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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-11-20-2001-02-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to GLP as well as Guidelines.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to
Guideline:
other: japanese MITI guidelines for toxicity testing of chemicals
Principles of method if other than guideline:
A two-year oral gavage toxicity/oncogenicity study of Bisphenol A Diglycidyl Ether (BADGE) was initiated but study was terminated on test days 99 (males) or 101 (females) due to excessive toxicity noted in rats. Standard toxicologic parameters consistent with OECD guideline #408 were evaluatedon a subset of ten rats/sex/dose level.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
99.32% pure BADGE by area percent chromatography. Water content determined to be 0.02% by a Karl Fischer coulometric titration. Infrared spectroscopy confirmed the major component to be BADGE.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Male and female Fischer 344 rats (7 weeks of age) were received from a commercial supplier, evaluated for general health by a laboratory veterinarian, and were housed 2-3 per cage (suspended stainless steel cages with feed crock and pressure-activated nipple watering system) in rooms designed to maintain adequate environmental conditions and photocycle for rats. They were allowed to acclimate to the laboratory for approximately 3 weeks prior to the start of the study.

The animals were stratified by body weight by a computer randomization program and were assigned to dose groups. They were assigned unique animal identification numbers via subcutaneously-implanted transponders.

Animals were provided food and water ad libitum throughout the study. Food was analyzed for nutritional content and contaminants by the supplier. The water was obtained from a municipal water source, analyzed periodically for chemical parameters and biological contaminants by the municipal water department.

The laboratory operates under AALAC accreditation standards.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% Methocel A4M methyl cellulose ethers with 0.1% Tween 80
Details on oral exposure:
Four groups of 65 male and 65 female rats were orally gavaged with BADGE 7 days per week for 14 weeks as an aqueous suspension in 0.5% Methocel A4M methyl cellulose ethers with 0.1% Tween 80. Concentrations resulting in doses of 0, 50, 250, and 1000 mg/kg/day were used to evaluate systemic toxicity. Dose volume and concentration was calculated based on the most recent animal body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity, stability, and concentration of the dose solutions were verified throughtout the study.
Duration of treatment / exposure:
7 days per week for 14 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 250, and 1000 mg/kg/day
Basis:
other: nominal in dose solution
No. of animals per sex per dose:
65/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
The study was intended to be a 2-year chronic/oncogenicity study. The study was terminated at 99-101 days due to excessive toxicity at 250 and 1000 mg/kg/day dose levels and the study was reported as a subchronic investigation.

Four groups of 65 male and 65 female rats were orally gavaged with BADGE 7 days per week for 14 weeks as an aqueous suspension in 0.5% Methocel A4M methyl cellulose ethers with 0.1% Tween 80. Concentrations resulting in doses of 0, 50, 250, and 1000 mg/kg/day were used to evaluate systemic toxicity. Dose volume and concentration was calculated based on the most recent animal body weight. Homogeneity, stability, and concentration of the dose solutions were verified throughtout the study.

The first 10 rats/sex/dose were selected for urinalysis, hematology, clinical chemistry, necropsy, organ weights, and histopathology. Hematology, clinical chemistry, and urinalysis data were collected following 3 months on study.

Twice daily cageside clinical observations (mortality, morbundity, and feed & water check) monthly detailed clinical observations (replacement animals or first 15 animals/sex/dose only), weekly categorical (clinical) observations on all animals, eye exams (indirect ophthalmology pre-exposure and prior to termination), weekly body weights, weekly food consumption were measured. Hematology, clinical chemistry, urinalysis, and organ weights were evaluated at study termination.

Organs evaluated: brain, liver, kidneys, heart, adrenals, testes, epididymis, uterus, ovaries, and spleen.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Twice daily cageside clinical observations (mortality, morbundity, and feed & water check) monthly detailed clinical observations (replacement animals or first 15 animals/sex/dose only), weekly categorical (clinical) observations on all animals, eye exams (indirect ophthalmology pre-exposure and prior to termination), weekly body weights, weekly food consumption were measured. Hematology, clinical chemistry, urinalysis, and organ weights were evaluated at study termination.
Sacrifice and pathology:
The first 10 rats/sex/dose were selected for urinalysis, hematology, clinical chemistry, necropsy, organ weights, and histopathology. Hematology, clinical chemistry, and urinalysis data were collected following 3 months on study.

Organs evaluated: brain, liver, kidneys, heart, adrenals, testes, epididymis, uterus, ovaries, and spleen.
Other examinations:
no additional examinations
Statistics:
Means and standard deviations were calculated for all continuous data. Body weights, food consumption, organ weights, urine volume, urine specific gravity, clinical chemistry data, coagulation, and hematologic data were evaluated by a Bartlett's test for equality of variances. Based on the outcome of the Bartlett's test, exploratory data analysis was performed by a parametric or nonparametric ANOVA. If alpha = 0.05, the ANOVA were followed by a Dunnett's test with a Bonferroni correction for multiple comparisons to the control. Detailed Clinical Observations were statistically analyzed by a z-test of proportions comparing each treated group to the control. Statistical outliers were identified by a sequential test. Outliers may have been excluded for sound scientific reasons. Since the overall false positive rate (Type I errors) was greater than the nominal alpha levels, the interpretation of the data considered other factors such as the dose-response relationship and whether the results were consistent with other biological or pathological findings and historical control values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Analytical
Homogeneity was confirmed pre-study and before last dosing at high and low doses, and the concentration was confirmed pre-study, mid-way through the study, and prior to the last dose delivery to be 104.7-109.3% of the targeted doses at each dose level. Stability analyses demonstrated stability for 13-20 days in two tests.

Mortality
There was no significant treatment-related mortality through the 99-101 day treatment period. Six animals died or were euthanized in moribund condition before study termination at 14 weeks. Three died incidentally during dosing or blood collection, and three high-dose rats had moderate to severe acute tubular necrosis of the kidneys.

Observations
There were no treatment-related observations noted in cageside or clinical examinations, nor were there any noted during detailed clinical observations.

Ophthalmology
No treatment-related ophthalmology was noted. All observations were of variable frequency and did not occur in a dose-related manner.

Body Weights / Weight Gains
Treatment-related statistically-significant decreases in body weights and gains were noted in male and female rats given 250 and 1000 mg/kg/day (19.2% and 17.2% for males and females at 1000 mg/kg/day at day 98), and additionally in females given 50 mg/kg/day. The decrements in body weight were slowly progressive throughout the dosing period. The minimal decrement in female body weight decrease at 50 mg/kg/day was interpreted to be a non-adverse effect.

Food Consumption
Treatment-related statistically-significant decreases in food consumption were noted in male and female rats at 50, 250, and 1000 mg/kg/day.

Clinical Pathology
Minimal, but statistically-significant, decreases in prothrombin time were observed in males given 250 or 1000 mg/kg/day, and in females at 250 mg/kg/day. The alterations were interpreted to be non-adverse, and reflective of normal biologic variation. A significant dose-related decrease in red blood cell count, hematocrit, and hemoglobin were noted in males and females at 250 or 1000 mg/kg/day.

Clinical Chemistry
Statistically-significant increases in urea nitrogen (males and females at 250 and 1000 mg/kg/day), aspartate aminotransferase (males at 1000 mg/kg/day), cholesterol (males and females at all dose levels), total bilirubin (females at 1000 mg/kg/day), and calcium (females at 1000 mg/kg/day). The increased urea nitrogen was consistent with treatment-related renal toxicity. The other clinical chemistry effects were unaccompanied by histopathologic alterations, and were considered to be non-adverse.

Urinalysis
Increases in timed urine volume were noted in males and females at the high dose. Decreases in urine specific gravity were noted in males at the high dose, and females at the high and mid doses. The effects were consistent with treatment-related renal toxicity. In addition, some males and females at 250 and 1000 mg/kg/day had slight, treatment-related decreases in urine pH relative to controls.

Organ Weights
All alterations in organ weights were considered to be reflective of treatment-related decreases in body weights for animals given 250 or 1000 mg/kg/day. In males, decreases in absolute adrenal, heart, spleen weights, and increased relative brain and testes weights were identified. In females, decreases in absolute adrenal, heart, spleen, ovaries, and uterus weights, and increased relative brain weight were recorded.

Gross Pathology
The only treatment-related observation noted was increased cecum size in all males at mid and high doses, and in all females at the high dose.

Histopathology
Effects were noted in adrenal glands, cecum, ileum, kidneys, liver, testes, and uterus of animals at the mid and high dose. Adrenal glands of males at these dose levels had slightly decreased vacuolization of the cortex, relative to controls. The cecal effect was a very slight, diffuse hyperplasia of the mucosal epithelium of males at the mid and high dose, and in females at the high dose. Some males given 1000 mg/kg/day also had slight, diffuse hyperplasia of the ileum. Treatment-related kidney effects were slight, decreased hyalin droplet formation in proximal convoluted tubules of males at the mid and high dose, and slight vacuolization of of females given 250 and 1000 mg/kg/day. In addition, 2 males and 1 female died prior to study termination of moderate or severe acute necrosis of renal proximal tubules. There were 2 treatment-related liver effects: single eosiniphilic focus of altered hepatocytes in females given 1000 mg/kg/day, and altered tinctorial properties of centrilobular hepatocytes of males at the mid and high dose and females at the high dose. The testicular effect was characterized by a slight to moderate degeneration of the seminiferous tubules in all males given 1000 mg/kg/day. Uterine changes included a slight atrophy of the endometrium and myometrium of females given 1000 mg/kg/day, corresponding to a decrease in the mean uterus weight of animals in this group. There were no treatment-related histopathologic effects in any organs or tissues of males or females given 50 mg/kg/day.

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

There were no treatment-related histopathologic effects in any organs or tissues of males or females given 50 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
Based on alterations in body weights and serum cholesterol in rats given 50 mg/kg/day, a no-observed-effect (NOEL) was not determined. However, since these alterations were not associated with detrimental effects, the dose of 50 mg/kg/day was interpreted to be the no-observed-adverse-effect level (NOAEL).
Executive summary:

A two-year oral gavage toxicity/oncogenicity study of Bisphenol A Diglycidyl Ether (BADGE) was initiated in Fischer 344 rats. Groups of 65 Fischer 344 rats/dose level were dosed with BADGE suspended in a Tween** 80 and methylcellulose vehicle at dose levels of 0, 50, 250 or 1000 mg/kg/day. The study was terminated on test days 99 (males) or 101 (females) due to excessive toxicity noted in rats given 250 or 1000 mg/kg/day. Standard toxicologic parameters consistent with OECD guideline #408 were evaluated on a subset of ten rats/sex/dose level.

Progressive decreases in body weights and feed consumption, relative to controls, occurred throughout the study in males and females given 250 or 1000 mg/kg/day. By the end of the study, body weights of males and females given 1000 mg/kg/day were 19.2 and 10.9% lower than controls, respectively, and body weights of males and females given 250 mg/kg/day were 10.8 and 5.1% lower than controls, respectively. Body weights of females given 50 mg/kg/day were 3.2% lower than controls, while body weights of males given 50 mg/kg/day were comparable to controls throughout the study.

Treatment-related alterations in hematology (decreases in red blood cell count, hemoglobin concentration, and hematocrit), clinical chemistries, urinalysis, and organ weights occurred in rats given 250 or 1000 mg/kg/day. Some clinical chemistry and urinalysis alterations were indicative of renal toxicity, while organ weight alterations were reflective of lower feed consumption and body weights. The only treatment-related clinical

pathology alteration in male and female rats given 50 mg/kg/day was increased cholesterol. The only treatment-related gross pathologic observation was increased size of the cecum in males given 250 or 1000 mg/kg/day, and in females given 1000 mg/kg/day. Two males and one female given 1000 mg/kg/day that died prior to study termination had moderate to severe acute necrosis of renal proximal tubules. Treatment related histopathologic effects in surviving animals given 250 and/or 1000 mg/kg/day were noted in the adrenal glands, cecum, ileum, kidneys, liver, testes, and uterus. Males and females given 50 mg/kg/day had no treatment-related histopathologic effects.

Based on alterations in body weights and serum cholesterol in rats given 50 mg/kg/day, a no-observed-effect level (NOEL) was not determined. However, since these alterations were not associated with detrimental effects, the dose of 50 mg/kg/day was interpreted to be the no-observed-adverse-effect level (NOAEL).