes, parathyroids, and pituitary gland. The epididymes and testes were fixed in Bouin’s
solution before examining them.
Statistics:
Body weight, food consumption, hematological data, clinical biochemical data, hormonal data, spermatological data (sperm counts), organ weight and FOB data were analyzed by the Bartlett’s test for homogeneity of variance. When the variance was homogeneous at a significance level of 5%, one-way analysis of variance was performed. If a significant diVerence was found, the difference between the control group and each of the dosage groups was analyzed by Dunnett’s test. If the variance was not homogeneous, the Kruskal–Wallis test was used. If a significant difference was found, the difference between the control group and each of the dosage groups was analyzed by the nonparametric Dunnett’s test. FOB numerical data and spermatological data (sperm morphological data) were analyzed by the Kruskal–Wallis test. If a significant difference was found, the difference between the control group and each of the dosage groups was analyzed by the nonparametric Dunnett’s test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Decreased spontaneous locomotion, stained lower abdomen and around anus, white turbid urine, and soft stool were observed in male rats in the 500 mg/kg group. Stained around the nose, mouth, anus, and lower abdomen was observed in female rats in the 100 group and 500 mg/kg group, and decreased spontaneous locomotion and white turbid and reddish urine were observed in female rats in the
500 mg/kg group.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight and food consumption of male rats given 500 mg/kg bisphenol F and females given 20, 100, and 500 mg/kg bisphenol F were lower than in rats given vehicle.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The body weight and food consumption of male rats given 500 mg/kg bisphenol F and females given 20, 100, and 500 mg/kg bisphenol F were lower than in rats given vehicle.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Red blood cells, hemoglobin concentration, and hematocrit values were decreased in the female rats of the 500 mg/kg group, but no hematological changes were detected in the male rats given bisphenol F
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the male rats, decreased serum total cholesterol and blood urea nitrogen values were observed in the 100 and 500 mg/kg groups, decreased glutamic-oxaloacetic transaminase in the 500 mg/kg group, and increased γ-glutamyl transpeptidase and total bilirubin in the 500 mg/kg group. In the female rats, decreased serum total cholesterol, glucose and albumin values were observed in the 20, 100, and 500 mg/kg groups, decreased cholinesterase and a decreased albumin–globulin ratio in the 100 and 500 mg/kg groups, and increased alkaline phosphatase and γ-glutamyl transpeptidase in the 500 mg/kg group
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the male rats, increased liver, testis, brain, and thyroid weight was observed in the 500 mg/kg group. In the female rats, increased brain weight was observed in the 20, 100, and 500 mg/kg groups, increased kidney weight in the 100 and 500 mg/kg groups, and increased liver weight in the 500 mg/kg group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No dose-related gross changes were detected in either the male or female rats.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Dilatation of the subcapsular sinus of the mesenteric lymph nodes was detected in two male rats in the 500 mg/kg group. No other dose-related histopathological changes were detected in either the male or female rats.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Hormone analysis
In the male rats, decreased serum T3 and increased T4 values were observed in the 500 mg/kg group. In the femalerats, increased T4 values were observed in the 20, 100 and 500 mg/kg groups, and decreased T3 in the 500 mg/kg group.

Spermatological analysis
There were no abnormal spermatological Wndings.

Estrous cycles
No abnormal estrous cycles were detected in any of the groups.
Details on results:
It was concluded that the endocrine-mediated effects were not detected in young adult rats given bisphenol F.

The decrease in body weights of the male rats given 500 mg/kg and in the female given 20, 100, and 500 mg/kg was a toxic effect of bisphenol F.

Although no histopathological effect was detected in the liver in the 500 mg/kg group, the increased relative liver weight in both sexes and the changes in alkaline phosphatase, γ-glutamyl transpeptidase and total bilirubin in the male and/or female rats in this group were concluded to be the result of effects of bisphenol F on the liver.

Although a slight increase in relative kidney weight was observed in the female rats in the 100 and 500 mg/kg groups, with no clear dose-relationship and
reddish urine in the female rats in the 500 mg/kg group, no histopathological changes and no increases in the blood urea nitrogen and creatinine values were detected in these groups. It was therefore concluded that bisphenol F had no effect on the kidney.

The decreases in cholinesterase, total cholesterol, glucose, and albumin values and decrease albumin– globulin ratio in the male and female rats given
bisphenol F appeared to be due to a nutrition disorder related to the decreasing body weights. The decreased glutamic-oxaloacetic transaminase and blood urea values werenot considered to be important toxic changes.

Relative brain weight in the male and female rats in the 500 mg/kg group and relative testis weights in the 500 mg/kg group increased, but the absolute weights of these organs did not change significantly. These organ weight changes were related to the decreased body weights and were not toxic changes.

On the other hand, the decreased blood cells counts, hemoglobin concentration, and hematocrit values in the female rats in the 500 mg/kg group presented mild anemia.

Effect levels

Dose descriptor:
NOEL
Effect level:
< 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The data obtained in this study indicated that the no observed-effect level of bisphenol F is under 20 mg/kg per day.
Executive summary:

A 28-day repeated-dose toxicity study (OECD test guideline No. 407) on bisphenol F to determine whether it has endocrine-mediated properties.

Bisphenol F was orally administered at doses 0, 20, 100 and 500 mg/kg per day for at least 28 days, but no clear endocrine- mediated changes were detected, and it was concluded to have no endocrine-mediated effects in young adult rats. On the other hand, the main effect of bisphenol F was concluded to be liver toxicity based on clinical biochemical parameters and liver weight, but without histopathological changes. The no-observed-effect level for bisphenol F is concluded to be under 20 mg/kg per day since decreased body weight accompanied by decreased serum total cholesterol, glucose, and albumin values were observed in the female rats given 20 mg/kg per day or higher doses of bisphenol F.

The data obtained in this study indicated that the no observed-effect level of bisphenol F is under 20 mg/kg per day.