2,6-di-tert-butyl-p-cresol

Carcinogenicity

Description of key information

Weight of evidence: Experimental results from chronic and carcinogenicity oral studies using different species and published in scientific articles.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity

Type of information:

other: cohort study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Cohort study

Principles of method if other than guideline:

Cohort study

GLP compliance:

no

Duration of treatment / exposure:

For assessing BHA and BHT intake, both information on consumption of BHA and BHT-containing foods and brand names of these foods were needed. Consumption was assessed using the dietary section of the baseline questionnaire of the NLCS. The dietary section was a 150-item semiquantitative food frequency questionnaire concentrating on usual consumption of food and beverages during the year preceding the start of the study. Information on BHA and BHT content was obtained by chemical analysis of selected potential BHA and BHT-containing foods and by the use of two other information sources: a Dutch database of food additives for people with food intolerance and allergy (ALBA) and the Dutch Compendium of Foods and Diet Products (1989/1990).

In this study, the intake of BHA was 105 (range 2 -3220) µg/day and BHT was 351 (range 19 -2052) µg/day. Finding no association between BHA and BHT intake and stomach cancer risk incidence substantiates the hypothesis that low intake levels of BHA and BHT are not carcinogenic in humans.

Conclusions:

In this study, no clear evidence of an association between usual dietary intake of low levels of BHA and BHT and the risk of stomach cancer in humans was observed.

Executive summary:

In the Netherlands Cohort Study in which more than 120000 men and women aged 55 - 69 years were followed-up for 6.3 years, no significant association was found between stomach cancer risk and mean BHT (351μg/day) and butylated hydroxyanisole (BHA) intake (105μg/day) through the diet.

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the carcinogenic effects of the substance.

Principles of method if other than guideline:

BHT was fed to male and female mice for 2 years at dietary concentrations of 0, 1000, 2500 and 5000 ppm. Animals killed at the termination of the study and those dying or killed because of clinical disease were submitted to a detailed pathological examination.

GLP compliance:

no

Species:

mouse

Strain:

CF-1

Sex:

male/female

Route of administration:

oral: feed

Duration of treatment / exposure:

Two years

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

2 500 ppm (nominal)

Dose / conc.:

5 000 ppm (nominal)

Control animals:

yes, plain diet

Details on study design:

Four weeks after the start of the experiment 24 males and 24 females which had received 1000 ppm BHT were subsequently fed 2500 ppm BHT and 12 males and 12 females that had received 1000 ppm BHT were then fed 5000 ppm BHT. Eight weeks after the start of the experiment 12 males and 12 females receiving 1000 ppm BHT were fed 5000 ppm BHT.

Clinical signs:

no effects observed

Description (incidence and severity):

CLINICAL SIGNS AND MORTALITY
Throughout the two year period there were no overt signs of intoxication. The general health and behaviour of treated and control animals were similar.

Mortality:

not specified

Description (incidence):

No significant compound-related reduction in survival was recorded in any treatment group, although the 5000 ppm groups showed a somewhat lower survival over the last few months of the experiment for males and at the termination of the study for females.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

HISTOPATHOLOGY: NON-NEOPLASTIC
In animals killed or dying during the study, there was a broad spectrum of non-neoplastic diseases, but no compound-related changes were seen among the treatment groups. There was a relatively high incidence of renal disease, notably mild to marked glomerulosclerosis and a less marked incidence of pulmonary disease among both male and female animals. The female mice showed an increase in the incidence of splenic and vascular diseases over male animals. The only compound-related hepatic changes found following the feeding of BHT was an increase in centrilobular cytomegaly and karyomegaly.

In animals killed at the end of the study, there was a high incidence of both pulmonary and splenic disease in males and females and a high incidence of renal disease in males only. The animals killed at the end of the study showed an increase in centrilobular cytomegaly in both male and female treated groups and an increase in the incidence of zonal karyomegaly in the male treated groups, but an apparent decrease in the female treated mice compared with controls.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Male CF1 mice fed BHT showed a significant increase in the incidence of lung carcinomas. A significant increase was also seen in the incidence of combined lung adenomas and carcinomas in female mice fed BHT. There was a dose-related increase in benign gonad tumours in female mice fed 1000, 2500 and 5000 ppm BHT.

Relevance of carcinogenic effects / potential:

These effects are difficult to judge in that the group sizes are small and the level of significance is low. However, in order to get a significant result with small group sizes large differences are necessary, additionally the lung result carries added weight as it appears in both male and female animals.

Conclusions:

Due to the nature of the data, two types of tumour analyses have been employed, as such they represent the two extremes in the way of analysis and the "true analysis" will lie between them. Nevertheless, it is apparent that there is an increased risk of a lung tumour at the higher treatment groups (carcinoma for males and total for females). A similar result occurs for gonad tumours in females.

Executive summary:

BHT was fed to male and female mice for 2 years at dietary concentrations of 0, 1000, 2500 and 5000 ppm. Animals killed at the termination of the study and those dying or killed because of clinical disease were submitted to a detailed pathological examination. Male CF1 mice fed BHT showed a significant increase in the incidence of lung carcinomas. A significant increase was also seen in the incidence of combined lung adenomas and carcinomas in female mice fed BHT. There was a dose-related increase in benign gonad tumours in female mice fed 1000, 2500 and 5000 ppm BHT.

Reference 3

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the carcinogenic effects of the substance.

Principles of method if other than guideline:

Groups of 57 Wistar rats of each sex were maintained on diet containing 0.25 or 1% butylated hydroxytoluene (BHT) for 104 wk; control groups comprised 36 rats of each sex.

GLP compliance:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Nihon Rat Co., Saitama, Japan.
- Age at study initiation: 7 week old
- Weight at study initiation: Approximately 100-200 g
- Housing: The animals were housed five to a plastic cage using wood chips for bedding.
- Diet (e.g. ad libitum): Charles River basal diet, ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1 ºC
- Humidity (%): 55 ± 2 %

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

Daily

Dose / conc.:

0.25 other: % (nominal) in diet

Dose / conc.:

1 other: % (nominal) in diet

No. of animals per sex per dose:

57 animals per sex per dose

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

BODY WEIGHTS AND FOOD CONSUMPTION:
Body weights were recorded weekly and food consumption was measured at regular intervals.

HAEMATOLOGY:
After 104 weeks, animals were anaesthetized with ether and blood samples were collected for blood smears, red and white blood cell counts and measurements of haemoglobin and haematocrit.

CLINICAL CHEMISTRY:
Glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), alkaline phosphatase, cholinesterase, γ-glutamyl transferase (γ-GTP), total protein, albumin/globulin, the thymol turbidity test, total cholesterol, triglyceride, β-lipoprotein, blood urea nitrogen, creatinine, uric acid, total bilirubin, sodium, potassium, chloride and inorganic phosphate.

Sacrifice and pathology:

Animals were then killed and the liver, spleen and kidneys were weighed. Selected organs and tissues were fixed in buffered formalin and embedded in paraffin, and sections were stained with haematoxylin and eosin for histological examination. Rats that died or became moribund during the experiment were also autopsied and those that survived for more than 69 weeks, the time when the first tumour appeared, were included in the effective numbers.

Statistics:

The data were subjected to analyses of variance and the differences between the means were tested with Student's t-test. Comparison between groups was made where applicable by the chi-square test.

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

CLINICAL SIGNS AND MORTALITY
A significant increase in mortality among high-dose males was seen after wk 96.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males at the lower dose level, a significant reduction in bodyweight gain was noted up to wk 36, but thereafter it tended to recover. The mean body weight of males given BHT at the higher dose level was consistently less than that of controls and the difference was significant up to wk 60. The low-dose females showed reduced body-weight gain at wk 12 and 48. A significant reduction was evident among the high-dose females throughout almost all of the experiment.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

No persistent reduction in mean food intake was seen in rats of either sex given BHT, but it tended to decrease at wk 84 in animals given BHT at the 0.25% level, at wk 62 to 84 in the 1%-level males and at wk 62, 78 and 96 in the 1%-level females.

Haematological findings:

not specified

Description (incidence and severity):

At wk 104 the red-blood-cell (RBC) count of females given both levels of BHT was significantly higher than that in controls. The white-blood-cell (WBC) count was significantly lower than that of controls in rats of both sexes at the low-dose level and in female rats at the high-dose level. These changes were not dose related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The serum triglyceride level in treated male rats was significantly lower than that in controls. Levels were increased compared with those in controls in the case of γ-GTP in male rats and total cholesterol in female rats and total cholesterol in male rats at the 0.25% BHT level.

Description (incidence and severity):

Increases in both absolute and relative liver weights were observed in treated rats of both sexes. A reduction in absolute and relative spleen weights was evident in treated females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

On autopsy, some animals, especially those that died during the experiment, showed various degrees of acute or chronic pneumonia, which was the cause of death in a few animals. The kidneys of both treated and untreated groups of both sexes showed chronic nephropathy: the glomerular basement membrane was thickened, tubules were dilated with colloidal casts, and stromal fibrous tissue had proliferated with slight lymphocytic infiltration. In some females the uterine cavity was dilated with a suppurative or necrotic exudate resulting from inflammatory stenosis of the cervical canal. The testicular gland was atrophic in some male animals. However, since these changes were found in both BHT-treated and control groups, they were probably not caused by BHT.

Histopathological findings: non-neoplastic:

not specified

Description (incidence and severity):

A significant reduction in the value of triglyceride in male rats, and increases in the levels of γ-GTP in male rats and in total cholesterol in rats of both sexes treated with BHT were observed, but no significant morphological changes in the Iiver attributable to BHT were detected.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of tumours was slightly, but not significantly, higher in BHT treated groups than in controls. The incidence of hyperplastic nodules and of pancreatic carcinomas in female rats, and of pituitary adenomas or adenocarcinomas in both males and females treated with BHT were higher than those in controls. The increase in the incidence of pituitary adenomas in females given 0.25% BHT diet was significant, but the incidence of pituitary adenomas was not dose-related, and the total incidence of pituitary tumours was not significantly different from that in controls. Thus these tumours do not seem to have been induced by BHT treatment.

Relevance of carcinogenic effects / potential:

Tumours were present in some animals given BHT, but their incidence was similar to that in the controls.

Conclusions:

It is concluded that BHT at levels of 0.25% and 1% in the diet, has no carcinogenic effect in rats.

Executive summary:

Groups of 57 Wistar rats of each sex were maintained on diet containing 0.25 or 1% butylated hydroxytoluene (BHT) for 104 wk; control groups comprised 36 rats of each sex. Treated rats of both sexes showed reduced body-weight gain, relative spleen weight and white-blood-cell count while in the males there was also a reduction in serum triglyceride. BHT-treated animals of both sexes showed increased relative liver weight and total blood cholesterol but increases in red-blood-cell count could be seen only in females and only the males showed increasedγ-glutamyltransferase. No significant histological changes were observed in the liver or haematopoietic system to explain these haematological and biochemical changes. Tumours were found in the liver, pancreas, mammary glands, uterus, pituitary gland, adrenal glands and in some other organs of some of the treated rats, but their incidence was not significantly different from that in controls. It is concluded that BHT at levels of 0.25% and 1% in the diet, has no carcinogenic effect in rats.

Reference 4

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the carcinogenic effects of the substance.

Principles of method if other than guideline:

Butylated hydroxytoluene (BHT) was orally administered at concentrations of 1% and 2% of the diet to B6C3F1 mice for 104 weeks. Treated animals underwent a 16-week recovery period prior to pathological examination.

GLP compliance:

not specified

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

Daily

Post exposure period:

16 weeks

Dose / conc.:

1 other: % (nominal)

Remarks:

Doses / Concentrations:
1% (1640 and 1750 mg/kg/day for male and female, respectively)

Dose / conc.:

2 other: % (nominal)

Remarks:

2% (3480 and 4130 mg/kg/day for male and female, respectively)

No. of animals per sex per dose:

50 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on a previous subacute toxicity test to determine the maximum tolerated dose.

Observations and examinations performed and frequency:

BODY WEIGHT AND FOOD CONSUMPTION: Yes
- Time schedule for examinations: The amount of the diet consumed per cage and the body weight of each mouse were measured once every other week for the first 12 weeks and then once every 4 weeks until the end of the 104-week treatment period.

Sacrifice and pathology:

GROSS PATHOLOGY AND HISTOPATHOLOGY: Yes
At necropsy all major visceral organs and all tumours observed at this time were weighed and prepared for microscopic examination.

Statistics:

The incidences of mice with various tumours and the survival times of the mice with tumours in the treated groups were compared with those of the control group, using the chi-square test or t-test.

Mortality:

mortality observed, treatment-related

Description (incidence):

Males had a dose-related change in survival throughout the study, but no difference in survival occurred between treated and control females until week 88, when the survival of BHT-treated mice increased compared to controls. At week 104, the percent survivals for males were 74 (high-dose), 64 (low dose),and 40% (control). For females, the values w ere 89, 8 1, and 58 %, respectively. Of the mice, 89% of BHT-treated males, 64% of control males, 91% of BHT-treated females, and 82% of female controls were considered effective and incorporated into the results.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average body weights of both male and female mice given BHT showed a dose-related reduction in comparison with those of the control mice.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

The average daily intakes of BHT were 59 and 57 mg/day for males and females fed 1% BHT, respectively, and were 116 and 118 mg/day for males and females of the high-dose group.

Histopathological findings: non-neoplastic:

not specified

Description (incidence and severity):

Females treated with BHT had a significantly decreased incidence of neoplasms compared to controls and the survival time of mice with neoplasms was increased. Survival times of males did not differ between treatment and control groups. The control incidences of neoplasms were 84% in males and 85% in females, and mice of this group survived for an average of 108 to 109 weeks. For mice of the low-dose group, 86 and 75% of males and females had neoplasms, respectively. The mean survival times were 110 and 114 weeks, respectively. For males and females of the high dose group, 81 and 55% had neoplasms, respectively. The mean survival times were 114 and 118 weeks, respectively. Neoplasms of the liver, lungs, hematopoietic system, integumentary system, reproductive system (females), pancreas, esophagus, and small intestine were detected; however, only the incidence of hepatocellular adenomas in males of the high-dose group was significant.

Relevance of carcinogenic effects / potential:

Limitations of this study are the rather high doses used, although their quantity is somewhat unclear due to the assumed inhomogeneous distribution of BHT in the feed, the long duration of exposure and an increased survival in males with increasing dose. The liver tumour incidence in control animals was elevated in comparison to earlier studies. In addition, the mouse strain used is known to have a relatively high rate of spontaneous hepatocellular tumours, especially in males, and a high variance of occurrence in different studies.

Conclusions:

Females treated with BHT had a significantly decreased incidence of neoplasms compared to controls and the survival time of mice with neoplasms was increased. Neoplasms of the liver, lungs, hematopoietic system, integumentary system, reproductive system (females), pancreas, esophagus, and small intestine were detected; however, only the incidence of hepatocellular adenomas in males of the high-dose group was significant.

Executive summary:

Butylated hydroxytoluene (BHT) was orally administered at concentrations of 1% and 2% of the diet to groups of B6C3F1 mice (50 animals per sex per group) for 104 weeks. Treated animals underwent a 16-week recovery period prior to pathological examination. Females treated with BHT had a significantly decreased incidence of neoplasms compared to controls and the survival time of mice with neoplasms was increased. Neoplasms of the liver, lungs, hematopoietic system, integumentary system, reproductive system (females), pancreas, esophagus, and small intestine were detected; however, only the incidence of hepatocellular adenomas in males of the high-dose group was significant. Limitations of this study are the rather high doses used, although their quantity is somewhat unclear due to the assumed inhomogeneous distribution of BHT in the feed, the long duration of exposure and an increased survival in males with increasing dose. The liver tumour incidence in control animals was elevated in comparison to earlier studies. In addition, the mouse strain used is known to have a relatively high rate of spontaneous hepatocellular tumours, especially in males, and a high variance of occurrence in different studies.

Reference 5

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the carcinogenicity of the substance.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Dose-ranging study. Eight male and 64 female rats were employed. Following receipt, animals were allowed to acclimatize for 2 weeks. At the end of the acclimatization period, 16 female and two male rats were allocated at random to either a control group or one of three BHT treatment groups. The animals were offered diets containing sufficient BHT to ensure intakes of 500, 750 or 1000 mg/kg body weight/day. Male rats were allowed access to female rats for 3 weeks commencing 5 weeks after first offering the BHT-containing diet. Dams continued to receive the dose of BHT to which they had been randomly allocated throughout pregnancy and lactation. Dams and the majority of pups were killed at weaning (21 days after birth). In the case of the dams, the liver, kidneys, adrenals, thyroid, spleen, pancreas and lungs were taken from all animals. An autopsy examination was carried out on one pup per litter and liver, kidneys and adrenals were taken and examined microscopically.
Main study: 28 male and 200 female rats were employed. Following receipt, animals were allowed to acclimatize for 2 weeks. At the end of the acclimatization period, animals were allocated at random to a control group or one of three BHT treatment groups. Each treatment group comprised seven virgin male and 50 virgin female rats. The animals were offered diets containing sufficient BHT to ensure intakes of 25, 100 and 500 mg/ kg body weight/day. Male rats were allowed access to female rats at times commencing 5 weeks after first offering the BHT-containing diet and continuing until sufficient pregnancies were confirmed to ensure a sufficient number of pups of the F1 generation. Throughout pregnancy and lactation, dams continued
to receive the dose of BHT to which they had been allocated. Groups of five dams from each dose level were killed and examined at the estimated day 19 or 20 of gestation. Livers and other tissues were removed at this time and prepared for biochemical and histopathological examination. Foetuses were removed by caesarean section, weighed, examined for developmental abnormalities and killed by decapitation. The bodies of five foetuses per dam were fixed for histological examination. The livers of the remaining pups were removed and either fixed for ultrastructural examination or pooled and homogenized for biochemical studies. The uterus of the dam was examined for resorption sites. Groups of five dams from each dose level were
killed at weaning, together with their litters at 21 days after birth. Only the liver was removed from the dams for histological examination. Liver, kidneys, thyroid and adrenals were excised from at least four pups per dam for histological examination. The liver was removed from sufficient pups to ensure that 5 g liver was available for homogenization and subsequent biochemical analysis. At this point all F0 males were culled and the liver, spleen and kidneys were removed for histological examination. In addition, five control dams and five dams from the group receiving 500mg BHT/kg body weight/day were examined and compared with five dams receiving control diet, and five receiving 500 mg BHT/kg body weight/day that had failed to become pregnant. The dams were culled and the livers removed for light- and electron microscopy and for biochemical analysis.

GLP compliance:

not specified

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bantin and Kingman (Hull, UK).
- Housing: The animals were housed in polypropylene cages with sterilized sawdust as bedding
- Diet (e.g. ad libitum): standard rodent breeding diet (CRM, Labsure, Manea, Cambs, UK), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
The concentration of BHT in the diet was adjusted every 2 weeks to maintain a constant intake when expressed on a body weight basis.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Main experiment: F0: Male rats were allowed access to female rats at times commencing 5 wk after first offering the BHT-containing diet and continuing until sufficient pregnancies were confirmed to ensure a sufficient number of pups of the FI generation. Throughout pregnancy and lactation, dams continued to receive the dose of BHT to which they had been allocated. Groups of five dams from each dose level were killed and examined at the estimated day 19 or 20 of gestation (time point 1). Groups of five dams from each dose level were killed at weaning, together with their litters at time
point 2 (21 days after birth). At this point all F0 males were culled. Groups of 60 pups from each dose group were killed at weaning (21 days after birth), and at 4 and 22 weeks post-weaning.

Frequency of treatment:

Daily

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

F0

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

F0

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

F0

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

F1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

F1

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

F1

No. of animals per sex per dose:

Each treatment group comprised seven virgin male and 50 virgin female rats.

Control animals:

yes, plain diet

Details on study design:

Dose-ranging study: The animals were offered diets containing sufficient BHT to ensure intakes of 500, 750 or 1000 mg/kg body weight/day.

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND BODY WEIGHTS:
The rats were examined daily and weighed weekly.

Sacrifice and pathology:

Groups of five dams from each dose level were killed and examined at the estimated day 19 or 20 of gestation (time point 1). Livers and other tissues
were removed at this time and prepared for biochemical and histopathological examination. Foetuses were removed by caesarean section, weighed, examined for developmental abnormalities and killed by decapitation. The bodies of five foetuses per dam were fixed for histological examination. The livers of the remaining pups were removed and either fixed for ultrastructural examination or pooled arid homogenized for biochemical studies. The uterus of the dam was examined for resorption sites.

Groups of five dams from each dose level were killed at weaning, together with their litters at time point 2 (21 days after birth). Only the liver was removed from the dams for histological examination. Liver, kidneys, thyroid and adrenals were excised from at least four pups per dam for histological examination. The liver was removed from sufficient pups to ensure that 5 g liver was available for homogenization and subsequent biochemical analysis. At this point all F0 males were culled and the liver, spleen and kidneys were removed for histological examination. In addition, five control dams and five dams from the group receiving 500 mg BHT/kg body weight/day were examined and compared with five dams receiving control diet, and five receiving 500 mg BHT/kg body weight/day that had failed to become pregnant. The dams were culled and the livers removed for light- and electron microscopy and for biochemical analysis. Pups from dams receiving 25, 100 or 500 mg BHT/kg body weight/day were weaned onto diets containing sufficient BHT to ensure intakes of 25, 100 or 250 mg/kg body weight/day. Groups of 60 pups from each dose group were killed at weaning (21 days after birth), and at 4 wk and 22 wk post-weaning.

Statistics:

Statistical analysis of data was performed using Student's t-test.

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

As expected from the dose-ranging study, rats treated with BHT at doses of 25, 100 and 500 mg/kg body weight/day showed no significant differences in weight gain or food consumption during pregnancy and lactation, compared with untreated control rats.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

As expected from the dose-ranging study, rats treated with BHT at doses of 25, 100 and 500 mg/kg body weight/day showed no significant differences in weight gain or food consumption during pregnancy and lactation, compared with untreated control rats.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Biochemical analysis was carried out on the livers of lactating and non-lactating rats treated with BHT at a dose of 500 mg/kg body weight/day and of corresponding control groups of lactating and non-lactating females fed control diet. Glucose-6-phosphatase levels were lower in non-lactating
BHT-fed females than in controls. Total glutathione levels in BHT-fed lactating dams were approximately one-third of those found in the other three groups, this decrease being statistically significant. No such changes were observed in non-lactating females treated with BHT. Glutathione S-transferase activity was significantly raised in both groups receiving BHT but more markedly so in the lactating BHT-treated dams. Total cytochrome P-450 levels were again significantly raised in both BHT-treated groups when compared with their respective controls. Specific isoenzymes of cytochrome P-450 were assayed and it was found that 7-ethoxyresorufin O-deethylase activity was significantly reduced in lactating females receiving BHT. 7-Pentoxyresorufin O-depentylase activity was significantly raised in both test groups. The greatest induction was in the livers of lactating dams receiving BHT.

Biochemical studies on foetal livers showed no systematic differences from controls for the parameters examined (glucose-6-phosphatase,
total glutathione, glutathione S-transferase and epoxide hydrolase).

Biochemical studies showed increased activity of drug-metabolizing enzymes in pups from the BHT-treated dams. Glutathione S-transferase, 7-ethoxyresorufin O-deethylase, benzphetamine N-demethylase and epoxide hydrolase activities were significantly higher than those of control animals. There was no alteration in hepatic glutathione, but there did appear to be a dose-related fall in glucose-6-phosphatase activity at a dose of 100 mg BHT/kg body weight/day or greater.

Urinalysis findings:

not examined

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):

The livers of dams treated with BHT at 500 mg/kg body weight/ day and examined at either day 19 or 20 of gestation in the main study appeared to be enlarged, although the results did not achieve statistical significance. Non-pregnant female rats treated with 500 mg/kg body weight/day showed a significant liver enlargement. The liver/body weight ratio of pregnant and lactating rats was consistently greater than that of controls. At day 21 of lactation, the livers of lactating females on control diet showed the expected physiological hypertrophy. BHT treatment resulted in a further dose-dependent increase in the liver weight of lactating dams.
There was no significant difference in the liver/body weight ratio in foetuses.

There was a statistically significant difference in the liver/body weight ratio between weanling pups of control and BHT-treated dams. At 4 and 22 wk after weaning, the liver/body weight ratio of the top dose group was approximately 10% greater than that of controls and the body weight was significantly below that of control animals. There were no differences in the body weight and in liver/body weight ratio between test and controls in the intermediate and low dose groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At autopsy a dose-related reduction in fat on the body wall and around the kidneys and adrenals was recorded when dams were examined 21 days after the birth of the pups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of lactation, the livers of dams treated with 100 or 500 mg BHT/kg body weight/day showed a dose-dependent ceiitrilobular cell enlargement. The livers of rats receiving 25 mg BHT/kg body weight/ day were indistinguishable from those of controls. No treatment-related changes were seen in any other tissues, apart from the thyroids where there was some evidence for hyperactivity at doses of 100 mg/kg body weight/day and above. Electron microscopic examination of liver sections from dams given 500 mg BHT/kg body weight/day confirmed a marked, dose-dependent proliferation of the smooth endoplasmic reticulum, indicating that the vacuoles observed by light microscopy were formed by dilation of the endoplasmic reticulum. Hypertrophy and dilation of the bile canaliculai were also found following treatment with BHT.

Examination of the livers by light- and electron microscopy of foetuses from dams treated with BHT did not reveal any histopathological effects when compared with those foetuses from untreated control dams.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Relevance of carcinogenic effects / potential:

No tumours were found in this two-generation carcinogenicity study under test conditions that were almost identical to those in the Olsen et al. (1986) study, with the following exceptions: (i) only male F1 Wistar rats exposed to BHT; (ii) exposure up to conventional period of 22 months (104 weeks). The slightly higher rates of altered hepatic foci and nodules in the high-dose group (250 mg/kg bw/day) may be considered as markers for a possible late tumour development if the study would have been extended.

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Histological examination showed no significant differences between the liver of control and treated animals, apart from slight dilation of the sinusoids in treated animals. Other tissues examined showed no significant changes with the exception of the thyroid, where mild hyperactivity was observed at doses of 100 and 250 mg/kg body weight/day, and the adrenal, where some hypertrophy of the cells in the zona fasciculata vias observed at the same doses. Electron microscopic examination of the livers from groups treated with 250 mg/kg body weight/day showed proliferation of the smooth endoplasmic reticulum, which is compatible with the centrilobular eosinophilia seen under the light microscope and with the induction of cytochrome P-450. There was also dilation of sinusoids and some loss of glycogen. Large vacuoles of unknown origin were seen within the hepatocytes. Finally, osmiophilic material was seen within the lumen of the bile canaliculi. Biochemical studies suggested no induction of cytochrome P-450 4 wk post-weaning. The only significant increase in activity was seen 22 wk post-weaning in rats treated with 250 mg BHT/kg body weight/day. Benzphetamine N-demethylase was not induced at either 4 or 22 wk post-weaning. However, there was a significant induction of ethoxyresorufin O-deethylase 4 wk postweaning following BHT administration at all dose levels. However, induction of this enzyme was significant only at a dose of 100 mg BHT/kg body weight/day when examined 22 wk post-weaning. Glutathione S-transferase and epoxide hydrolase activities were significantly elevated in rats treated with 250 mg BHT/kg body weight/day at both time points and in rats treated with 100 mg BHT/kg body weight/day examined 4 wk after weaning only. Glucose 6-phosphatase activity was reduced in rats treated with both 100 and 250 mg BHT/kg body weight/day at both time points and total glutathione in animals examined 4 and 22 wk after weaning.

Conclusions:

No tumours were found in this two-generation carcinogenicity study.

Executive summary:

In the main experiment the F0 generation were fed 0, 25, 100 and 500 mg/kg body weight/day. Their offspring (F1 generation) were weaned on diets containing the same amount of BHT as the respective parents, apart from the group given the highest dose level (500 mg/kg body weight/day). This dose level was reduced to 250 mg/kg body weight/day at weaning in order to conform with previously published findings. The pups from the dams given the highest dose level were maintained on a dietary concentration of 250 mg/kg body weight/day for the entire study. A group of age-matched non-pregnant females was also studied and the results obtained compared with those from pregnant dams. Pups from all groups were examined at day 20 of gestation, at weaning (21 days after birth), and at 4 and 22 wk post-weaning. There were no effects on fertility and no increase in foetal abnormalities at any dose of BHT. Dams receiving BHT at a nominal dose of 500 mg/kg body weight/day showed liver enlargement accompanied by induction of pentoxyresorufin O-depentylase and glutathione S-transferase, and proliferation of the endoplasmic reticulum. Pups from these dams were of the same weight at birth as controls but lost weight during the lactation period. This deficit was not recovered by the time the experiment was terminated. Hence, in two independent studies, the only significant finding in rats treated with BHT in utero and during lactation was that the weight gain of pups during lactation was less than expected when dams received at least 500 mg BHT/kg body weight/day. No tumours were found in this two-generation carcinogenicity study under test conditions that were almost identical to those in the Olsen et al. (1986) study, with the following exceptions: (i) only male F1 Wistar rats exposed to BHT; (ii) exposure up to conventional period of 22 months (104 weeks). The slightly higher rates of altered hepatic foci and nodules in the high-dose group (250 mg/kg bw/day) may be considered as markers for a possible late tumour development if the study would have been extended.

Reference 6

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the carcinogenic effects of the substance.

Principles of method if other than guideline:

Carcinogenicity study: 50 mice per sex per dose were exposed to BHT at concentrations of 3000 and 6000 ppm during 107 -108 weeks, receiving daily doses.The pathologic evaluation consisted of gross and microscopic examination of major tissues, major organs, and all gross lesions. The tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

GLP compliance:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: NCI Frederick Cancer Research Center (Frederick, Md.)
- Age at study initiation: approximately 6 weeks old.
- Weight at study initiation: males: 18-22 g; females: 17-21 g
- Housing: Animals were housed within the test facility for 2 weeks and were then assigned four rats of the same sex to a cage (polycarbonate cages 11-1/2 x 7-1/2 x 5 inches).
- Diet (e.g. ad libitum): Presterilised Wayne Sterilizable Lab Meal, ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: Two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24 ºC
- Humidity (%): 44-55%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): Lighting was provided automatically on a 12-hour-per-day cycle.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Test diets containing BHT were prepared every 1 to 1-1/2 weeks in 6-to 12-kg batches at appropriate doses.
- Mixing appropriate amounts with (Type of food): A known weight of the chemical was first mixed with an equal weight of autoclaved Wayne® Sterilizable Lab Meal containing 4% fat (Allied Mills, Inc., Chicago, 111.), using a mortar and pestle. The Wayne® Sterilizable Lab Meal contained 4% fat but no added BHT (Drews, 1978). The mixing was continued with second and third additions of feed, and final mixing was performed with the remaining quantity of feed for a minimum of 15 minutes in a Patterson-Kelly® twin-shell blender with an intensifier bar.
- Storage temperature of food: The diets were stored at 7 C until used.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

107-108 weeks

Frequency of treatment:

7 days per week

Dose / conc.:

3 000 ppm (nominal)

Remarks:

c.a. 450 mg/kg-bw/day

Dose / conc.:

6 000 ppm (nominal)

Remarks:

c.a. 900 mg/kg bw/day

No. of animals per sex per dose:

50 animals per sex per dose

Control animals:

yes

Details on study design:

-Dose selection rationale: Based on the results obtained in a subchronic study. Groups of five rats of each sex were fed diets containing BHT at the following doses: 3100, 6200, 12500, 25000 and 50000 ppm for 7 weeks, followed by 1 week of observation, and groups of five control animals of each species and sex were administered basal diet only.

Observations and examinations performed and frequency:

All animals were observed twice daily. Observations for sick, tumor-bearing, and moribund animals were recorded daily. Clinical examination and palpation for masses were performed each month, and the animals were weighed at least once per month.

Sacrifice and pathology:

Moribund animals and animals that survived to the end of the bioassay were killed using CCL and necropsied. The pathologic evaluation consisted of gross and microscopic examination of major tissues, major organs, and all gross lesions. The tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following tissues were examined microscopically: skin, lungs and bronchi, trachea, bone marrow (femur), spleen, lymph nodes (mesenteric and submandibular), thymus, heart, salivary glands (parotid, sublingual, and submaxillary), liver, pancreas, esophagus, stomach (glandular and nonglandular), small and large intestines, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, testis, prostate, uterus, ovary, brain (cerebrum and cerebellum), and all tissue masses. Peripheral blood smears also were made for all animals, whenever possible.

Necropsies were also performed on all animals found dead, unless precluded in whole or in part by autolysis or cannibalization. Thus, the number of animals from which particular organs or tissues were examined microscopically varies and does not necessarily represent the number of animals that were placed on study in each group.

Statistics:

Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958). Animals were statistically censored as of the time that they died of other than natural causes or were found to be missing; animals dying from natural causes were not statistically censored. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone's (1975) extensions of Cox's methods for testing for a dose-related trend. One-tailed P values have been reported for all tests except the departure from linearity test, which is only reported when its two-tailed P value is less than 0.05.

The incidence of neoplastic or nonneoplastic lesions was given as the ratio of the number of animals bearing such lesions at a specific anatomic site (numerator) to the number of animals in which that site is examined (denominator). In most instances, the denominators included only those animals for which that site was examined histologically.

The purpose of the statistical analyses of tumor incidence is to determine whether animals receiving the test chemical developed a significantly higher proportion of tumors than did the control animals. The one-tailed Fisher exact test (Cox, 1970) was used to compare the tumor incidence of a control group with that of a group of dosed animals at each dose level. When results for a number of dosed groups (k) are compared simultaneously with those for a control group, a correction to ensure an overall significance level of 0.05 may be made. The Bonferroni inequality (Miller, 1966) requires that the P value for any comparison be less than or equal to 0.05/k.

The Cochran-Armitage test for linear trend in proportions, with continuity correction (Armitage, 1971), was also used.

Description (incidence):

In male mice, the result of the Tarone test for dose-related trend in mortality is significant (P = 0.005), but in the negative direction. In females, the result of the Tarone test is not significant. In male mice, 46/50 (92%) of the high-dose group, 43/50 (86%) of the low-dose group, and 12/20 (60%) of the control group lived to the end of the bioassay. In female mice, 45/50 (90%) of the high-dose group, 41/50 (82%) of the low-dose group, and 17/20 (85%) of the control group lived to the end of the bioassay.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of dosed male and female mice were lower than those of corresponding controls throughout the bioassay, and were dose related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Tissue masses occurred at comparable incidences in dosed and control groups.

Description (incidence and severity):

Focal hepatocytomegaly was characterized by well-demarcated areas of slightly enlarged hepatocytes. Typically, the cytoplasm of the hepatocytes was more eosinophilic and mildly to severely vacuolated. The edges of these foci were continuous with the surrounding hepatocytes, and there was little or no compression of the adjacent hepatic parenchyma. Multifocal hepatocytomegaly was used to describe less well-demarcated areas of hepatocytic enlargement and cellular change. The hepatocytes within these areas usually were vacuolated or had a slightly more eosinophilic staining quality than the surrounding liver parenchyma. The term "hepatocellular adenoma" was used to describe focal areas of hepatocellular proliferation which compressed the adjacent hepatic parenchyma. Within these foci, there was increased cellular pleomorphism, and mitotic figures were sometimes present.

Description (incidence and severity):

Hepatocellular carcinomas were characterized by poorly circumscribed areas of proliferating hepatocytes. The cells were basophilic and extremely variable in size, and the cytoplasm varied from being finely vacuolated to containing large, clear vacuoles or large eosinophilic-staining bodies. Nuclear atypia and mitotic figures were common. These growths compressed the adjacent liver parenchyma, but usually had areas of invasion into the adjacent liver lobules. Metastatic nodules of cells having similar morphologic characteristics were found in the lungs of three control and three low-dose male mice. Angiosarcomas were characterized by large, cavernous blood-filled spaces lined by proliferating spindle cells that invaded the adjacent liver parenchyma.
The alveolar/bronchiolar adenomas were characterized by circumscribed masses of well-differentiated cuboidal epithelial cells resting on a thin, fibrovascular stroma. These masses often compressed the surrounding pulmonary parenchyma, and on occasion protruded into the lumen of a bronchiole or elevated the pleura. The alveolar/bronchiolar carcinomas were usually large in size and less circumscribed than the adenomas; they usually invaded the surrounding lung parenchyma. The cells stained more basophilic, were piled up on one another, and showed cellular pleomorphism. In several of the mice with alveolar/bronchiolar adenocarcinomas, the pulmonary parenchyma adjacent to the tumor contained intra-alveolar mononuclear or multinucleated cells containing richly eosinophilic-staining cytoplasmic material.

Relevance of carcinogenic effects / potential:

The incidence of alveolar/bronchiolar carcinomas or adenomas in low-dose female mice is significantly higher (P = 0.009) than that in the control group, but the incidence in the high-dose group is not significant. Historical records at this laboratory indicate that female control mice had an incidence of alveolar/ bronchiolar carcinomas or adenomas of 21/440 (4.7%), compared with 1/20 (5%) in the female controls in this study, 16/46 (35%) in the low-dose group, and 7/50 (14%) in the high-dose group. The result of the Cochran-Armitage test also is not significant.

Significant results in the negative direction are observed in the incidence of tumors of the liver in male mice and in the incidence of sarcomas of multiple organs in female mice.

Table 7.7-02: Incidence of carcinomas and adenomas in mice

	Males			Females
	Control	3000 ppm	6000 ppm	Control	3000 ppm	6000 ppm
No. of animals with tissues examined	20	50	49	20	46	50
Alveolar/bronchiolar carcinoma	5 (25%)	12 (24%)	7 (14%)	1 (5%)	4 (9%)	4 (8%)
Adenoma	2 (10%)	9 (18%)	10 (20%)	0 (0%)	12 (26%)	3 (6%)

Conclusions:

Based on the histopathologic examination, under the conditions of this bioassay, the administration of BHT was associated with a high incidence of nonneoplastic hepatocellular changes in dosed male B6C3F1 mice compared with controls. Alveolar/bronchiolar carcinomas or adenomas occurred in the female mice at a significant incidence in the low-dose group (P =0.009) but not in the high-dose group, and the incidences were not significantly dose related (control 1/20, low-dose 16/46, high-dose 7/50).

Executive summary:

In a carcinogenicity study, 50 mice per sex per dose were exposed to BHT at concentrations of 3000 and 6000 ppm (ca. 450 and 900 mg/kg bw/day) during 107 -108 weeks, receiving daily doses.The pathologic evaluation consisted of gross and microscopic examination of major tissues, major organs, and all gross lesions. The tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Based on the histopathologic examination, under the conditions of this bioassay, the administration of BHT was associated with a high incidence of nonneoplastic hepatocellular changes in dosed male B6C3F1 mice compared with controls. Also, there was an increased incidence of lung tumors in the female mice. Alveolar/bronchiolar carcinomas or adenomas occurred in the female mice at a significant incidence in the low-dose group (P =0.009) but not in the high-dose group, and the incidences were

not significantly dose related (control 1/20, low-dose 16/46, high-dose 7/50). Thus, these lung tumors in the females cannot clearly be related to the administration of the BHT. No tumors occurred in either male or female rats at incidences that were significantly higher in dosed groups than in corresponding control groups. Nonneoplastic lesions that may have been related to the administration of the test chemical included focal alveolar histiocytosis at increased incidences in the dosed female rats and various lesions of the liver at increased incidences in the dosed male mice.

Reference 7

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the carcinogenic effects of the substance.

Principles of method if other than guideline:

Carcinogenicity study: 50 animals per sex per dose were exposed to BHT at concentrations of 3000 and 6000 ppm during 105 weeks, receiving daily doses. The pathologic evaluation consisted of gross and microscopic examination of major tissues, major organs, and all gross lesions. The tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

GLP compliance:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: NCI Frederick Cancer Research Center (Frederick, Md.)
- Age at study initiation: approximately 6 weeks old.
- Weight at study initiation: males: 90-105 g; females: 80-95 g
- Housing: Animals were housed within the test facility for 2 weeks and were then assigned four rats of the same sex to a cage (polycarbonate cages 19 x 10-1/2 x 8 inches).
- Diet (e.g. ad libitum): Presterilised Wayne Sterilizable Lab Meal, ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: Two weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24 ºC
- Humidity (%): 44-55%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): Lighting was provided automatically on a 12-hour-per-day cycle.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Test diets containing BHT were prepared every 1 to 1-1/2 weeks in 6-to 12-kg batches at appropriate doses.
- Mixing appropriate amounts with (Type of food): A known weight of the chemical was first mixed with an equal weight of autoclaved Wayne® Sterilizable Lab Meal containing 4% fat (Allied Mills, Inc., Chicago, 111.), using a mortar and pestle. The Wayne® Sterilizable Lab Meal contained 4% fat but no added BHT (Drews, 1978). The mixing was continued with second and third additions of feed, and final mixing was performed with the remaining quantity of feed for a minimum of 15 minutes in a Patterson-Kelly® twin-shell blender with an intensifier bar.
- Storage temperature of food: The diets were stored at 7 C until used.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

105 weeks

Frequency of treatment:

7 days per week

Dose / conc.:

3 000 ppm (nominal)

Remarks:

c.a. 225 mg/kg-bw/day

Dose / conc.:

6 000 ppm (nominal)

Remarks:

c.a. 450 mg/kg-bw/day

No. of animals per sex per dose:

50 animals per sex per dose

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on the results obtained in a subchronic study. Groups of five rats of each sex were fed diets containing BHT at the following doses: 6200, 12500, 25000 and 50000 ppm for 7 weeks, followed by 1 week of observation, and groups of five control animals of each species and sex were administered basal diet only.

Observations and examinations performed and frequency:

All animals were observed twice daily. Observations for sick, tumor-bearing, and moribund animals were recorded daily. Clinical examination and palpation for masses were performed each month, and the animals were weighed at least once per month.

Sacrifice and pathology:

Moribund animals and animals that survived to the end of the bioassay were killed using CCL and necropsied. The pathologic evaluation consisted of gross and microscopic examination of major tissues, major organs, and all gross lesions. The tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following tissues were examined microscopically: skin, lungs and bronchi, trachea, bone marrow (femur), spleen, lymph nodes (mesenteric and submandibular), thymus, heart, salivary glands (parotid, sublingual, and submaxillary), liver, pancreas, esophagus, stomach (glandular and nonglandular), small and large intestines, kidney, urinary bladder, pituitary, adrenal, thyroid, parathyroid, testis, prostate, uterus, ovary, brain (cerebrum and cerebellum), and all tissue masses. Peripheral blood smears also were made for all animals, whenever possible.

Necropsies were also performed on all animals found dead, unless precluded in whole or in part by autolysis or cannibalization. Thus, the number of animals from which particular organs or tissues were examined microscopically varies and does not necessarily represent the number of animals that were placed on study in each group.

Statistics:

Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958). Animals were statistically censored as of the time that they died of other than natural causes or were found to be missing; animals dying from natural causes were not statistically censored. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone's (1975) extensions of Cox's methods for testing for a dose-related trend. One-tailed P values have been reported for all tests except the departure from linearity test, which is only reported when its two-tailed P value is less than 0.05.

The incidence of neoplastic or nonneoplastic lesions was given as the ratio of the number of animals bearing such lesions at a specific anatomic site (numerator) to the number of animals in which that site is examined (denominator). In most instances, the denominators included only those animals for which that site was examined histologically.

The purpose of the statistical analyses of tumor incidence is to determine whether animals receiving the test chemical developed a significantly higher proportion of tumors than did the control animals. The one-tailed Fisher exact test (Cox, 1970) was used to compare the tumor incidence of a control group with that of a group of dosed animals at each dose level. When results for a number of dosed groups (k) are compared simultaneously with those for a control group, a correction to ensure an overall significance level of 0.05 may be made. The Bonferroni inequality (Miller, 1966) requires that the P value for any comparison be less than or equal to 0.05/k.

The Cochran-Armitage test for linear trend in proportions, with continuity correction (Armitage, 1971), was also used.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs occurred at comparable incidences in dosed and control groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

The result of the Tarone test for dose-related trend in mortality is not significant in either sex. In male rats, 36/50 (72%) of the high-dose group, 39/50 (78%) of the low-dose group, and 13/20 (65%) of the control group lived to the end of the bioassay. In females, 39/50 (78%) of the high-dose group, 37/50 (74%) of the low-dose group, and 13/20 (65%) of the control group lived to the end of the bioassay.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of dosed male and female rats were lower than those of corresponding controls throughout the bioassay, and this depression was dose related.

Histopathological findings: non-neoplastic:

not specified

Description (incidence and severity):

Several inflammatory, degenerative, and proliferative lesions commonly seen in aged F344 rats occurred with approximately equal frequency in dosed and control animals. Focal alveolar histiocytosis in the lung was observed in both dosed and control animals, but this lesion was most often observed in the high-dose female rats. This lesion consisted of focal aggregates of large mononuclear cells within the alveolar lumen. These cells contained abundant foamy vacuolated cytoplasm.

Histopathological findings: neoplastic:

not specified

Description (incidence and severity):

A variety of neoplasms commonly seen in aged F344 rats occurred with approximately equal frequency in dosed and control rats. In the male rats, interstitial-cell tumors of the testes and pheochromocytomas of the adrenal were the most frequently observed neoplasms. In the female rats, fibroadenomas of the mammary gland and endometrial stromal polyps of the uterus were observed frequently.

Relevance of carcinogenic effects / potential:

In each sex, the results of the Cochran-Armitage test for dose-related trend in the incidence of tumors and the results of the Fisher exact test comparing the incidence of tumors in each dosed group with that in the control group are not significant in the positive direction. However, significant results in the negative direction are observed in the incidence of adenomas of the pituitary in female rats.

Table 7.7-01: Incidence of focal alveolar histiocytosis in rats

	Males			Females
	Control	3000 ppm	6000 ppm	Control	3000 ppm	6000 ppm
No. of animals with tissues examined	20	49	49	18	48	49
Focal alveolar histiocytosis	1 (5%)	4 (8%)	7 (14%)	2 (11%)	12 (25%)	21 (43%)

Conclusions:

Based on the histopathologic examination, the administration of BHT at the doses used in this bioassay did not induce either neoplastic or nonneoplastic lesions in the F344 rat, with the possible exception of focal alveolar histiocytosis in the females.

Executive summary:

In a carcinogenicity study, 50 rats per sex per dose were exposed to BHT at concentrations of 3000 and 6000 ppm (ca. 225 and 450 mg/kg bw/day) during 105 weeks, receiving daily doses. The pathologic evaluation consisted of gross and microscopic examination of major tissues, major organs, and all gross lesions. The tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Based on the histopathologic examination, the administration of BHT at the doses used in this bioassay did not induce either neoplastic or nonneoplastic lesions in the F344 rat, with the possible exception of focal alveolar histiocytosis in the females.

Reference 8

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the carcinogenicity of the substance.

Reason / purpose for cross-reference:

other: EFSA Opinion

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Groups of 40, 40 and 60 F0 rats of each sex were fed from 7 wk of age to the end of the lactation period (females) on the semi-synthetic diet with BHT added at levels providing intakes of 25, 100 or 500 mg/body weight/day, respectively. A fourth group of 60 F0 rats of each sex was given control diet. The F0 rats were mated after 13 wk of dosing and groups of 100, 80, 80 or 100 F1 rats of each sex were formed from 40, 29, 30 and 44 litters, respectively. After mating, the male F0 rats were left out of the study; the females were omitted after weaning. Because of an adverse effect on the kidney (Meyer et al. 1978) in the female F0 rats, the concentration of BHT given to the highest dose group was lowered to 250 mg/kg body weight/day for
the F1 generation. Body weight was recorded weekly until the rats were 31 wk old and subsequently every second week. Food consumption was measured weekly. Blood samples from 20 F1 males and females in the control and highest dose group were drawn from the orbital plexus under CO2 anaesthesia after 9, 19, 43 and 108 wk. Haematocrit and haemoglobin were determined in whole blood, and red and white blood cell and differential white cell counts were made. Glucose, blood urea nitrogen, free and total cholesterol, triglycerides and phospholipids were measured in serum. All F1 rats were inspected regularly for the presence of tumours. The study was terminated by killing the surviving rats at 141-144 wk of age. Gross and microscopic pathology was performed on these animals as well as on those that were killed or died during the entire study. Specimens from the liver, kidneys, heart, lungs, brain, spleen, pituitary gland, thyroid, thymus (if any), pancreas, adrenals, testes, ovaries, seminal gland, uterus, mesenteric and axillary lymph nodes, salivary gland, gastro-intestinal tract (six levels), urinary bladder, spinal cord, peripheral nerve, skeletal muscle, bone, skin, mammary gland, eye and Harderian gland were fixed in 10% neutral buffered formalin and embedded in paraffin, and sections were stained with haematoxylin and eosin for histological examination. Other appropriate staining methods were used for selected specimens. Animals that survived beyond wk 43, the time when the first tumour appeared in the spleen of a male rat in the high-dose group, were included in the 'effective numbers'.

GLP compliance:

not specified

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mollegaards breeding Centre Ltd, LI. Skensved.
- Age at study initiation: 7 weeks
- Housing: stainless-steel wire cages (two males or females/cage)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 4 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1 ºC
- Humidity (%): 60 ± 5%
- Air changes (per hr): 6-8 times/h
- Photoperiod (hrs dark / hrs light): electric light from 21.00 to 09.00 h

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual levels of BHT in the prepared diets were a few percent less than the added amounts.

Duration of treatment / exposure:

BHT was administered in the diet to male and female Wistar rats at doses of 0, 25, 100 or 500 mg/kg bw/day (F0 generation) until mating (week 13) and, in the case of female rats, until the end of the lactation period. Groups of the F1 generation received the above doses until the age of 141 - 144 weeks, with the exception of 250 instead of 500 mg/kg bw/day because of nephrotoxic effects in F0 female rats in the highest dose group.

Frequency of treatment:

Daily

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Because of an adverse effect on the kidney (Meyer et al. 1978) in the female F0 rats, the concentration of BHT given to the highest dose group was lowered to 250 mg/kg body weight/day for the F1 generation.

No. of animals per sex per dose:

Groups of 40, 40 and 60 F0 rats of each sex at levels providing intakes of 25, 100 or 500 mg/body weight/day, respectively. A fourth group of 60 F0 rats of each sex was given control diet. The F0 rats were mated after 13 wk of dosing and groups of 100, 80, 80 or 100 F1 rats of each sex were formed from 40, 29, 30 and 44 litters, respectively.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

Body weight was recorded weekly until the rats were 31 wk old and subsequently every second week. Food consumption was measured weekly. Blood samples from 20 F1 males and females in the control and highest dose group were drawn from the orbital plexus under CO2 anaesthesia after 9, 19, 43 and 108 wk. Haematocrit and haemoglobin were determined in whole blood, and red and white blood cell and differential white cell counts were made. Glucose, blood urea nitrogen, free and total cholesterol, triglycerides and phospholipids were measured in serum. All F1 rats were inspected regularly for the presence of tumours.

Sacrifice and pathology:

The study was terminated by killing the surviving rats at 141-144 wk of age. Gross and microscopic pathology was performed on these animals as well as on those that were killed or died during the entire study. Specimens from the liver, kidneys, heart, lungs, brain, spleen, pituitary gland, thyroid, thymus (if any), pancreas, adrenals, testes, ovaries, seminal gland, uterus, mesenteric and axillary lymph nodes, salivary gland, gastro-intestinal tract (six levels), urinary bladder, spinal cord, peripheral nerve, skeletal muscle, bone, skin, mammary gland, eye and Harderian gland were fixed in 10% neutral buffered formalin and embedded in paraffin, and sections were stained with haematoxylin and eosin for histological examination. Other appropriate staining methods were used for selected specimens. Animals that survived beyond wk 43, the time when the first tumour appeared in the spleen of a male rat in the high-dose group, were included in the 'effective numbers'.

Statistics:

Student's t test was used for biochemical, haematological and other biological data for F1 rats. The Armitage-Cochran test for linear trend was used for litterwise analysis of pre-weaning mortality. Data on mortality and tumour incidence in different groups were analysed according to Peto, Pike, Day et al. (1980). A test for intra-litter correlation was performed according to Grice, Munro & Krewski (1981).

Clinical signs:

no effects observed

Description (incidence and severity):

F1: BHT adminstration had no effect on the clinical appearance or behaviour of the animals.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

F1 rats given BHT survived longer than the controls. By wk 104, 86% of males and females in the high-dose group had survived compared with 70% of the males and 69% of the females in the control group, and 44% of males and 39% of females in the high-dose group survived to termination (at wk 141-144 of age) compared with 16% of control males and 17% of control females. In both sexes significant differences (P < 0.001) in longevity were seen. The higher mortality up to 2 yr of age among control rats compared with those in treated rats mainly originated in males from inflammation of the bladder, often associated with stones, and in females from earlier occurrence of nephropathy and pituitary tumours.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F0: Male and female rats dosed with 500 mg BHT/kg/day showed a statistically significant (P < 0.001) reduction in body-weight gain, compared with the controls, from wk 6 of treatment, and this persisted throughout their life. During the lactation period the pups in the BHT-treated groups showed a dose-related depression of body-weight gain. Thus the body weight of pups at weaning was 5, 7 and 41% lower than that of the controls in the groups given 25, 100 and 500 mg BHT/kg/day, respectively.

F1: The dose-related depression of the mean body weight in the test groups compared with the controls at the end of the lactation period persisted throughout the study in both sexes. The lower body weights in F1 rats given 250 mg BHT/kg differed from the control values by up to 21% for males and 16% for females. In the 100-mg/kg group these differences were up to 11% (males) and 10% (females) and in the 25-mg/kg group up to 7% (males) and 5% (females).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

F0: No differences in food consumption were noted between BHT-treated and control rats.

F1: No reduction in average food consumption was seen in any group given BHT.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

F1: The haematological findings showed no persistent changes that could be attributed to BHT.

Clinical biochemistry findings:

not specified

Description (incidence and severity):

F1: blood levels of cholesterol and phospholipids were higher in female rats treated with the highest level of BHT than in the controls, at least in the first year. Both sexes showed lower levels of triglycerides in the treated than in control rats at wk 19, 43 and 108.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

F1: A slight reddish discoloration of the urine in males in the high dose group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

F1: BHT adminstration had no effect on the clinical appearance or behaviour of the animals.

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):

The incidences of hepatocellular adenomas and carcinomas in males and of hepatocellular adenomas in females were higher in F1 rats treated with BHT than in the control. All hepatocellular tumours were found incidentally. The first carcinoma in the treated rats was observed at wk 132 in a male in the highest dose group. The rest of the carcinomas were observed when the study was terminated. The only carcinoma in the controls was found in a male rat at 117 wk of age. The first adenoma was observed in a male in the high-dose group after 115 wk, but most adenomas in both sexes were found at termination (wk 141-144).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Among the non-neoplastic lesions in the liver, a dose-related increase in the incidence of bile-duct proliferation and cysts was found in males and of focal cellular enlargement in females. Nephropathy and fibrosis of the heart were less frequent in BHT-treated rats than in the controls. The other non-neoplastic lesions occurred incidentally and showed no relationship to BHT treatment.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The lesions identified by the terms nodular hyperplasia and hepatocellular adenoma used in this study are identical to those described for hyperplastic foci and areas and for hepatic cell adenoma, respectively. Basophilic adenomas were seen occasionally, but eosinophilic adenomas predominated. The hepatocellular carcinomas consisted of basophilic hepatocytes forming a trabecular pattern. In some carcinomas, projection of irregular cords without endothelial lining was seen in dilated sinusoids. However, no metastases of any carcinomas were detected grossly or microscopically.

Other effects:

no effects observed

Relevance of carcinogenic effects / potential:

Dose-related increases in hepatocellular carcinomas and hepatocellular adenomas in male Wistar rats exposed in utero, during lactation and thereafter up to 144 weeks were reported. Increased tumour rates in female rats were only statistically significant for hepatocellular adenomas when tested for trend. Most tumours were detecte in the animals examined at terminal sacrifice at 141-144 weeks, an exposure time that exceeds the duration of conventional studies.

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: neoplastic

Remarks on result:

other: See cross reference to EFSA opinion.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

BHT induced benign and malignant hepatocellular neoplasms in Wistar rats of both sexes under the conditions of this study. A dose-response relation was noted and was most pronounced in the males. In the high-dose group, the overall numbers of F1 rats with a malignant tumour (males and females) or with multiple tumours (females) were slightly higher, but not to a statistically significant degree, than in the controls. Most of the neoplasia was found in animals at least 2.5 year old.

Executive summary:

Groups of 60, 40, 40 and 60 F0 Wistar rats of each sex were fed a semi-synthetic diet containing butylated hydroxytoluene (BHT) in concentrations to provide intakes of 0, 25, 100 or 500 mg/kg body weight/day, respectively. The F0 rats were mated and groups of 100, 80, 80 or 100 F1 rats of each sex were formed from 40, 29, 30 and 44 litters, respectively. After weaning, the highest dose (500 mg BHT/kg/day) was lowered to 250 mg/kg/day for the F1 rats. The numbers of litters of ten or more pups at birth decreased with increasing BHT dose. At weaning, treated F1 rats had lower body weights than the controls, the extent of the reduction being dose related; the effect, which persisted throughout the study, was most pronounced in the males. The survival of BHT-treated F1 rats of both sexes was significantly better than that of the controls. No significant changes attributable to BHT treatment were found in the haematological parameters. F1 females on the highest dose showed an increase in serum cholesterol and phospholipids, and serum triglycerides were reduced in this group in both sexes. Dose-related increases in the numbers of hepatocellular adenomas and carcinomas were statistically significant in male F1 rats when all groups together were tested for heterogeneity or analysis for trend. The increase in hepatocellular adenomas and carcinomas in treated female F1 rats was only statistically significant for adenomas. All hepatocellular tumours were detected when the F1 rats were more than 2 year old. Tumours were found in many other organs of some of the treated rats, but their incidence was not significantly different from that in controls.

Reference 9

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the carcinogenic effects of the substance.

Principles of method if other than guideline:

Groups of approximately 50 male and 50 female B6C3F1 mice were given butylated hydroxytoluene (BHT) at concentrations of 200, 1000 and 5000 ppm in their diet for 96 weeks followed by a basal diet for 8 weeks and were then killed. Similar groups of male and female controls were given basal diet throughout the 104 weeks.

GLP compliance:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of BHT in the diet was measured by gas-liquid chromatography on eight occasions during the feeding period.

Duration of treatment / exposure:

96 weeks

Frequency of treatment:

Daily

Post exposure period:

Basal diet for 8 weeks

Dose / conc.:

200 ppm (nominal)

Dose / conc.:

1 000 ppm (nominal)

Dose / conc.:

5 000 ppm (nominal)

No. of animals per sex per dose:

Groups of 51 or 52 mice of each sex per dose

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

BODY WEIGHT AND FOOD CONSUMPTION: Yes
- Time schedule for examinations: The animals were weighed weekly and their food consumption was measured at less frequent but regular intervals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Terminal blood samples.
- How many animals: 31 mice of each sex in each group
- Parameters: red and white cell counts, platelet counts and determination of haemoglobin, haematocrit, glutamic-oxal-acetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), alkakine phosphatase, total bilirubin, total cholesterol, total protein, the albumin/globulin ration, blood urea nitrogen and glucose.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Terminal blood samples.
- How many animals: 31 mice of each sex in each group
- Parameters: glutamic-oxal-acetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), alkakine phosphatase, total bilirubin, total cholesterol, total protein, the albumin/globulin ration, blood urea nitrogen and glucose.


URINALYSIS: Yes
- Time schedule for collection of urine: just before the animals were killed.
- How many animals: 31 mice of each sex in each group
- Parameters: specific gravity, pH, presence or absence of protein, glucose, ketones, bilirubin, occult blood and urobilinogen.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes (lungs, liver, kidneys, brains, hearts, spleens, salivary glands, testes, ovaries and pituitary glands).

HISTOPATHOLOGY: Yes (all resected organs were fixed in 10% buffered formalin and processed for histological examination).

Statistics:

The data were subjected to analyses of variance and the differences between the means were tested by Student´s t test. The incidences of tumours in different groups were compared by the chi-square test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

Most of the deaths occurring later in all groups were due to malignant lymphomas. The number of deaths among mice of both sexes treated with BHT were similar to those in the control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of females given 1000 and 5000 ppm BHT were lower than those of the controls during the treatment period and the differences were statistically significant from week 12 to week 104 for the 5000 ppm group and from week 48 to week 84 for the 1000 ppm group. Males given 5000 ppm showed a statistically significant reduction in weight gain throughout almost all the experiment, but their body weights increased to the level of the controls by week 104.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no consistent deviations from normal in the rates of food consumption by males or females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related changes were observed.

Relevance of carcinogenic effects / potential:

No significantly higher incidence of any tumour was observed in BHT-treated groups than in controls.

Conclusions:

It was concluded that BHT at a level of 200, 1000 or 5000 ppm in the food had no carcinogenic effect in B6C3F1 mice of either sex.

Executive summary:

Groups of approximately 50 male and 50 female B6C3F1 mice were given butylated hydroxytoluene (BHT) at concentrations of 200, 1000 and 5000 ppm in their diet for 96 weeks followed by a basal diet for 8 weeks and were then killed. Similar groups of male and female controls were given basal diet throughout the 104 weeks. Females fed the two highest doses and males given the high dose had decreased body weight gain, although feed consumption by both sexes did not differ from controls. BHT had no adverse effect on survival rates, did not cause changes in haematologic parameters and had no effect on features of serum and urine. Neoplasms were

observed in the lungs, liver, lymph nodes, and spleen in both test and control mice, but were considered unrelated to BHT treatment. It was concluded that BHT at a level of 200, 1000 or 5000 ppm in the food had no carcinogenic effect in B6C3F1 mice of either sex.

Reference 10

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Although the study was not conducted according to the recommended guidelines, it provides scientific valid information to assess the carcinogenicity of the substance.

Principles of method if other than guideline:

Experiment 1: One-hundred and forty one rats were divided into 6 groups. A group of 36 rats was given the basal diet with no addition (group 1-1), while groups of 21 rats were fed diets containing 100 ppm (group 1-2), 300 ppm (group 1-3), 1000 ppm (group 1-4), 3000 ppm (group 1-5)
or 6000 ppm BHT (group 1-6) for 76 weeks. Four randomly selected rats from each group were killed at 12, 36, 48 and 76 weeks to measure the number of altered hepatocellular foci. The remaining animals were killed at 76 weeks.
Experiment 2: Eighty-one rats were divided equally into 3 groups. The rats in each group were administered a 1 : 1 solution of dimethylsulphoxide and physiological saline by a single gavage to provide vehicle controls for carcinogen administration to other groups not reported here. One week later, 27 rats were given the basal diet with no addition (group 2-1) and 27 were fed 12,000 ppm BHT (group 2-2) for 110 weeks. All animals were killed at 110 weeks. Complete autopsies were performed on all animals. At autopsy, livers were weighed and slices from each lobe were taken and fixed in 10% neutral buffered formalin. Sections were stained with haematoxylin and eosin and tested for iron (experiment 1) to determine the presence of iron storage-deficient lesions. Tumours and lesions in other organs were submitted for histology.

GLP compliance:

not specified

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY, USA
- Age at study initiation: approximately 6 weeks (Experiment 1) and 11 weeks of age (Experiment 2).
- Weight at study initiation: 150 g (Experiment 1) and 200 g (Experiment 2).
- Housing: Animals were kept in polycarbonate cages (3 rats/cage) with hardwood chips for bedding.
- Diet (e.g. ad libitum): NIH-07 diet (Zeigler Brothers, Inc., Gardners, PA, USA) ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2ºC
- Humidity (%): 50 ± 5%
- Air changes (per hr): 12/h
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
Mixing various concentrations of BHT into the powdered NIH-07 diet was carried out in a V-blender after premixing the chemicals into a small amount of diet in a feed mixer. The diets were prepared every 4 weeks and stored at 4ºC until use.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Experiment 1: 76 weeks
Experiment 2: 110 weeks

Frequency of treatment:

Daily

Dose / conc.:

100 ppm (nominal)

Remarks:

Experiment 1
Equivalent to about 7.5 mg/kg bw/day.

Dose / conc.:

300 ppm (nominal)

Remarks:

Experiment 1
Equivalent to about 23 mg/kg bw/day.

Dose / conc.:

1 000 ppm (nominal)

Remarks:

Experiment 1
Equivalent to about 75 mg/kg bw/day.

Dose / conc.:

3 000 ppm (nominal)

Remarks:

Experiment 1
Equivalent to about 225 mg/kg-bw/day.

Dose / conc.:

6 000 ppm (nominal)

Remarks:

Experiment 1
Equivalent to about 450 mg/kg bw/day.

Dose / conc.:

12 000 ppm (nominal)

Remarks:

Experiment 2: 12000 ppm BHT (ca. 900 mg/kg bw/day)

No. of animals per sex per dose:

Experiment 1: A group of 36 male rats was given the basal diet, while groups of 21 male rats were fed diets containing 100, 300, 1000, 3000 or 6000 ppm.
Experiment 2: 27 male rats were given the basal diet and 27 male rats were fed 12,000 ppm.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

Body weights

Sacrifice and pathology:

Rats were killed by carbon dioxide asphyxiation. Complete autopsies were performed on all animals. At autopsy, livers were weighed and slices
from each lobe were taken and fixed in 10% neutral buffered formalin. Sections were stained with haematoxylin and eosin and tested for iron (experiment 1 ) to determine the presence of iron storage-deficient lesions. Tumours and lesions in other organs were submitted for histology.

Other examinations:

To demonstrate a deficiency in iron storage in cells of altered hepatocellular foci, rats were iron-loaded with sc injections of 12.5 mg elemental iron/100 g body weight (Nonemic brand, iron-dextran 100 mg elemental iron/ml) in the inguinal regions, alternating sides 3 times/week for 2 weeks prior to killing.

Statistics:

Differences between groups in body and liver weights and the various liver neoplasms were analysed using Student's t-test. The incidences of neoplasms in the various groups were compared using Fisher's exact probability test.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In experiment 1, all scheduled rats survived for up to 76 wk. In experiment 2, the survival of rats in all groups gradually decreased after 84 wk, but there was no significant difference between the BHT exposed groups and the control group at 110 wk.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Feeding of BHT at concentrations of 3000 and 6000 ppm for 76 weeks and at 12,000 ppm for 110 weeks significantly reduced the body-weight gainscompared with those in the controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

BHT at 6000 ppm produced a significant increase in the absolute and relative liver weights, whereas 12,000 ppm BHT significantly decreased the absolute liver weight without affecting the relative liver weight; this was due to reduced body weight.

Gross pathological findings:

not specified

Description (incidence and severity):

Feeding of BHT at doses of 100-6000 ppm did not induce any altered foci by 36 weeks. The number of iron storage-deficient altered foci at 48 and 76 weeks was slightly, but not significantly, higher in BHT-treated groups than that in untreated controls, but the incidence and area of altered foci in the group fed 12,000 ppm BHT for 110 weeks were slightly decreased. The incidence of hepatic cell neoplasms in the group fed 6000 ppm BHT for 76 weeks was slightly increased, and that in the group given 12,000 ppm BHT for 110 weeks was significantly lower, but neither was significantly different compared with the control group.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Epithelial hyperplasia of the non-glandular squamous portion of the stomach (the forestomach) was graded as mild when basal cells occupied up to one-third of the thickness of the epithelium, moderate when basal cells occupied one-half of the thickness of the epithelium, or severe when nearly the entire epithelium was composed of basal cells. Mild hyperplasia was observed in all control and treated groups.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Neoplasms of the testes were the most frequent, followed by those in the adrenal gland, haematopoietic organs, pancreas, subcutis, thyroid gland and preputial gland. Other than gastric tumours, the incidences of other tumours were not increased in the BHT exposed groups. Histologically, all neoplasms other than those in the stomach, were similar to the cryptogenic neoplasms found in this strain of rats.

Relevance of carcinogenic effects / potential:

No hepatocellular carcinomas were detected in the male Fischer 344 rats exposed to up to 900 mg/kg bw/day and there was no BHT-related trend in increase in hepatocellular adenomas, which were detected in all dose groups including controls.

Conclusions:

In the experiments reported here, BHT exerted no carcinogenic effect in rats. In the present study, there was not an enhancement of altered hepatocellular foci, which is significant because hepatocellular neoplasm enhancing agents and carcinogens often display these effects.

Executive summary:

In this long-term feeding study, which was not designed as typical chronic study, but focused on the possible development of hepatocellular foci, male Fischer 344 rats were fed diets containing 100, 300, 1000, 3000 and 6000 ppm BHT, which was equivalent to about 7.5, 23, 75, 225 and 450 mg/kg bw/day. After random selection, part of the animals was sacrificed at 12, 36 and 48 weeks and examined, while the remaining animals were exposed for 76 weeks. In the two highest dose groups, body weight gain was reduced. Only rats fed 6000 ppm BHT revealed a significantly increased liver weight. After the different treatment periods the number of phenotypically altered hepatic foci (AHF) was not significantly different compared with respective controls. Similar findings were obtained in a second study (Williams et al. 1990a), in which rats received diets containing 0 and 12000 ppm BHT (ca. 900 mg/kg bw/day) for 110 weeks, though the incidence of AHF was slightly decreased. With regard to the examined parameters, i.e. body and liver weight and histopathology, the NOAEL derived from the first study was 1000 ppm BHT (ca. 75 mg/kg bw/day). No hepatocellular carcinomas were detected in the male Fischer 344 rats exposed to up to 900 mg/kg bw/day and there was no BHT-related trend in increase in hepatocellular adenomas, which were detected in all dose groups including controls.

Reference 11

Endpoint:

carcinogenicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Review of carcinogenicity studies

Principles of method if other than guideline:

Review: no data provided on the test method.

GLP compliance:

not specified

A number of chronic carcinogenicity bioassays were conducted in mice and rats by oral administration in the diet. In one mouse study, there was no difference in tumour incidence among exposed and control groups. In another mouse study, 7500 ppm BHT increase the number of lung tumours (Clapp et al., 1974). When larger number of animals were used by the same investigators, this finding was not confirmed (Clapp et al., 1978). In one of the two other mouse studies, alveolar/bronchiolar neoplasms were present in the mid (3000 ppm) but not in the high (6000 ppm) dose BHT group (NCI, 1979). In the other, BHT at up to 5000 ppm did not induce any tumours (Shirai et al., 1982). In one study in Wistar rat, no increase in tumour incidence was seen with BHT at 10000 ppm (Deichmann et al., 1955). In the same rat strain, an increased incidence of pituitary adenoma in females was present at the lower (2500 ppm) but not at the higher (10000 ppm) dose level (Hirose et al., 1981) but the incidence of this and all other tumours was not significantly different from that in controls. The pituitary tumour finding also was not confirmed in a subsequent study (Olsen et al., 1986) in Wistar rats. In an unique study by Olsen et al., (1986), BHT was administered to Wistar rats prenatally, during nursing and for 144 weeks. F0 generation rats of each sex were fed BHT at 0, 375, 1500 or 7500 ppm in the diet for 13 weeks prior to mating. After mating, the F0 males and after weaning the F0 females were removed from the study. After weaning F1 rats were continued on the same doses, except that the top dose was lowered to 3750 ppm because of concern of nephrotoxicity seen in F0 females at 7500 ppm. BHT at 3750 ppm, but not lower doses, increased the incidences of benign hepatocellular adenoma in both sexes and of hepatocellular carcinoma only in males. Increases in liver neoplasia were found only at exposure that exceeded the maximum tolerable dose and only after a duration beyond that of conventional studies. In this study, there was a 41% reduction in the body weight of F1 pups in high dose at weaning and a 21% reduction in males and 16% reduction in females in body weight in high dose rats. With such drastic growth rate reduction there is likely to be under-utilization of protein and triglycerides in the liver with gluconeogenesis and hypermetabolism leading to glutathione depletion, cell death and hepatocellular compensatory hyperplasis. Such effects may in part explain the hepatocellular neoplasia. Additionally, the very high mortality in the control animals compromised the statistical comparison to BHT exposure groups. Liver neoplasia was not observed in any other study in rats, including in the sameWistar strain receiving 10000 ppm (Hirose et al., 1981) or F344 rats in which BHT exposure at 3000 and 6000 ppm was not associated with any tumours (NCI, 1979).

Overall, these data do not provide convincing evidence that BHT has carcinogenic activity in either mice or rats.

Conclusions:

Overall, the available data do not provide convincing evidence that BHT has carcinogenic activity in either mice or rats.

Executive summary:

A number of chronic carcinogenicity bioassays were conducted in mice and rats by oral administration in the diet. In one mouse study, there was no difference in tumour incidence among exposed and control groups. In another mouse study, 7500 ppm BHT increase the number of lung tumours (Clapp et al., 1974). When larger number of animals were used by the same investigators, this finding was not confirmed (Clapp et al., 1978). In one of the two other mouse studies, alveolar/bronchiolar neoplasms were present in the mid (3000 ppm) but not in the high (6000 ppm) dose BHT group (NCI, 1979). In the other, BHT at up to 5000 ppm did not induce any tumours (Shirai et al., 1982). In one study in Wistar rat, no increase in tumour incidence was seen with BHT at 10000 ppm (Deichmann et al., 1955). In the same rat strain, an increased incidence of pituitary adenoma in females was present at the lower (2500 ppm) but not at the higher (10000 ppm) dose level (Hirose et al., 1981) but the incidence of this and all other tumours was not significantly different from that in controls. The pituitary tumour finding also was not confirmed in a subsequent study (Olsen et al., 1986) in Wistar rats. In an unique study by Olsen et al., (1986), BHT was administered to Wistar rats prenatally, during nursing and for 144 weeks. F0 generation rats of each sex were fed BHT at 0, 375, 1500 or 7500 ppm in the diet for 13 weeks prior to mating. After mating, the F0 males and after weaning the F0 females were removed from the study. After weaning F1 rats were continued on the same doses, except that the top dose was lowered to 3750 ppm because of concern of nephrotoxicity seen in F0 females at 7500 ppm. BHT at 3750 ppm, but not lower doses, increased the incidences of benign hepatocellular adenoma in both sexes and of hepatocellular carcinoma only in males. Increases in liver neoplasia were found only at exposure that exceeded the maximum tolerable dose and only after a duration beyond that of conventional studies. In this study, there was a 41% reduction in the body weight of F1 pups in high dose at weaning and a 21% reduction in males and 16% reduction in females in body weight in high dose rats. With such drastic growth rate reduction there is likely to be under-utilization of protein and triglycerides in the liver with gluconeogenesis and hypermetabolism leading to glutathione depletion, cell death and hepatocellular compensatory hyperplasis. Such effects may in part explain the hepatocellular neoplasia. Additionally, the very high mortality in the control animals compromised the statistical comparison to BHT exposure groups. Liver neoplasia was not observed in any other study in rats, including in the sameWistar strain receiving 10000 ppm (Hirose et al., 1981) or F344 rats in which BHT exposure at 3000 and 6000 ppm was not associated with any tumours (NCI, 1979). Overall, these data do not provide convincing evidence that BHT has carcinogenic activity in either mice or rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Mode of Action Analysis / Human Relevance Framework

In evaluating the available studies, BHT was not clearly carcinogenic. An exception is the study conducted by Olsen et al. (1986). It has been discussed (Bomhard et al. 1992; WHO 1996; Williams et al. 1999) whether the specific test conditions of this study and not BHT itself resulted in the increased tumour rates. This has been mainly based on the results of the other two-generation study (McFarlane et al. 1997). Taken together, the following two factors are considered as causative non-genotoxic factors:

1.- Liver enlargement accompanied by induction of mixed function oxidase enzyme: there is experience from other studies, e.g. with phenobarbitone, that lifetime duration of such an adaptive response can result in the development of tumours as a "late" phenomenon.

2.-Malnutrition: In both two-generation studies, body weight gain was significantly reduced in the progeny of the high-dose parental generation throughout the entire exposure periods. It has been discussed, but not examined, that a deficiency of the phospholipid phosphatidyl choline resulted from malnourishment during the lactation period. Since choline was not exogeneously supplied with the feed, a long-term choline deficiency finally may have caused an increase in liver cancer as known from the literature.

It is probable that both factors, alone or in combination, were responsible for the formation of tumours in the study conducted by Olsen et al. (1986). In any case, both explanations imply that a tumorigenic response should be expected to occur only after a relatively long exposure period. As a matter of fact, most tumours were detected in the animals examined at terminal sacrifice at 141 -144 weeks, an exposure time that exceeds the duration of conventional studies by about 40 weeks. However, no tumours were observed in the McFarlane (1997) study that terminated after 22 months.

In the Netherlands Cohort Study (Botterweck et al. 2000) in which more than 120000 men and women aged 55 - 69 years were followed-up for 6.3 years, no significant association was found between stomach cancer risk and mean BHT (351 μg/day) and butylated hydroxyanisole (BHA) intake (105 μg/day) through the diet. For comparison, the acceptable daily intake (ADI) established

by the FAO/WHO Joint Expert Committee on Food Additives is 0 - 0.3 mg/kg bw/day for BHT (WHO 1996).

Justification for classification or non-classification

Taking into account all the available information on carcinogenicity for the weight of evidence analysis, it is concluded that the substance BHT is not classified as a carcinogen in accordance with the CLP Regulation.

Additional information

Weight of evidence: Experimental results from chronic and carcinogenicity oral studies using different species and published in scientific articles.