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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK
- Age at study initiation: less than 7 weeks old at start of dosing
- Weight at study initiation: start mean male groups - 178.6, 179.3, 176.4 and 176.1g
start mean female groups - 140.3, 136.9, 139.9 and 141.9g
- Fasting period before study: no
- Housing: The animals were housed in a single, exclusive room, The animals were housed in groups of five in stainless steel mesh cages of size 55 x 34 x
20 cm, floor area 1870 cm2.
- Diet: ad libitum, Each batch of diet was analysed for specific constituents.
- Water:ad libitum, The water was periodically analysed for specific contaminants.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70
- Air changes (per hr): air-conditioned to provide a minimum of 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours dark.
Han Wistar rats of the HsdBRL: strain (about 28 days of age) were obtained from Harlan UK in order to provide 40 healthy animals of each sex. All
animals were given a clinical inspection for ill health on arrival. They were acclimatised for about 2 weeks and veterinary inspection was performed
before the start of dosing. At randomisation their body weight was within 20% of the overall mean for each sex. The animals were assigned to
treatment groups and individually identified by an ear tattoo.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
coconut oil
Details on oral exposure:
Four groups of 10 male and 10 female rats were orally gavaged with BFDGE 7 days per week for at least 13 weeks as a suspension in coconut oil.
Generally the test article was well tolerated when dosed orally by gavage at 10, 50 or 250 mg/kg/day. The analysis of formulations showed that all
formulations were accurately prepared. The test article was not detected in any control formulation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity were determined by the Sponsor. Analysis of the formulations for achieved concentration was performed in Weeks 1, 7 and 13.
Duration of treatment / exposure:
7 days per week for 13 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 50 and 250 mg/kg/day
Basis:
other: nominal in dose suspensions
No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Four groups of 10 male and 10 female rats were orally gavaged with BFDGE 7 days per week for at least 13 weeks as a suspension in coconut oil.
Concentrations resulting in doses 0, 10, 50 and 250 mg/kg/day were used to evaluate systemic toxicity. Homogeneity, stability and concentration of the dose
suspensions were verified. All animals were observed for signs of ill health or overt toxicity. Individual body weights were recorded
before treatment on the first day of dosing, at weekly intervals and before necropsy. The amount of food consumed by each cage of animals was
determined weekly. Consumption was calculated as g/animal/week. Ophthalmoscopy investigations were performed on all animals pre-treatment
and on control and high dose animals in Week 12. Blood samples (for haematology and clinical chemistry) were withdrawn from all animals in Week 13.
Samples were collected by orbital sinus puncture under a halothane anesthesia after an overnight period without food. Samples were also
taken from the abdominal aorta of the decedent at necropsy. Bone marrow smears were prepared at necropsy. All animals were subject to necropsy.
An intraperitoneal sodium pentobarbitone overdose was given prior to exsanguination. A full macroscopic examination was performed under the
general supervision of a pathologist and all lesions were recorded. Particular attention was paid to the stomach from all animals and the testes and
seminal vesicles from males. Some of the organs (adrenals, brain, heart, kidney, liver, spleen, pituitary, testes with epididymides, thyroids with
parathyroids and thymus) were dissected free from fat and other contiguous tissue and weighed before fixation. All collected tissues from all
animals were preserved in the appropriate fixatives.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: after dosing and than 30 minutes, 1 hour, 2 hours and 4 hours after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing, at weekly intervals and before necropsy

FOOD CONSUMPTION
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/kg body weight/week: 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: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all animals pre-treatment and on control and high dose animals in Week 12

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in Week 13
- Anaesthetic used for blood collection: Yes halothane anaesthesia
- Animals fasted: Yes
- How many animals: all animals
- Parameters: haemoglobin concentration, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin
concentration, reticulocytes, total and differential white cell count, platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in Week 13
- Animals fasted: Yes
- How many animals: all animals
- Parameters: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, sodium, potassium, calcium, inorganic phosphorus, chloride, total protein, albumin, globulin, albumin/globulin ratio, total cholesterol, glucose, urea, total bilirubin, creatinine

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

BONE MARROW SMEARS: full myelogram was performed for all animals
Post-dosing clinical observations were performed immediately after dosing and than 30 minutes, 1 hour, 2 hours and 4 hours after dosing. Each
animal was given a detailed physical examination at weekly intervals. All animals were examined at the beginning and the end of the working day to
ensure the animals were in good health.
Individual body weights were recorded before treatment on the first day of dosing, at weekly intervals and before necropsy. The amount of food
consumed by each cage of animals was determined weekly. Consumption was calculated as g/animal/week. Ophthalmoscopy investigations were
performed on all animals pre-treatment and on control and high dose animals in Week 12.
Blood samples (3x 0.5 mL nominal) were withdrawn from all animals in Week 13.
Haematology – the following parameters were determined on blood taken into EDTA anticoagulant: haemoglobin concentration, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocytes, total and differential white cell
count, platelet count. The following parameters were determined on blood taken into trisodium citrate anticoagulant: prothrombin time and activated partial thromboplastin time. A full myelogram was performed on bone marrow smears for all animals.
Clinical chemistry – the following parameters were determined on plasma derived from whole blood collected into lithium heparin anticoagulant:
aspartate aminotransferase, alkaline phosphatase, alanine aminotransferase, sodium, potassium, calcium, inorganic phosphorus, chloride, total
protein, albumin, globulin, albumin/globumin ratio, total cholesterol, glucose, urea, total bilirubin and creatinine.
Sacrifice and pathology:
Organ weights – the following organs were weighted: adrenals, brain, heart, kidney, liver, spleen, pituitary, testes with epididymides, thyroids with
parathyroids and thymus.
Histopathology was performed on following tissues from control and high dose animals and decedent: adrenals, aorta, brain, caecum, colon,
duodenum, heart, ileum, jejunum, kidneys, lacrimal glands, liver, lungs, mandibular lymph nodes, mesenteric lymph nodes, oesophagus, optic nerves,
ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerves, seminal vesicles, spinal cord cervical, lumbar and thoratic, spleen,
sternum with bone marrow, stomach, testes, epididymides, thyroids, parathyroids, trachea, urinary bladder, uterus, vagina and all gross lesions.
The lungs and all gross lessions were examined from all animals.
Statistics:
Body weight gains, necropsy body weight, haematology, clinical chemistry and myelogram data were analysed using one-way analysis of variance
(ANOVA), separately for each sex. Pairwise comparisons with control were made using Dunnett´s test. A regression test was performed to determine whether there was a linear relationship between increasing dose and response. Where it showed a significant result (P<0.05) and any of the pairwise comparisons were also significant, the regression result was not reported. Levene´s test for equality of variances between the groups was also performed and in all cases this provided no evidence of heterogeneous variances (P>0.01).
Food consumption was not statistically analysed because there were less than 4 cages of animals/sex/group. This would have provided too few values for meaningful statistical analysis.
Organ weights were analysed using Analysis of Covariance (ANCOVA) and Dunnett´s test, using the necropsy body weight as covariate. This analysis depends on the assumption that the relationship between the organ weights and the covariate is the same for all groups and the validity of this
assumption was tested. Levene´s test for equality of variances across the groups was also performed for all organ weights and in all cases this
provided no evidence of heterogeneous variances (P>0.01).

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:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
One high dose female was killed in extremis during Week 12 of the study. The animal was found in a twisted position, unable to coordinate its movements and had splayed hindquarters. It also had an enlarged/protruding right eye, urine staining around the vulva and staining around the mouth. All investigations performed on this animal after its death: haematology, clinical chemistry, necropsy and organ weights, revealed no differences from the results of the control animals in Week 13. There were no gross or microscopic findings that could be related to the neurological signs or to treatment, and its demise was considered incidental.

Clinical signs
Salivation and paddling immediately past-dose were observed in all groups including controls. Piloerection and raised tails immediately post-dose later became apparent in all dosed groups including controls. By Week 8, most animals that were dosed with test article had increased activity immediately post-dose. Additionally, the high dose animals developed a high stepping gait immediately post-dose from Week 6. In the absence of any microscopic neurological findings, the significance of these latter two clinical signs is unclear.

Body weights/ weight gains
All animals gained weight during the study. Weight gain in high dose males was decreased (P<0.01) compared with controls up to approximately Week 7. From Week 7 to the end of the study the reduction in weight gain in high dose males ceased but the body weights remained consistently less than
controls.

Food consumption
During the first seven weeks of the study the high dose males consumed less food than controls. Than from Week 8 the high dose males consumed
approximately the same of food as controls. The intermediate and low dose males consumed comparable amounts of food as controls throughout
the study.

Haematology
APTT clotting time increased in a dose-related manner in the male animals. There was a slight decrease in reticulocyte count (percent and absolute) in the high dose females. However, in the absence of any other changes in the high dose female red cell parameters, the importance of this reduction is unclear.

Myelograms
There were a few minor statistically significant differences in the myelogram parameters. Marginally higher percentages of early erythroblasts and
eosinophils were present in the bone marrow of treated males. Similarly, the percentages of intermediate erythroblasts and eosinophils were slightly
increased in the bone marrow of treated females. Findings were considered not to be of toxicological importance.

Clinical chemistry
Group mean aspartate aminotransferase and alanine aminotransferase levels were slightly elevated in the high dose female group. However, the
large standard deviations, which were due to animal number 80, means that these were not true group mean increases and were considered not to be of toxicological importance.

Organ weights
There was a significant increase in liver weight adjusted for body weight of high dose males (P<0.001) and high dose females (P<0.001) by
approximately 16% and 19%, respectively. However, when the relevant absolute organ weights are compared there is little or no difference in weight.
Therefore, it can be concluded that the statistical significances are as a result of the difference in terminal body weight. The adrenal weight adjusted
for body weight of the high dose females was approximately 17% (P<0.05) less than controls. The absolute adrenal weight was also decreased.

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 250 mg/kg bw/day (nominal)
Based on:
test mat.
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 was no effect on the females body weight, food consumptions and haematology. There were no findings of toxicological importance in the ophthalmoscopy or at necropsy and histopathology of either sex.

Applicant's summary and conclusion

Conclusions:
Minor effects observed at 250 mg/kg/day were judged to have no adverse health effect consequences Therefore the NOAEL for this oral 90-day rat study is 250 mg/kg/day.
Executive summary:

The objective of this O.E.C.D. 408 Testing Guideline study was to determine the toxicity of the test article, Bisphenol F Diglycidylether (BPFDGE)  following oral (gavage) administration to rats for at least 13 weeks. Minor effects observed at 250 mg/kg/day were judged to have no adverse health effect consequences. Therefore, the NOAEL for this oral 90-day rat study is 250 mg/kg/day.