Shale oils

Repeated dose toxicity: oral

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

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic 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 408. The main deviations from the current test guidelines include a lack of thyroid hormone measurements and neurobehavioral investigations, as well as a limited number of organs weighed and examined at the terminal investigations.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

In conjunction with the 2-year carcinogenicity study, groups of 10 animals per sex, per dose, received doses of test material, by oral gavage, for 5 days a week over a period of 90 days. The animals were observed daily and their behaviour and clinical signs were examined. Body weights, food and water consumption was measured at least weekly. Following the end of the exposure period the animals were sacrificed by exsanguination from the abdominal aorta. Blood, urine and tissues were taken for investigations on standard toxicological parameters. All animals were subjected to macroscopic examination, organ weights were recorded, and tissue samples were processed for further histopathological investigation. The purpose of this 90-day kill was to reassure the appropriateness of the dose levels chosen for the chronic carcinogenicity study.

GLP compliance:

yes

Limit test:

no

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 %.

Species:

rat

Strain:

Wistar

Remarks:

SPF Riv:TOX

Sex:

male/female

Details on test animals or test system 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. Animals were 6 weeks of age at study initiation (following acclimation).
- 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 (standard SSPTox food contains 4.55% soy oil on a weight basis).
- Water: ad libitum (tap water - public drinkingwater, WMN, Utrecht)
- Acclimation period: 10 days

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

Route of administration:

oral: gavage

Details on route of administration:

The length of the gavage needle used assured exposure of the distal half of the oesophagus (a known target-site for carcinogenesis).
Application was always in the morning for all groups and took about 1.5 - 2.5 hours.

Vehicle:

soya oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved in soy oil. At the start of treatment the test material was applied in 0.4 and 0.5 mL soy oil per female and male rat, respectively. Upon body weight gain and herewith amount of test material per rat these volumes were adjusted to 0.6 and 0.75 mL, respectively.
Solubility of test material in soy oil was ≈ 30 g/l.
Fresh solutions were prepared weekly.
Dosing was on the basis of extrapolated mean group weights (per dose, per sex).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test material solutions in use were regularly verified for their benzo[a]pyrene content.

Duration of treatment / exposure:

90-days

Frequency of treatment:

5 days per week
(Rats were not dosed at some national or christian celebration days)

Dose / conc.:

3 mg/kg bw/day (nominal)

Remarks:

Due to several corrections (e.g. purity) actually achieved dose levels were somewhat lower, i.e. 2.9 ± 0.3 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Due to several corrections (e.g. purity) actually achieved dose levels were somewhat lower, i.e. 9.6 ± 1.0 mg/kg bw/day

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Due to several corrections (e.g. purity) actually achieved dose levels were somewhat lower, i.e. 29 ± 3 mg/kg bw/day

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

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were examined daily for behaviour and clinical symptoms and by palpation.

BODY WEIGHT: Yes
- Time schedule for examinations: every week (week 1 - 8), every 2 weeks thereafter

FOOD CONSUMPTION: Yes
- Time schedule for examinations: twice weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of sacrifice blood was taken under ether narcosis by orbita puncture.
- Parameters examined included: WBC, RBC, Hb, Ht, MCV, MCHC, PLT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of sacrifice blood was taken under ether narcosis by orbita puncture.
- Parameters examined included: ALAT, ASAT, γ-GT, LD, creatinine

URINALYSIS: Yes
- Time schedule for collection of urine: On the day before sacrifice the animals received their last dose, and were allocated in metabolism cages for collection of urine.
- Parameters examined included: volume, creatinine and protein

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

TERMINAL PROCEDURES: Prior (1h ± 15') to euthanasia the rats received an i.p. injection of 2 mL buffered saline containing 20 mg BrdU/mL. The animals were sacrificed by exsanguination from the abdominal aorta.

GROSS PATHOLOGY: Yes
The following organs were sampled: brain; pituitary; thyroid (attached to larynx); thymus; heart; lungs (after formalin infusion); liver (two lobes); spleen; pancreas; kidneys; adrenals; ovaries / testes; uterus / prostate with seminal vesicles; sciatic nerve; quadriceps muscle; gastro-intestinal tract (stomach opened and spread on paraffin wax disks; swiss rolls from oesophagus, duodenum; jejunum; ileum; caecum; colon and rectum); lymph nodes (axillary, mesenteric and mandibular); ventral abdominal skin (including subcutaneous mammary tissue) and femur.
All tissues were fixed in 4% neutral buffered formaldehyde.

ORGAN WEIGHTS: Yes
Liver, thymus and spleen were weighted.

HISTOPATHOLOGY: Yes
For initial histological examination the following tissues were designated as protocol organs: liver, stomach, esophagus, thymus, lung, spleen and mesenteric lymph node.
Other organs were archived separately for backup purposes (possible unexpected tumour yields) and possible other future research, in view of the unique character of the material.
Histological specimens of protocol organs were processed routinely (paraffin embedding and H&E staining of 5 μm sections). From the stomach, a putative target organ and selected for morphometric counting of BrdU labelling, two samples were taken perpendicular to the limiting ridge, containing both fore- and glandular stomach. Other samples were processed according to Standard Operation Procedures of the Laboratory of Pathology.
Initially only control and high dose groups animals were examined. Organs displaying changes that could be dose-related, were further analysed by "blind reading", and if a difference was apparent, also the intermediate dose groups were studied.
Morphometrical analysis of the forestomach was carried out for the prevalence of S-phase epithelial cells displaying BrdU incorporation after immunohistochemical staining. From each section of the forestomach five representative samples (if possible), each over the length of the monitor were counted, and the total per stomach was expressed in relation to the surface of the corresponding muscularis mucosae. The latter was done to compensate for variation in stretching of the stomach wall. Morphometry was performed using the IBAS 2000 image analysis system (Kontron Bildanalyse GmbH, Eching,
Germany).

Statistics:

(Morphometrical analysis of the forestomach) the data were analysed statistically by ANOVA for treatment and by Student-t test for group wise-comparison.

Clinical signs:

no effects observed

Description (incidence and severity):

No remarkable effects were observed during this 3-months treatment period, either on behaviour or upon handling.

Mortality:

no mortality observed

Description (incidence):

There were no differences in survival between controls and test material-treated animals within the 3-month period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Only at the highest dose tested (30 mg/kg bw), the test material appeared to affect body weight gain in males only, i.e. from week 10 onwards. At the same dose level females appear to be less sensitive for this effect.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Treatment with the test material appeared to have no significant effect on food consumption.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Treatment with the test material appeared to have no significant effect on 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 RBC count and Hb (both sexes), treatment with the test material did not have any significant effects on the investigated haematological parameters.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related group-differences, nor dose-response relationships could be discerned for ALAT, ASAT, LD, or creatinine. For γ-GT a small dose-related decrease in activity was found in males. The treatment-related small increase in creatinine levels observed in the range-finding study with males could not be reproduced under the present conditions.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinary volume was increased in males at the highest B[a]P-dose tested, though in the absence of a clear dose-response relationship. Urinary creatinine levels appeared increased at this treatment level in males and females. Both male and female rats showed a dose-related increase in urinary protein levels. In the absence of further investigations, underlying mechanisms are yet unresolved.

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):

Clear target-organs of toxicity were the liver and thymus i.e. increased liver- and decreased thymus weights; changes in weight of liver and thymus were generally confined to the highest dose level, except for the liver of males, in which also 10 mg/kg bw induced a substantial (15%) weight increase. Spleen weights were only just significantly increased (P < 0.05) at the top dose in both sexes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A consistent finding in most animals was a discolouration of the mandibular lymph nodes, and occasionally other regional lymph nodes (axillary). This discolouration varied from reddish to brown / grey, although in the table this discolouration was named brown / red. This phenomenon was attributed to minute local haemorrhages caused by manipulation (gavage), which was conveyed to the regional lymph node. Other changes occurred at a low incidence and were consistent with common background pathology.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

FORESTOMACH:
The epithelium of the forestomach of the 30 mg/kg group in particular demonstrated minimal changes that could be summarised as "basal cell disturbance". The diagnosis basal cell disturbance was based on the combination of various features that occurred either focal or diffuse. These features were: increase in number of basal cells, mitotic figures and remnants of necrotic cells; occasionally early nodule development; infiltration by inflammatory cells (mainly histiocytes); capillary hyperaemia, often in combination with the previous changes. Intermediate dose groups were also studied, and the changes were scored by "blind reading". Although difficult to determine by this way and based on data from only a limited number of animals, the lowest dose without effect could be estimated as 3 mg/kg for males and 10 mg/kg for females, or 3 mg/kg for sexes combined. The nodular hyperplasia, probably an indication of early neoplasia, was found in one animal of the 30 mg/kg group in either sex.
Morphometry was carried out for mitotic cells as a discrete parameter for the changes listed above. It can be concluded that a steep increase in mean number of mitotic cells was found with increasing dose, although the vatiation was considerable. ANOVA demonstrated a significant relation with dose, and Student-t revealed statistical significance (two-tailed) for groups treated with 3 and 10 mg/kg bw, respectively, when compared to controls.

THYMUS:
Thymus atrophy was diagnosed in relation to the structure of the thymic lobes, i.e. the relative quantity, shape and distribution of cortical and medullary areas. In animals displaying thymus atrophy, the proportion of medullary areas was increased with irregular boundaries. The incidence of this slight thymus atrophy in the 30 mg/kg groups appeared increased compared to the controls. For this reason also the intermediate groups were evaluated; this finding was recorded by blind reading. The incidence in these groups were nil or insignificantly low. Therefore the lowest dose without effect can be established at 10 mg/kg.

LIVER:
All animals contained a few to many small inflammatory cell foci consisting mainly of macrophages, occasionally surrounding a necrotic cell. When this lesion was quantified (categories 0-5; 5-10 and >10 foci per 2 liver sections), no difference between control and top dose group was apparent. In one male of both control and 30 mg/kg group a necrotic area was seen in the liver.
Slight fatty vacuolation was seen in a significant proportion of both males and females, with a slightly higher incidence in high dose males (when “vacuolation” and “fatty vacuolation” are combined). The distribution was more or less zonal, and occurred mainly in the periportal (zone I) areas. In a few females (1 in control, 2 in the top dose group) the vacuolation occurred more focally and was more of the glycogen type. This change was suggestive for early clear-cell foci.

LUNG:
Minimal inflammatory changes (inflammatory cell foci, histiocytosis, increased septal cellularity) occurred at a low incidence, although the number of animals affected was higher in the 30 mg/kg females (6/10 vs 2/10). This, as well as the arterial mineralisation observed is considered as a common background lesion.

OESOPHAGUS:
Changes in the oesophagus consisted of degeneration and regeneration of muscle fibers, and focal inflammation of the muscular wall. Although the covering mucosa appeared intact, these changes are considered as a result of blunt traumatic damage due to gavage application of the test material. These lesions occurred occasionally and were not related to test material exposure.

SPLEEN:
Occasional changes such as increased haemosiderin pigment, hyperaemia, lymphoid depletion or reticulum cell hyperplasia, were observed. Although these changes were found exclusively in treated animals, the incidence was too low to be considered significant.
No changes were observed in mesenteric lymph nodes and glandular stomach.

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Conclusions:

This study identified the forestomach and thymus as target organs for toxicity with minimal effects at the higher dose levels. Consequently, there was no indication for a need to change the dose regimen for the subsequent carcinogenicity study.

Executive summary:

A sub-chronic repeated dose toxicity study was conducted to help inform on dose selection for a subsequent carcinogenicity study. This 90 -day 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, for 5 days a week over a period of 90 days. The animals were observed daily and their behaviour and clinical signs were examined. Body weights, food and water consumption was measured at least weekly. Following the end of the exposure period the animals were sacrificed by exsanguination from the abdominal aorta. Blood, urine and tissues were taken for investigations on standard toxicological parameters. All animals were subjected to macroscopic examination, organ weights were recorded, and tissue samples were processed for further histopathological investigation. The purpose of this 90-day kill was to reassure the appropriateness of the dose levels chosen for the chronic carcinogenicity study.

In particular the forestomach was considered a target organ, in which ultimately the development of tumours was expected. A possible persistent irritation with frank hyperplasia induced by the compound would reduce the value of the tumour induction for extrapolation purposes, and possibly reduce the life span of the rats. Fortunately, the hyperplasia was only minimal and could be characterised as “basal cell disturbance”, and only in two out of 20 animals from the high dose group a small hyperplastic nodule was found, one of which showing dysplasia indicative of early neoplasia. However, due to the small size (no grossly observable changes) and the random sampling, this figure is probably underestimated. Further analysis of the basal cell hyperplasia was carried out by morphometric counting of BrdU incorporation. This confirmed the minimal changes observed by histopathology, and in addition provided data that could be analysed statistically by which a significant increase was found from 10 mg/kg onwards.

Another phenomenon was the atropy of the thymus, a confirmation of the reduction of thymus weight observed at necropsy, which occurred only at the top-dose.

Despite the clear increase in liver weight by the test material, examination of histopathology at the microscopic level, as well as of clinico-chemical parameters (ALAT, γGT, LDH) did not reveal any signs of hepatocellular toxicity.

In conclusion, this study identified the forestomach and thymus as target organs for toxicity with minimal effects at the higher dose levels. Consequently, there was no indication for a need to change the dose regimen.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: 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 451. The stduy was conduced as a combined sub-chronic toxicity/carcinogenicity study. The study summary of 3-month repeated dose toxicity study is reported separately under section 7.7. The main deviations from the current test guidelines include a limited number of organs examined at the terminal investigations.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of 52 animals per sex, per dose, received doses of test material, by oral gavage, for 5 days a week over a period of 104 weeks . The animals were observed daily and their behaviour and clinical signs were examined. Body weights, food and water consumption was measured at least weekly. Following the end of the exposure period the animals were sacrificed and were subjected to macroscopic examination, additionally tissue samples were processed for further histopathological investigation.

GLP compliance:

yes

Specific details on test material used for the study:

B[a]P was from Serva (Heidelberg, Germany) and had a purity of 98.6 ± 0.4%.

Species:

rat

Strain:

Wistar

Remarks:

SPF Riv:TOX

Sex:

male/female

Details on test animals or test system 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. Animals were 6 weeks of age at study initiation (following acclimation).
- 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 (standard SSPTox food contains 4.55% soy oil on a weight basis).
- Water: ad libitum (tap water - public drinkingwater, WMN, Utrecht)
- Acclimation period: 10 days

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

Route of administration:

oral: gavage

Vehicle:

soya oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved in soy oil under heating (up to 60°C) and ultrasonic vibration.
Fresh solutions were prepared every week (week 1 - 8), every 2 weeks (week 9-18), and every 3 weeks (19-104), respectively.
The test material appeared to be stable under conditions of dissolution and storage (once dissolved). The test material solutions were prepared as follows. First, mean group weights were determined (per sex, per dose). Then, the mean group weight was estimated at half the dosing-period by simple extrapolation on the group’s growth curve (this ‘extrapolated’ mean group weight was used to prepare the solutions; this resulted in some slight initial overdosing).

DETAILS ON ROUTE OF EXPOSURE:
The length of the gavage needle used assured exposure of the distal half of the oesophagus (a known target-site for carcinogenesis).
Application was always in the morning for all groups and took about 1.5 - 2.5 hours.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test material solutions in use were regularly verified for their benzo[a]pyrene content.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

5 days per week
(Rats were not dosed at some national or christian celebration days)

Dose / conc.:

3 mg/kg bw/day

Remarks:

Due to several corrections (e.g. purity) actually achieved dose levels were somewhat lower, i.e. 2.9 ± 0.3 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

Remarks:

Due to several corrections (e.g. purity) actually achieved dose levels were somewhat lower, i.e. 9.6 ± 1.0 mg/kg bw/day

Dose / conc.:

30 mg/kg bw/day

Remarks:

Due to several corrections (e.g. purity) actually achieved dose levels were somewhat lower, i.e. 29 ± 3 mg/kg bw/day

No. of animals per sex per dose:

52 rats (per dose, per sex)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: doses were selected on the basis of findings from short-term, and sub-chronic repeated dose toxicity studies (please see relevant robust study summaries in section 7.5.1 for further information).
- Rationale for animal assignment: animals were ad random assigned to the various dose groups by standardised procedures

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were examined daily for behaviour and clinical symptoms and by palpation.

BODY WEIGHT: Yes
- Time schedule for examinations: every week (week 1 - 8), every 2 weeks (week 9-18), or every 3 weeks (week 19-104), respectively.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: twice weekly during the first 18 weeks, and once every two weeks for the remaining period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice weekly during the first 18 weeks, and once every two weeks for the remaining period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

TERMINAL PROCEDURES: All animals were subjected to complete necropsy after death, or intercurrent or terminal sacrifice. The rats were killed by exsanguination from the abdominal caval vein under ether narcosis. From week 32 onwards exsanguination was performed under inhalation anaesthesia with CO2 / O2.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
After macroscopic inspection the following organs were collected for further microscopic analysis: brain, pituitary, heart, thyroid, submandibular salivary glands, lungs (including trachea and infused with fixative), stomach (mounted on paraffin wax disks after opening along curvature maior and flushing), oesophagus, duodenum, jejunum, ileum, caecum, colon, and rectum (swiss rolls), thymus, kidneys, urinary bladder (after instillation with fixative), spleen, mesenteric, axillary and mandibular lymph nodes, liver, pancreas, adrenals, sciatic nerve, biceps femoris muscle, femur (diaphysis), vertebral column and head (after flushing the nasal cavity), skin including mammary tissue (ventral abdominal strip adjacent to midline, swiss rolled), ovaries/uterus or testis/accessory sex glands.
In addition, all gross abnormalities, in particular masses and lesions suspected of tumourous nature were sampled. After fixation (and for vertebral column, femur and skull: after subsequent decalcification in formic acid 20% as adequate) samples were trimmed, processed, and paraffin wax embedded. From the head, transverse sections (5) of the nose were taken, as well as a transverse section at the level of the auditory canal. Sections (5 μm) were cut and stained routinely with haematoxylin and eosin (H&E). Fixation, paraffin wax embedding, sectioning, staining, and all other histotechnical procedures were performed according to the Standard Operating Procedures of the Laboratory of Pathology of the Institute.

Clinical signs:

not specified

Description (incidence and severity):

Condition and behaviour, with particular emphasis on animal welfare aspects, remained reasonably well.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In animals exposed to the test material, treament of both males and females resulted in a dose-related decrease in survival. Most intercurrent mortalitites were from euthanasia due to poor condition, ususally related to tumours (mainly in liver and stomach), while significant non-neoplastic effects were not observed.
The mortality in the highest dosed groups was 100% after about 70 weeks. This mortality was mainly due to sacrifice for humane reasons when the rats became emaciated, with distended abdomen in which frequently one ore more palpable masses were present in the cranial area (liver). Despite the presence of palpable masses, the condition and behaviour, with particular emphasis on animal welfare aspects, remained reasonably well.
Liver tumours were the most predominant tumour type encountered in this study in terms of morbidity and mortality.
In the control animals, survival after 104 weeks was about 65% and 50% in males and females, respectively. In these animals by far the main cause of death was tumour development in the pituitary, which is consistent with earlier findings in our historical controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Only at the highest dose tested (30 mg/kg bw), treatment with the test material appeared to affect body weight gain in males only, i.e. from week 10 onwards. At the same dose level females appear to be less sensitive for this effect. Accordingly, it was concluded that adequate top-dose levels that were originally chosen were adequate, i.e. around or just below the maximum tolerated dose (MTD).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with the test material apparently had no significant effect on food consumption. For some short periods within the 104 weeks of treatment small (i.e. < 10%) statistically significant differences between the various treatment groups and controls were observed, though without a clear relationship to dose. An exception appears to be the more sustained reduction of food consumption though again of less than 10% of high dosed males starting at about week 36.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Treatment with the test material had no major effect on water consumption in females. Water consumption in males appeared to be dose-relatedly increased, starting from week 13 onwards. High-dose males showed an increased consumption of water (more than 50%) briefly before their withdrawal from the study. It was noted that high-dose males also showed a decreased food consumption, and a decreasing body weight during this period. There is not yet a satisfactory explanation for these observations.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See histopathological findings: neoplastic.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination showed only minor changes such as slight reactive (proliferative) changes in the mucosa of the forestomach.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment with the test material for 2 years resulted in a dose-dependent increase in tumour incidence in a variety of organs and/or tissues. Clearly the most prominent carcinogenic effects of the test material were observed in the liver and forestomach, both organs with a low spontaneous tumour incidence in this rat strain, as illustrated by the incidence found in the concurrent controls of the present study. At the two highest dose levels multiple tumours in the liver was a common finding, together with tumour development in the forestomach. Besides these two target sites, treatment with the test material also induced soft tissues sarcomas at various sites (oesophagus, skin, mammary), as well as tumours of the auditory canal, skin, and oral cavity. Tumours were also observed in the small intestine (at the top dose; especially males), and in the kidney (the two highest dose levels, males only).

IN-LIFE OBSERVATIONS:
Next to the palpable liver tumours, frequently tumours were seen on the base of the ear which eventually could result in ulceration or eruption through the auditory canal. Often these occurred bilateral. In the intermediate groups, subcutaneous tumours were often found in the neck area between pharynx and axillary region. When these tumours compromised feeding or mobility, or became ulcerated, the animal was necropsied and the tumour was qualified as fatal.

NECROPSY:
Gross changes in the highest dose group included masses in the liver (99/104), papilliferous thickening or masses in the forestomach (89/104) and nodules in the lungs suspect for metastatic spread. Also, frequently masses in the skin (back / head / neck / lips) or at the basis of the auditory canal were found. In a number of cases (predominantly in groups 3 and 4) typically masses were found in the neck / prescapular or mediastinal area. Ususally these tumours were closely associated with the esophagus and / or neck musculature. Occasionally masses were observed on other organs and tissues, such as kidney, small intestine, etc. Other gross findings occurred occasionally or in accordance with common age and strain related background pathology. Histopathological observations for some major tumours will be briefly presented below.

LIVER:
Masses in the liver were found in the intercurrently died rats, the first case observed in week 35 (male, highest dose). These were most often found in the middle and right lateral lobes, but in advanced cases multiple tumours were found in multiple lobes. Tumours were often pleomorphic: solid, cystic, necrotic areas, haemorrhagic areas. In advanced cases, adhesions were formed with surrounding organs and omentum, and intraabdominal metastatic spread and ascites could be present. Liver tumours were the most frequent cause of death (i.e. indication for euthanasia). In the lower dose group, a much lower incidence and manifestation at a later time point was observed.
Tumours of the liver were usually complex, multiple and malignant in nature. A large proportion had metastasised to the lungs (59 of 150) and some to the abdominal cavity. The growth pattern and cytology were highly variable, while strikingly metastases were cytomorphologically often well differentiated. Therefore, cytomorphology was not a powerful criterion in discriminating between benign and malignant tumours. Malignant liver tumours were often associated with cystic structures that could be either of hepatocellar or cholangiocellular origin. Since these cholangiocellular lesions usually did not occur separately, but within or associated with malignant liver tumours, they have not been monitored as a separate entity with a few exemptions where they were clearly separated.
Histologically only the most progressed lesion was scored, thereby encompassing overdiagnosing. Thus, since tumours were usually multiple, if carcinoma was scored, this implies the concomitant presence of adenomas and foci of cellular alteration. Scoring was quantified as one, few or multiple.
Foci of cellular alteration (mainly clear cell type) were scored in tumour free livers, and quantified as one, few or multiple.

FORESTOMACH:
Masses in the forestomach were seen in most animals (86%) from the high dose group, and in 65 and 14% of the lower dose groups. These appeared as isolated small protrusions with a papilliferous surface. The common site of origin was the apex of the forestomach, and in advanced cases multiple masses became confluent. In some cases (pre)perforative local peritonitis had occurred, occasionally with evidence of intra-abdominal spread. In a few cases, the forestomach tumours were considered to be fatal, as decided upon presence of peritonitis and / or metastatic spread.
Lesions in the forestomach consisted of (multi)focal hyperplasia of basal cells, followed in advanced cases by papillomas, which ultimately could have resulted in invasive squamous cell carcinomas. In a few instances metastatic spread into the abdominal cavity and organs or lungs had occurred.
Forestomach lesions could be monitored in a time-sequence fashion swing to the intercurrent sacrifices. Small and /or early lesions were characterised by focal or confluent multiple hyperplasia of basal cells. In more advanced lesions hyperplasia of the squamous component, including squamous cell papilloma, became apparent. Invasive growth usually occurred by the squamous component (squamous cell carcinoma), in some cases resulting in perforation, contact metastasis in the abdomen, and lung and liver metastases. Consequently, the diagnosis of squamous cell carcinoma implicitly signifies the presence of both papilloma and basal cell hyperplasia, and the diagnosis papilloma implicitly includes the presence of basal cell hyperplasia.
The glandular stomach or oesophageal mucosa were generally unaffected.

SOFT TISSUE:
Most sarcomas were found in the subcutis of neck, prescapular or axillary area, often involving the oesophagus, thyroid or neck muscles by invasion. For this reason most have been entered under “oesophagus” or “skin and mammary”. Occasionally this type of tumour was found at other locations (chest wall, groin, abdominal cavity (3x), stomach wall (2x), uterus / ovary (2x) and nasal cavity. These sarcomas typically revealed a variety of characteristics within the same mass, such as rhabdo-myosarcoma, fibrosarcoma, malignant fibrous histiocytoma, undifferentiated sarcoma, etc. When one feature predominated, this served as the basis of classification. In a proportion of these tumours transparant spaces were observed with material suggestive of oil. These spaces were covered by thin elongated fibroblast or macrophage like cells, but without florid inflammatory response.

AUDITORY CANAL:
The tumours were histologically composed of squamous or sebaceous cells with the range of intermediate types, although in the larger tumours the squamous component predominated. These lesions apparently arose from the pilo-sebaceous units (sebaceous glands, including the Zymbal gland) in the auditory canal. In some cases a normal Zymbal gland existed in association with a tumour. This lesion was usually not preceded by (diffuse) hyperplasia, but focal dysplasia, hyperplasia or cystic change occurred instead. Malignant tumours of the auditory canal tended to infiltrate the masticatory musculature, and occasionally the skull and brain. Metastasis to the lung was found once.

ORAL CAVITY:
Tumours at this site usually were derived from pilosebaceous tissue, normally present as clusters in the lip commissure. In many cases, including control animals, hyperplasia of the units was found, often with abscesses, dilation of hair shafts / glandular outlets. The incidence of these hyperplastic and inflammatory lesions appeared not to be treatment related. Sizeable tumours usually were of the squamous or mixed squamous / sebaceous cell type, and were often multiple / bilateral, and dose-related increased.

SKIN:
Skin tumours were basically characterised as arising from epidermis (squamous cell papilloma, carcinoma or keratoacanthoma) or appendages (basal, hair follicle or sebaceous cell types and mixtures thereof). For that reason the diagnostic differentiation was not always unequivocal and was based upon the most prominent or most malignant component. In fact it may be realistic to pool these varieties of skin / appendage. These tumours were often multiple and variable, therefore the total number of tumours may seem overestimated when related to number of tumour bearing animals.

KIDNEY:
Tumours of the kidney mainly included cortical adenoma and occurred in the two highest dose group males. These benign tumours were usually small and could only be diagnosed incidentally by careful histopathology. A clear distinction with hyperplasia is not always feasible, even with the commonly accepted criteria (IARC / SNTP), since there is a gradual continuum between hyperplasia and adenoma. Therefore the lesions might be merged. Since the observation of these lesions is particularly chance-dependent, this lesion is under this protocol (one section of either kidney) most probably underscored. Occasionally adenocarcinoma, and tumours of pelvis or ureter were found.

SMALL INTESTINE:
Tumours of the small intestine were observed mainly in the top dose males and were sometimes multiple. Malignant tumours occasionally metastasised to the liver and were often associated with obstruction and / or peritonitis.

Description (incidence and severity):

See the table.

Relevance of carcinogenic effects / potential:

The B[a]P treatment, lasting for two years, resulted in a dose-dependent increase in tumour incidence in a wide spectrum of organs and tissues, the most prominent clearly being the liver and rumen, both being organs with a low spontaneous tumour incidence in this strain. Liver tumours were also responsible for the high mortality rate at the highest dose level in both sexes.The results show that only the top dose in males may possibly be regarded as exceeding the MTD, and therefore, the carcinogenic effects observed in the other dose groups are considered to be relevant for extrapolation to humans. A number of oesophagus- associated tumours were observed in the present study, but these were sarcomas instead of carcinomas, and attributed to accidental local tissue deposition of gavaged material (injection site sarcomas). The incorporated DNA adduct test showed no evidence of the possibility that either the total number (i.e. intensity) of adducts or the presence of specific adducts are responsible for tumourigenesis.

Dose descriptor:

LOAEL

Effect level:

3 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: neoplastic

mortality

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

other: Gastrointestinal tract, hepatobiliary

Organ:

liver

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Table. Incidences of some major treatment-related neoplasms in female rats treated with B[a]P. The most advanced stage of lesions is scored.

		Females				Males
Site	Dose (mg/kg bw/kg)	0	3	10	30^#1	0	3	10	30^#1
forestomach	Examined	52	51	51	52	52	52	52	52
	squamous cell papilloma	1	3	20***	25***	0	7*	18***	17***
	squamous cell carcinoma	0	3	10**	25***	0	1	25***	35***
Liver	Examined	52	52	52	52	52	52	52	52
	hepatocellular adenoma	0	2	7*	1	0	3	15***	4
	hepatocellular carcinoma	0	0	32***	50***	0	1	23***	45***
auditory canal	Examined#2	0	1	0	20	1	0	7	33
	squamous cell papilloma	0	0	0	1	0	0	0	4
	carcinoma#3	0	0	0	13**	0	0	2	19***

#1 note that this group had a significantly shorter lifetime;

#2: these tissues were examined only when abnormalities were observed upon macroscopic examination;

#3: composite tumours of squamous and sebaceous cells apparently arisen from the pilosebaceous units / "Zymbal glands".

*p<0.01, **p<0.001 ***p<0.00001,

Conclusions:

This study demonstrated the carcinogenic potential of the test material in a variety of organs when administered by gavage to rats. Major target organs were liver, forestomach and epidermal structures, of which the liver is considered the most relevant for human risk assessment in terms of pathogenesis and sensitivity.

Executive summary:

A study was conducted to provide an estimation of the cancer risks associated with the actually experienced daily exposure to dietary PAH in the Netherlands. Benzo[a]pyrene was considered to be a representative indicator of PAH. This carcinogenicity study in rats orally exposed to benzo[a]pyrene, was considered to adequately represent the carcinogenic fraction of PAH.

During the study, groups of 52 animals per sex, per dose, received doses of test material, by oral gavage, for 5 days a week over a period of 104 weeks . The animals were observed daily and their behaviour and clinical signs were examined. Body weights, food and water consumption was measured at least weekly. Following the end of the exposure period the animals were sacrificed and were subjected to macroscopic examination, additionally tissue samples were processed for further histopathological investigation.

Findings from the study clearly showed benzo[a]pyrene to be a potent carcinogen upon chronic oral administration. Tumours were induced at multiple sites in both sexes of rats, i.e. liver, forestomach, auditory canal, oral cavity, skin, and intestines, and additionally the kidney in males, and the mammary and oesophagus in females. The most potent carcinogenic effects of benzo[a]pyrene under the present conditions were observed in the liver and forestomach, both organs with a low spontaneous incidence in this rat strain. The lowest dose level associated with a significantly inceased tumour response is 10 mg/kg bw/day for females and 3 mg/kg bw/day for males.

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

Test material information

Constituent 1

Reference substance name:

Benzo[def]chrysene

EC Number:

200-028-5

EC Name:

Benzo[def]chrysene

Cas Number:

50-32-8

Molecular formula:

C20H12

IUPAC Name:

Benzo(a)pyrene

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 or test system 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 / concentrations

Doses / concentrations 1

Dose / conc.:

1.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 4

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.