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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
not reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Method and results were sufficiently described, similar to OECD-guideline 407. The main deviations from the current test guidelines include a lack of neurobehavioral investigations and a limited number of organs weighed and examined at the terminal investigations.
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 10 animals per sex, per dose, received doses of test material, by oral gavage, 5 days a week. The animals were observed daily and their behaviour and clinical signs were examined. Body weights, food and water consumption was measured weekly. During the sixth week of exposure the animals were killed by exsanguination under ether anesthesia and blood, urine and tissues were taken for investigations on standard toxicological parameters. All animals were subjected to macroscopical examination, organ weights were recorded, and tissue samples were processed for further histopathological investigations. In addition, liver enzyme induction was monitored by EROD (ethoxyresurfin-O-deethylase) activity in plasma.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
The dose-range finding study was performed with B[a]P from Janssen Chimica (Beerse, Belgium), which had a purity of 97.7 %.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
SPF Riv:TOX
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: the animals were bred at and derived from the Animal Facility at the Institute
- Age at study initiation: 4-5 weeks at acclimation initiation, 6 weeks of age at the initiation of dosing
- Housing: animals were housed in macrolon cages with a wire floor, two per cage
- Diet: ad libitum (SSP-Tox, Hope Farms BV, Woerden). From the start of treatment the applied food contained a reduced amount of soy oil to compensate for the soy oil used to administer the test material.
- Water: ad libitum (tap water - public drinkingwater, WMN, Utrecht)
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 40 - 72 %

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The length of the gavage needle used assured exposure of (at least the caudal part of) the oesophagus (a known target-site for carcinogenesis). Application was always in the morning for all groups and took about 0.5 -1 hour.
Vehicle:
soya oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: The test material was dissolved in soy oil in volumes of 0.4 and 0.5 mL per female and male rat, respectively. The test material was dissolvable in soy oil up to ≈ 30 g/L.
Fresh solutions were prepared weekly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of B[a]P under conditions of dissolution and storage (once dissolved) was verified. Due to several corrections (e.g. purity) actually achieved dose levels were a few percent below those targeted. No detailed analytical method was reported.
Duration of treatment / exposure:
5 weeks
Frequency of treatment:
5 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Groups of 10 animals (per dose, per sex)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: animals were ad random assigned to the various dose groups by standardised procedures

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included assessment of behaviour and clinical symptoms

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during the sixth week of exposure the animals were killed by exsanguination under ether anesthesia and blood, urine and tissues were taken for investigations on standard toxicological parameters
- Parameters examined included: Hb, Ht, RBC, MCV, MHC, MCHC, WBC, PLT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during the sixth week of exposure the animals were killed by exsanguination under ether anesthesia and blood, urine and tissues were taken for investigations on standard toxicological parameters
- Parameters examined included: GGT, ASAT, ALAT, LDH, and kreatinine

URINALYSIS: Yes
- Time schedule for collection of urine: during the sixth week of exposure the animals were killed by exsanguination under ether anesthesia and blood, urine and tissues were taken for investigations on standard toxicological parameters
- Parameters examined included: pH, protein, glucose, ketone, bilirubin, blood, nitrate, and urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were subjected to macroscopical examination.
- Organ weights: liver, lung, thymus, spleen, kidneys, adrenals, and ovaries

HISTOPATHOLOGY: Yes. All animals of the highest dose groups and controls were histopathologically examined for their oesophagus, stomach, duodenum, liver, kidneys, spleen, thymus, lung and mammary gland (females only). In case of abnormalities the intermediate dose-groups were examined additionally.
Other examinations:
Liver enzyme induction was monitored by EROD (ethoxyresurfin-O-deethylase) activity in plasma

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No effects were observed during this 5-week treatment period, either on behaviour or upon handling.
Mortality:
no mortality observed
Description (incidence):
None of the animals died within the 5-week treatment period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no differences in body weight development between controls and test material-treated animals within the 5-week period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
An increase in food consumption with treatment was observed for females at ≥ 5 mg/kg bw/day , whereas for males a decrease was found without a dose-response relationship. There are no explanations for the observed changes in food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
The treatment did not affect water-consumption by females, but it did reduce consumption by males (except for the highest dose), which was about 12% in excess over control value. There are no explanations for the observed changes in water consumption.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Apart from some non-statistical, small, dose-related decreases in Hb (both sexes), and RBC counts (males), treatment with the test material did not have any significant effects on investigated haematological parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test material did not have any significant effects on GGT, ASAT, and ALAT values. The observation of slight haemolysis within a substantial number of samples paralleled the variably increased LDH values. A small increase with treatment was observed on creatinine levels in males only.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Of the urinalysis parameters investigated, treatment with the test material only induced some increase in nitrite concentration in females.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Treatment with the test material did not have any significant effects on organ-weights of lung, spleen, kidneys, adrenals, ovaria, and testis (data not shown). A reduction in thymus weight (in both sexes at 15 and/or 50 mg/kg bw/day) and an increase in liver weight (in both sexes at 50 mg/kg bw/day) was observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects could be discerned after the 5-week exposure period. Occasionally, some common background changes were obeserved; single cases of petechiae at the thymus, hydronephrosis, and a dilated uterus.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
FORESTOMACH: Small hyperplastic responses were observed in the basal layer of the epithelium, without any pattern and to a variable extent. This response was characterised by an increased number of cells with an oval rather than rounded nucleus and oriented perpendiculary to the basement membrane, or cells clearly showing hyperthrophy. Also, the regular occurrence of mitotic figures in the basal as well as suprabasal layer, vacuolization within and around the nucleus, nuclear polymorphy, atypia, and eosinophilic nucleoli reflected this mitotic response. Furthermore, the border with the lamina propria sometimes was irregular and not distinct due to nests of proliferating epithelial cells and/or influx of predominantly mononucleated inflammatory cells. A dose-response relationship was observed at and above 15 mg/kg bw/day.

LIVER: No clear treatment-related changes were observed in this organ. Therefore, intermediate dose-groups were not examined.

OESOPHAGUS: Inflammatory lesions, muscle regeneration, and atrophy were observed in a number of cases. As these are unusual, and show no relation with treatment with the test material, it is assumed that they are secondary to trauma by gavage application. Local mucosa appeared normal, probably due to a higher regerative capability.

THYMUS: An increased incidence of brown pigmentation of red pulp (hemosiderin) was observed in treated animals of both sexes. Because of the very small difference (as compared to controls), and the absence of any further support for this (e.g. haematological changes) intermediate groups were not examined.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Liver enzyme induction (EROD): treatment with the test material had a very pronounced effect on liver microsomal EROD activity. The induction level at a dose of 50 mg/kg bw appears to be the maximal achievable in male rats (under the present treatment conditions); a level which is about 36 times control values. Induction was already apparent (i.e. about 5 times increase) at the lowest tested dose of 1.5 mg/kg bw. In females the highest induction level appeared not to be reached at 50 mg/kg bw. In the absence of treatment with the test material (during the weekends) EROD activity appeared to rapidly decline to almost control values, indicating the dynamics of the induction mechanism.

Effect levels

Remarks on result:
not measured/tested

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The study was conducted as a range-finding study for a subsequent 2-year carcinogenicity study. On the basis of findings from this study a top dose of 30 mg/kg bw/day was taken forward for the carcinogenicity study.
Executive summary:

A short term repeated dose toxicity study was conducted to help inform on dose selection for a subsequent carcinogenicity study. This range-finding study was conducted under GLP conditions.

During the study, groups of 10 animals per sex, per dose, received doses of test material, by oral gavage, 5 days a week. The animals were observed daily and their behaviour and clinical signs were examined. Body weights, food and water consumption was measured weekly. During the sixth week of exposure the animals were killed by exsanguination under ether anesthesia and blood, urine and tissues were taken for investigations on standard toxicological parameters. All animals were subjected to macroscopical examination, organ weights were recorded, and tissue samples were processed for further histopathological investigations. In addition, liver enzyme induction was monitored by EROD (ethoxyresurfin-O-deethylase) activity in plasma.

On the basis of findings from this study, particularly in relation to effects noted on basal cell hyperplasia in the forestomach as well as the effects upon liver- and thymus-weight, doses of 3, 10 and 30 mg/kg bw/day were proprosed for the carcinogenicity study. In order to verify the observed forestomach proliferation, morphometrical analysis of the forestomach was incorporated into a 90-day repeated dose toxicity stduy in rats using the BrdU-incorporation technique (see a robust study summary under section 7.5.1). This study essentially showed similar results.