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

Endpoint summary

Description of key information

Weight of evidence

Long-term exposure to BHT can result in functional and histological changes of lung, liver, kidneys and thyroid. Higher sub-acute and sub-chronic doses of BHT can cause death of mice or rats, either due to severe lung damage or massive haemorrhages. In the case of chronic oral exposure, liver is the main target and thyroid is a indirect target. Doses above 25 mg BHT/kg bw/day result in thyroid hyperactivity, enlargement of the liver, induction of several liver enzymes. These hepatic effects, together with the formation of preneoplastic foci and nodules are discussed as causative factors in the mechanism of non-genotoxic carcinogenicity of BHT. As derived from rat studies, a dose of 25 mg BHT/kg bw/day can be considered as reliable NOAEL for chronic exposure.The haemorrhagic effects of high repeated doses of BHT seen in certain strains of mice and rats, but not in other species, may be related to its ability to interact with prothrombin and vitamin K.

Comprehensive in vivo studies, including generation reproduction toxicity studies, demonstrate that the liver is the primary target with increased liver weight and phase 1 and phase 2 liver enzyme activity. The observed thyroid hyperactivity in rodents is a consequence of BHT dependent liver enzyme activation. This secondary mechanism on the thyroid is confirmed in recent comprehensive evaluation by EFSA (2012) See 7.7. EFSA 2012.

 

The administration of BHT to piglets for fattening for 35 consecutive days up to 1500 g/tonne feed (ca. 47.2-61.0 mgBHT/kg bw), did not cause any adverse effects under the test conditions. No adverse effects were observed related neither to the thyroid hormones (TSH, T3, T4) nor hepatic function (GOT, GPT, GGT).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Acceptable to be used in a weight of evidence approach.

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

Limit test:

no

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 oral 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

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

Description (incidence and severity):

There were no significant differences in overall mating success between rats treated with BHT and those receiving control diet.

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

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

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:

The results of these studies give no indication that BHT, at dietary levels up to 1000 mg/kg body weight/day, has any systemic effects on adult male or female rats.

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.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

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

Limit test:

no

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.

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):

0: 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

Description (incidence and severity):

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

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:

effects observed, treatment-related

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:

not specified

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

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: neoplastic

Remarks on result:

other: See cross reference.

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. The lowest dose used in this study (25 mg/kg bw/day) can be considered as the NOAEL.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 September 2013 - 15 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

The aim of the study was to evaluate the tolerance of weaned piglets to Butylated Hydroxytoluene (BHT) as technological feed additive (E 321). The trial was carried out with 144 weaned piglets weighing 7.66 (± 0.89) kg/head at the start of the study. The feeding treatments were: 1) Control group T1: animals fed basal diet; 2) T2: animals fed basal diet (T1) supplemented with BHT at 150 g/tonne feed (recommended dose); 3) T3: animals fed basal diet (T1) supplemented with BHT at 1000 g/tonne feed (6.7x
recommended dose) and 4) T4: animals fed basal diet (T1) supplemented with BHT at 1500 g/tonne feed (10x recommended dose). The feeds were issued to the appropriate pens for 42 consecutive days (between day 0 and day 42 of the trial). Each feeding treatment was replicated in 9 pens (5 pens of castrated males and 4 pens of female piglets) with 4 animals of the same gender per pen. The data recorded during the feeding phase were live weight (LW) at 0, 14 and 42 days from the start of the study; average daily gain (ADG), daily feed intake and feed:gain ratio (F:G) during the periods 0-14; 14-42 and 0-42 days from the start of the study; blood haematology and biochemistry parameters at D0 and D43.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

pig

Strain:

other: Goland x Italian Duroc boar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Az. Agr. Guida di Valtulini Cristina – Fabio e Giovanna S.S. Soc. Agricola, Cascina Guida – 26020 Cappella Cantone (CR), Farm code: 076 CR 032.
- Age at study initiation: 28 days of age approx.
- Weight at study initiation: 7.66 (± 0.89) kg
- Fasting period before study:
- Housing: Grouped, 4 piglets per pen (total of 36 pens). The animals were placed in 5 cleaned, disinfected weaning rooms. In rooms 1 to 4 six pens are aligned in one row with a lateral corridor, 1.0 m wide. In room 5 there are 12 pens arranged in two rows separated by a corridor of 1.0 m wide. For animal health reasons, the weaning rooms are managed according to an “all in – all out” system, with at least 7 days between weaning batches.
- Diet (e.g. ad libitum): Ad libitum (steel feeders).
- Water (e.g. ad libitum): Ad libitum (nipple drinkers).
- Acclimation period: 4 days (medicated feed with amoxicillin and colisting).

DETAILS OF FOOD AND WATER QUALITY: See Table no. 1 below.

ENVIRONMENTAL CONDITIONS :
- Temperature (°C): Within specifications.
- Humidity (%): Within specifications.
*The temperature and relative humidity was recorded every 30 minutes during each day of the trial by a computerised automatic system.
- Air changes (per hr): The ventilation rate varied from 0 m3/hour to the maximum ventilation rate required, according to the desired temperature and the age of the
piglets.
- Photoperiod (hrs dark / hrs light): During the whole study period the lighting scheme was natural.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test producttreated feeds differed only in test product addition from the T1 control feed. The test product was included as dry product by mixing with part of the basal diet. This mixture was added to the rest of the basal diet. All control feeds (those containing no test products) were made before the test product containing feeds.

Duration of treatment / exposure:

42 days (pre-starter period from day 0 to 14 and starter period from day 14 to 42).

Frequency of treatment:

Daily

Dose / conc.:

0 other: g/tonne feed

Dose / conc.:

150 other: g/Tonne feed (recommended use level)

Remarks:

ca. 4.6-6.5 mgBHT/kg bw

Dose / conc.:

1 000 other: g/Tonne feed (6.7 x recommended use level)

Remarks:

ca. 29.7-42.1 mgBHT/kg bw

Dose / conc.:

1 500 other: g/tonne feed (10 x recommended use level)

Remarks:

ca. 47.2-61.0 mgBHT/kg bw

No. of animals per sex per dose:

20 castrated pigs and 16 females per dose (divided in 4 replicates each).

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Not available.
- Rationale for animal assignment (if not random): Random.
- Rationale for selecting satellite groups: Not applicable.
- Post-exposure recovery period in satellite groups: Not applicable.
- Section schedule rationale (if not random): Random.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily, in the morning and in the afternoon.
- Parameters: General health status, ensuring constant feed and water supply as well as checking for the correct temperature and ventilation.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Health/illness and diarrhoea was scored daily. Culling and mortality alongside the reason for culling and probable cause of mortality were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual and mean pen weight of the animals at day 0, 14, and 42.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Mean pen feed intake at days 14 and 42 for to calculate the feed intake in the periods D0-D14, D14-D42 and D0-D42

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes. Mean daily gain and feed:gain ratio, calculated per pen

HAEMATOLOGY: Yes / No / Not specified
- Time schedule for collection of blood: At time 0 and at the end of the trial (D43).
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: At time 0: randomly 20 piglets in total, 10 per sex. At D43: 12 piglets per treatment, 6 per sex.
- Parameters checked: RBC, Haemoglobin, Haematocrit, MCV, MCH, MCHC, WBC, Lymphocytes, Monocytes, Neutrophils, Eosinophils, Basophils, Platelets, reticulocyte count, RPI, MPV, PCT, PDW.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood: At time 0 and at the end of the trial (D43).
- Animals fasted: Not specified
- How many animals: At time 0: randomply 20 piglets in total, 10 per sex. At D43: 12 piglets per treatment, 6 per sex.
- Parameters checked: Glucose, Calcium, Inorganic P, Cholesterol, Triglycerides, Phospholipids, Uric acid, Urea, Creatinine, Lactate dehydrogenase, Alkaline Phosphatase, GOT (Aspartate Transaminase), GPT (Alanine Transaminase), Total Bilirubin, GGT, haptoglobin, serum albumin, serum total protein, blood clotting: quick/INR value, prothrombin time (PT), thyroid hormones (TSH, T3, T4).

OTHER:
DIETARY ANALYSIS:
- Time schedule: Nine representative samples of at least 250 g were taken from each diet and for each growing period: 3 in the beginning, 3 in the middle and 3 in the end of the production process (during the bagging-of the feeds). These 9 samples were pooled to 3 samples in such a way that 1 sample from the beginning, middle and end were pooled together and thoroughly mixed.
- Parameters checked: Feed moisture, crude protein content, crude fat content, crude fibre content, crude ash, starch, digestible energy.

Statistics:

All data were analysed by the GLM (General Linear Model) procedure of SAS (SAS, 2002-2008, release 9.2) using ANOVA (Analysis of Variance) as the main statistical test. Student’s “t” Test was used to compare the means of each group. The level of significance to indicate differences stated in the ANOVA model was P≤0.05 when the difference was statistically significant, while 0.05

Clinical signs:

no effects observed

Description (incidence and severity):

The general performance parameters were good and similar to those expected under commercial rearing of weaned piglets in Italy. The veterinarian considered piglet health and husbandry to be generally good with no mortality and normal consistency of the faeces.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically-significant differences were found for live weight and average daily gain among feeding treatments. See Table no. 2. below.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant differences were found for daily feed intake among feeding treatments in the all phases of the study. See Table no. 3 below.

Food efficiency:

no effects observed

Description (incidence and severity):

No statistically significant differences were found for feed: gain ratio among feeding treatments in the all phases of the study. See Table no. 3 below.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No statistical differences were found among feeding treatments with the exception of haemoglobin and haematocrit that were higher in T2 vs. T1, T3 and T4 groups (respectively 11.14 vs. 10.23, 10.46 and 10.46 %; P=0.0457 and 38.60 vs. 35.73, 36.23 and 35.94 %; P=0.0432) but no difference occurred between T1 and T4 group. Tendentially higher was WBC in T2 vs. T1 and T3 groups (9.90 vs. 8.27 and 8.27 1,000/ml respectively; P=0.0503). All measured blood haematological parameters on D0 and D43 were within the respective reference ranges. See Table no. 4.
For MCV and MCH statistically significant (treatment x gender) interactions were found. See Table no. 5. In females, both MCV and MCH parameters resulted higher in the T3 and T4 vs. T1 group (MCV: 60.53 and 59.65 vs. 56.23 fl; P=0.0225) (MCH: 17.72 and 17.32 vs. 16.28 pg; P=0.0222). See Table no. 6. In castrated males, MCV resulted higher in the T1 and T2 vs. T3 group (respectively 59.43 and 61.13 vs. 54.20 fl; P=0.0354) and MCH resulted tendentially higher in the T2 and T4 vs. T3 group (respectively 17.53 and 17.13 vs. 15.43 pg; P=0.0618). See Table no. 7.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistical significant differences were found for lactate dehydrogenase higher in T4 vs. T1, T2 and T3 groups (respectively 370.58 vs. 334.50, 344.92 and 335.08 U/l; P = 0.0066), while for triglycerides, GPT and serum total protein the differences were only tendentially. Triglycerides were tendentially lower in T2 and T3 vs. T1 group (respectively 72.67 and 71.67 vs. 80.17 mg/dl; P = 0.0976). GPT was tendentially higher in T2 and T4 vs. T1 group (respectively 44.75 and 44.17 vs.
35.42 U/l; P = 0.0959). Serum total protein was tendentially higher in T4 vs. T1, T2 and T3 groups (respectively 7.81 vs. 7.41, 7.44 and 7.47 g/dl; P = 0.0739). All measured blood biochemical parameters on D0 and D43 were within the respective reference ranges. For none of the blood biochemical parameters statistically significant (treatment x gender) interactions were found. See Table no. 8 below. It is important to point out that no differences were observed between the control and the treatment groups regarding neither the thyroid hormones (TSH, T3, T4) nor hepatic function related enzymes (GOT, GPT, GGT). Individual results for thyroid hormones have been included in Table no. 9.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 61 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant adverse effects observed at the highest dose tested.

Remarks on result:

other: 1500 g/tonne feed (ca. 47.2-61.0 mgBHT/kg bw)

Key result

Critical effects observed:

no

Table 1. Feed analysis:

BHT, mg/kg (mean ± standard deviation; n=4)
Experimental period	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000 g/ton. feed	T4 BHT 1500 g/ton. feed
D0 - D14	Not detected	149.2± 5.4	1007.3± 40.0	1424.9± 50.2
D14 - D42	Not detected	149.5±4.6	1005.6± 26.5	1413.8± 75.4

The actual BHT content was within the nominal values and within the tolerance limits given.

Table 2. Body weight:

Zootechnical performance: live weight and average daily gain (mean; n = 36)
Experimental period	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000g/ton. feed	T4 BHT 1500 g/ton. feed	Treatment Effect (P)	Standard error of the mean
Live weight (kg)
D0	7.55	7.83	7.60	7.68	0.5815	0.1458
D14	10.31	10.56	10.26	10.53	0.6433	0.2008
D42	20.76	20.89	20.27	20.93	0.2064	0.2400
Average daily gain (g)
D0-D14	196.63	195.04	189.88	203.57	0.7952	9.6637
D14-D42	373.31	386.71	357.34	371.43	0.4259	12.2961
D0-D42	314.42	322.82	301.52	315.48	0.3358	8.1768

Table 3. Food intake and feed:gain ratio:

Daily feed intake and feed:gain ratio (mean, n = 9)
Experimental period	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000g/ton. feed	T4 BHT 1500 g/ton. feed	Treatment Effect(P)	Standard error of the mean
Feed intake (g)
D0-D14	274.78	282.86	263.53	301.87	0.1819	12.3017
D14-D42	677.36	678.39	639.36	673.22	0.3470	17.4249
D0-D42	543.17	546.55	514.08	549.44	0.1324	11.5380
Feed:Gain ratio
D0-D14	1.42	1.46	1.39	1.49	0.4217	0.0480
D14-D42	1.82	1.77	1.79	1.82	0.8459	0.0403
D0-D42	1.73	1.70	1.71	1.74	0.8188	0.0314

Table 4. Haematology:

Blood haematological parameters at D43 days from the start of the study (mean; n =12 [6 castrated males + 6 females])
	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000 g/ton. feed	T4 BHT 1500 g/ton. feed	Treatment effect (P)	Standard error of the mean
WBC (1,000/ml)	8.27 x	9.90 y	8.27 x	9.27 xy	0.0503	0.4789
RBC (1,000,000/ml)	6.20	6.47	6.32	6.07	0.1171	0.1187
Haemoglobin(%)	10.23a	11.14b	10.46a	10.46a	0.0457	0.2314
Haematocrit(%)	35.73a	38.60b	36.23a	35.94a	0.0432	0.7774
MCHC(%)	28.68	28.85	28.83	29.12	0.6196	0.2375
Neutrophils(%)	42.58	36.75	35.17	36.67	0.2665	0.8061
Lymphocytes(%)	45.38	50.50	53.08	51.00	0.3436	3.0663
Monocytes(%)	7.08	8.42	7.67	8.25	0.5848	0.7497
Eosinophils(%)	3.42	3.00	2.92	3.21	0.9151	0.5410
Basophils(%)	1.54	1.33	1.17	0.88	0.3939	0.2791
Platelets (1,000/mmc)	332.92	303.08	308.67	304.50	0.4431	14.6137
Reticulocyte(%)	0.94	0.97	0.98	1.01	0.6718	0.0390
RPI(%)	0.95	1.05	1.01	1.02	0.4108	0.0446
MPV (fl)	7.57	7.85	9.02	7.60	0.1606	0.5107
PCT(%)	0.40	0.35	0.44	0.40	0.5880	0.0452
PDW	20.09	20.19	21.09	19.86	0.2699	0.4654

WBC = white blood cells; RBC = red blood cells; MCHC = mean concentration of haemoglobin in erythrocytes; RPI = Reticulocyte production index; MPV = mean platelet volume; PCT = platelet crit (percent volume of the blood occupied by platelets); PDW = platelet distribution width.

a,b.: Different letters in the same row = differences significant (P<0.05)

x,y.: Different letter in the same row = differences tendentially significant (0.0.5 < P < 0.1)

Table 5. Haematology - MCV and MCH (treatment x gender)

Blood haematological parameters at D43 days from the start of the study (mean; n = 6)
	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000 g/ton. feed	T4 BHT 1500 g/ton. feed	Treatment effect (P)	Treatment * sex interaction (P)	Standard error of the mean
MCV (fl)	57.83	59.80	57.37	59.14	0.2281	0.0054	0.3092
MCH (pg)	16.55	17.24	16.58	17.23	0.2151	0.0099	0.2314

MCV = mean volume of erythrocytes; MCH = mean content of haemoglobin.

Table 6. Haematology - MCV and MCH (treatment x females)

Blood haematological parameters at D43 days from the start of the study - females (mean; n = 6)
	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000g/ton. feed	T4 BHT 1500 g/ton. feed	Treatment Effect (P)	Standard error of the mean
MCV (fl)	56.23a	58.47ab	60.53b	59.65b	0.0225	0.9334
MCH (pg)	16.28a	16.95ab	17.72b	17.32b	0.0222	0.3047

MCV = mean volume of erythrocytes; MCH = mean content of haemoglobin.

a,b.: Different letters in the same row = differences significant (P<0.05)

Table 7. Haematology - MCV and MCH (treatment x castrated males)

Blood haematological parameters at D43 days from the start of the study - castrated males (mean; n = 6)
	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000g/ton. feed	T4 BHT 1500 g/ton. feed	Treatment Effect (P)	Standard error of the mean
MCV (fl)	59.43b	61.13b	54.20a	58.63ab	0.0354	1.5871
MCH (pg)	16.82xy	17.53y	15.43x	17.13y	0.0618	0.5381

MCV = mean volume of erythrocytes; MCH = mean content of haemoglobin.

a,b.: Different letters in the same row = differences significant (P<0.05)

x,y.: Different letter in the same row = differences tendentially significant (0.05 < P < 0.1)

Table 8. Biochemistry

Blood biochemical parameters at D43 days from the start of the study (mean; n =12 [6 castrated males + 6 females])
	T1 Control	T2 BHT 150 g/ton. feed	T3 BHT 1000g/ton. feed	T4 BHT 1500 g/ton. feed	Treatment effect (P)	Standard error of the mean
Glucose (mg/dl)	89.17	94.33	93.83	94.58	0.6725	3.5603
Urea (mg/dl)	23.80	22.24	22.78	23.02	0.7618	13.0450
Uric acid (mg/dl)	0.34	0.32	0.34	0.32	0.8398	0.0177
Creatinine (mg/dl)	1.34	1.39	1.41	1.33	0.2992	0.0341
Cholesterol (mg/dl)	85.33	76.08	77.92	85.00	0.1618	3.5634
Triglycerides (mg/dl)	80.17y	72.67x	71.67x	73.83xy	0.0976	2.5601
Bilirubin (mg/dl)	0.13	0.14	0.13	0.12	0.8483	0.0119
GOT (U/l)	43.08	42.75	44.08	46.08	0.8780	3.1572
GPT (U/l)	35.42x	44.75y	42.00xy	44.17y	0.0959	2.8527
GGT (U/l)	28.22	28.95	28.40	32.24	0.2057	1.4945
Alkaline phosphate (U/l)	280.17	319.33	295.75	301.42	0.3014	14.4340
Lactate dehydrogenase (U/l	334.50a	344.92a	335.08a	370.58b	0.0066	7.8378
Calcium (mg/dl)	10.66	10.06	10.61	10.02	0.2558	0.2889
Inorganic P (mg/dl)	8.47	9.10	9.28	9.34	0.1128	0.2761
Phospholipides (mg/dl)	110.25	103.95	105.17	113.78	0.2255	3.7170
Haptoglobin (mg/dl)	1.12	1.13	1.12	1.12	0.9907	0.0290
Serum albumin (g/dl)	2.48	2.63	2.49	2.41	0.5113	0.1041
Serum total protein (g/dl)	7.41 x	7.44 x	7.47 x	7.81 y	0.0739	0.0173
INR value	0.96	0.97	0.95	0.95	0.6826	0.0099
Prothrombin value (sec)	10.73	10.72	10.83	10.80	0.9178	0.1320
TSH (mU/ml)	0.02	0.02	0.02	0.02	0.9474	0.0011
T3 (pg/ml)	2.47	2.44	2.48	2.56	0.9067	0.1171
T4 (ng/dl)	1.38	1.34	1.27	1.27	0.6250	0.0672

GOT = Aspartate Transminase; GPT = Alanine Transminase, GGT = gamma-glutamyl transpeptidase, INR value = International Normalised Ratio; TSH = Thyroid stimulating hormone; T3 = Triiodothyronine; T4 = Thyroxine.

a,b.: Different letters in the same row = differences significant (P<0.05)

x,y.: Different letter in the same row = differences tendentially significant (0.05 < P < 0.1)

Table 9: Individual results on T3, T4 and TSH (thyroid hormones):

Treatment	Sex	Room	Pen	FT3	FT4	TSH
T1	F	1	5	2.5	1.29	0.015
T1	F	1	5	2.1	1.27	0.019
T1	F	4	2	2.6	1.36	0.026
T1	F	4	2	2.9	1.06	0.021
T1	F	4	6	2.1	1.36	0.019
T1	F	5	5	2.7	1.6	0.017
T1	M	1	1	2	1.9	0.015
T1	M	1	1	2.2	1.6	0.018
T1	M	2	3	3.1	1.41	0.017
T1	M	5	1	2.5	1.15	0.021
T1	M	5	10	2.8	1.26	0.021
T1	M	5	10	2.1	1.26	0.028
T2	F	1	2	2.4	1.2	0.024
T2	F	2	4	2.5	1.26	0.025
T2	F	3	5	2.1	1.6	0.015
T2	F	3	5	2.3	1.15	0.018
T2	F	5	6	2.1	1.8	0.024
T2	F	5	6	2.4	1.4	0.015
T2	M	1	6	2.3	1.29	0.021
T2	M	1	6	2.2	1.31	0.024
T2	M	3	1	3	1.15	0.019
T2	M	4	3	3	1.19	0.016
T2	M	5	2	2.4	1.29	0.024
T2	M	5	9	2.6	1.41	0.015
T3	F	2	1	2.6	1.02	0.022
T3	F	2	1	2.6	1	0.021
T3	F	3	2	2.9	1.12	0.023
T3	F	4	4	3.1	1.26	0.019
T3	F	5	8	2	1.45	0.016
T3	F	5	8	2.5	1.66	0.021
T3	M	1	3	2	1.23	0.027
T3	M	1	3	2	1.22	0.018
T3	M	2	5	2.1	1.27	0.014
T3	M	3	6	2.5	1.81	0.019
T3	M	5	3	3.2	1.11	0.027
T3	M	2	12	2.3	1.12	0.015
T4	F	1	4	2.1	1.7	0.02
T4	F	1	4	2.5	1.36	0.019
T4	F	1	4	2.1	1.24	0.027
T4	F	2	6	3.1	0.99	0.022
T4	F	4	5	2.7	1.27	0.017
T4	F	5	11	2	1.15	0.021
T4	M	2	4	2.4	1.09	0.019
T4	M	2	2	3	1.35	0.015
T4	M	3	3	2.4	0.96	0.014
T4	M	4	1	2.1	1.45	0.022
T4	M	5	7	3.7	1.7	0.019
T4	M	5	2	2.6	1	0.016

Conclusions:

The administration of BHT to piglets for fattening for 35 consecutive days up to 1500 g/tonne feed (ca. 47.2-61.0 mgBHT/kg bw), did not cause any adverse effects under the test conditions. No adverse effects were observed related neither to the thyroid hormones (TSH, T3, T4) nor hepatic function (GOT, GPT, GGT).

Executive summary:

In the present study, the tolerance of weaned piglets to Butylated hydroxytoluene (BHT) at technological feed additive was evaluated (GLP study). The trial was carried out with 144 weaned piglets weighing 7.66 (± 0.89) kg/head at the start of the study. The feeding treatments were: 1) Control group T1: animals fed basal diet; 2) T2: animals fed basal diet (T1) supplemented with BHT at 150 g/tonne feed (recommended dose, ca. 4.6 -6.5 mgBHT/kg bw); 3) T3: animals fed basal diet (T1) supplemented with BHT at 1000 g/tonne feed (6.7x recommended dose, ca. 29.7 -42.1 mgBHT/kg bw) and 4) T4: animals fed basal diet (T1) supplemented with BHT at 1500 g/tonne feed (10x recommended dose, ca. 47.2 -61.0 mgBHT/kg bw). The feeds were issued to the appropriate pens for 42 consecutive days (between day 0 and day 42 of the trial). Each feeding treatment was replicated in 9 pens (5 pens of castrated males and 4 pens of female piglets) with 4 animals of the same gender per pen. The data recorded during the feeding phase were live weight (LW) at 0, 14 and 42 days from the start of the study; average daily gain (ADG), daily feed intake and feed:gain ratio (F:G) during the periods 0-14; 14-42 and 0-42 days from the start of the study; blood haematology and biochemistry parameters at D0 and D43. The piglets were considered of good health, no mortality was observed and the faeces were of normal consistency. No statistically significant differences were found for piglets performance (LW, ADG, daily feed intake and feed:gain ratio) among feeding treatments in all phases of the study. No statistical differences were found for haematological parameters at D43 among feeding treatments with the exception of haemoglobin and haematocrit that were higher in T2 vs. T1, T3 and T4 groups but no difference occurred between T1 and T4 group. Tendentially higher was WBC in T2 vs. T1 and T3 groups. Some statistically significant treatment * gender interactions were found for MCV and MCH and a statistical analysis for each gender was performed. In female pigs both MCV and MCH resulted higher in the T3 and T4 vs. T1 group while in castrated male pigs MCV resulted higher in the T1 and T2 vs. T3 group and MCH resulted tendentially higher in the T2 and T4 vs. T3 group. Concerning the biochemical parameters at D43 LDH was lower in T1, T2 and T3 vs. T4 group; triglycerides were tendentially higher in T1 vs. T2 and T3 groups; GPT was tendentially lower in T1 vs. T2 and T4 groups and serum total protein was tendentially higher in T4 vs. T1, T2 and T3 groups. All measured blood haematological and biochemical parameters were within the respective reference ranges. In conclusion, the administration of BHT to weaned piglets for 42 consecutive days up to 1500 g/tonne fed (ca. 47.2 -61.0 mgBHT/kg bw), did not cause significant adverse effects under the test conditions. It should be highlighted that no test item related adverse effects were observed regarding neither the thyroid hormones (TSH, T3, T4) nor the hepatic function (GOT, GPT, GGT).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Weight of evidence of several published studies of high quality (Klimish score=2). The time exposure ranges from a sub-acute to a chronic regimen.

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Weight of evidence: Experimental results from subacute and chronic oral studies using different species and published in scientific articles and one study report on short-term toxicity in male rats.

Long-term exposure to BHT can result in functional and histological changes of lung, liver, kidneys and thyroid. Higher sub-acute and sub-chronic doses of BHT can cause death of mice or rats, either due to severe lung damage or massive haemorrhages. In the case of chronic oral exposure, liver is the main target and thyroid is a indirect target. Doses above 25 mg BHT/kg bw/day result in thyroid hyperactivity, enlargement of the liver, induction of several liver enzymes. These hepatic effects, together with the formation of preneoplastic foci and nodules are discussed as causative factors in the mechanism of non-genotoxic carcinogenicity of BHT. As derived from rat studies, a dose of 25 mg BHT/kg bw/day can be considered as reliable NOAEL for chronic exposure.The haemorrhagic effects of high repeated doses of BHT seen in certain strains of mice and rats, but not in other species, may be related to its ability to interact with prothrombin and vitamin K.

 

Comprehensive in vivo studies, including generation reproduction toxicity studies, demonstrate that the liver is the primary target with increased liver weight and phase 1 and phase 2 liver enzyme activity. The observed thyroid hyperactivity in rodents is a consequence of BHT dependent liver enzyme activation. This secondary mechanism on the thyroid is confirmed in recent comprehensive evaluation by EFSA (2012) See 7.7. EFSA 2012.

The administration of BHT to chickens for fattening for 35 consecutive days up to 1500 g/tonne feed (ca. 47.2-61.0 mgBHT/kg bw), did not cause any adverse effects under the test conditions. No adverse effects were observed related neither to the thyroid hormones (TSH, T3, T4) nor hepatic function (GOT, GPT, GGT).

 

Repeated dose toxicity: via oral route - systemic effects (target organ):liver

Justification for classification or non-classification

As derived from rat studies, a dose of 25 mg BHT/kg bw/day can be considered as reliable NOAEL for chronic exposure. This value was derived from a two-generation carcinogenicity study (Olsen et al. 1986) where male and female rats were given BHT until the age of 141 -144 weeks. The criteria established under the CLP Regulation for classification of a substance as "Specific target organ toxicity — repeated exposure" indicate guidance values which refer to effects seen in a standard 90-day toxicity study conducted in rats. They can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration, using dose/exposure time extrapolation similar to Haber's rule for inhalation, which states essentially that the effective dose is directly proportional to the exposure concentration and the duration of exposure. Classification in Category 2 would be applicable, when significant toxic effects are seen to occur at or below the guidance values between 1 and 10 mg/kg bw/day for an oral exposure period of approximately 140 weeks (approximately 900 days). Since the NOAEL derived from the chronic study is 25 mg/kg/day, the substance is not classified as "Specific target organ toxicity — repeated exposure".