Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Weight of evidence: Experimental results from two or three generation studies using different species and published in scientific articles.

Based on the available information, the only effects on reproduction were lower numbers of litters of ten or more pups at birth at doses of 100 mg/kg bw/day and above. The NOAEL was 25 mg/kg bw/day in the rat.

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
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 toxicity for reproduction of the substance.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Bantin and Kingman Ltd (Hull, U.K.)
- Housing: During the first three weeks of BHT administration the males from each group will be housed individually, and the females from each
group will be housed in seven cages, each containing seven or eight animals.
- Diet (e.g. ad libitum): standard rodent breeding diet (CM, Labsure, Manea, Cambridge, U.K.), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: three weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12h light/12h dark cycle
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
Virgin male rats , even maintained on control diet , will not normally mate readily with virgin female rats. Hence, the males will be allowed access to untreated females at intervals during this three week period to encourage subsequent mating with the treated females. One male rat will mate with 3 untreated females. These females will be obtained in addition to those in the main part of the study. All these females w ill be discarded at the end of the 3 week period. After three weeks, six male and 48 female rats will be selected from each group. In the case of male rats any animal with poor mating success recorded during the first three weeks will be removed otherwise the removal will be done using a random numbers table.

One treated male will be placed in each cage with eight females that have been exposed to the same level of BHT. When palpation confirms that a female ra t is pregnant, that animal will be removed from the group and housed singly, while continuing to be exposed to the same level of BHT as previously. Food consumption will be estimated on a group basis. After 14 days, the males, and any non-pregnant females, will be removed and discarded, provided that a sufficient number of successful matings have occurred to supply the minimum number of pups required for the remainder of the study. Established mating records indicate thar 14 days is an adequate period in which to achieve this with the number of animals involved, but the time will be prolonged if necessary. Once a female becomes pregnant the estimated level of BHT is based on that administered just prior to pregnancy being confirmed.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Three weeks before mating, during mating, gestation and lactation.
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:
Treatment groups will be such that each comprises seven males and fifty females.
Control animals:
yes, plain diet
Parental animals: Observations and examinations:
- A measurement of blood clotting potential. (Thrombotest) will be made in all dams.

- Milk production: Five dams per dose group giving birth on the same day where possible [or within 24 hours] will be used in order to measure milk production. At the 15th day post partum the pups will be removed from the dam and weighed. After 2 hours the pups will be returned to their mother, allowed to suckle for 1 hour and then reweighed. The difference in weight between will allow a crude measurement of milk production.

Litter observations:
- On the 20th day of gestation, 5 pregnant female rats from each group will be anaesthetised, and the pups delivered by Caesarian section. The remaining females will be allowed to give birht ; 4 days after litterinf the pups will be examined and the litter sizes reduced to a maximum of 8, leaving the maximum number of males in each litter. Where the number of pups in a litter is less than 8, cross fostering of pups born on the same day [where possible] and in the same dose group will be take place otherwise the litter will be discarded. If only female pups require culling, then these will be chosen at random for removal. If a litter contains more than eight males, then underweight or evidently sickly animals will be removed first ; any further culling will be conducted by random selection.

At weaning, 5 dams and their litters will be selected at random from each dose group. The dams and four male pups will be killed. At this point, all of the other dams and any remaining female pups will also be discarded.

- Determination of foetal weights.

- The BHT concentration in the combined carcasses of the 4 foetuses per group used for electron microscopy will be measured.
Postmortem examinations (parental animals):
- Enumeration of implantation sites, resorption sites and the number of foetuses.

- Total weight and light microscopy of maternal livers.
Postmortem examinations (offspring):
- Light microscopy of 4 foetuses/dam, fixed intact and sectioned transversely to display the liver. Sections from all foetuses will be stained with haematoxylin and eosin. If necessary for interpretation, sections from rats in groups A and B (control and high dose) will be stained for lipid with Oil Red O (ORO), for glycogen by the periodic acid Schiffs (PAS) technique, and for reticulin. Sections from groups A and B (control and high dose) will be stained by imrnunocytochemical procedures for cytochrome P-450 and P-448 isoenzymes and for epoxide hydrolase.

- Biochemical assays to be carried out on foetal liver homogenates (some combination of foetal livers for biochemistry will be required, but this will be minimised as far as is practicably possible) are:
Glucose-6-phosphatase
Epoxide hydrolase
Glutathione S-transferase
Total cytochrome P-450
Ethoxyresorufin O-deethylase
Benzphetamine N-demethylase
Reduced glutathione
Total, microsomal and cytosolic protein

- Sections of the livers, kidneys, adrenals and thyroids will be fixed for histological examination.
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):
After the initial week all dose groups of both male and female rats receiving BHT showed weight gains equal to those of control animals.
Description (incidence and severity):
No differences in food consumption between test and control animals were observed.
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 studies on females showed a massive elevation of the cytochrome P450 isoenzymes showing pentoxyresorufin dealkylase (P450 II otherwise called P450) activity but little alteration in those showing ethoxyresoruphin O-deethylase (P450 I otherwise called P448) activity. There was also some induction of glutathione S-transferase. In the F0 generation biochemical and morphological studies were only undertaken on control females and females receiving doses of 500 mg/kg/day of BHT so that it is not possible to identify a no-effect limit.
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):
Examination of males and non-pregnant females of the F0 generation revealed that, at doses of 500 mg/kg/day or higher there was a dose-related increase in the liver weight. There was dose-dependent proliferation of smooth endoplasmic reticulum with the associated enlargement of centrilobular hepatocytes and centrilobular sinusoidal dilatation.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Examination of the livers from rats of the F0 generation showed hepatomegaly in BHT-treated male rats and in female rats examined before the onset of pregnancy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not specified
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
After the initial week all dose groups of both male and female rats receiving BHT showed weight gains equal to those of control animals.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No differences in food consumption between test and control animals were observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Examination of the livers from rats of the F0 generation showed hepatomegaly in BHT-treated male rats and in female rats examined before the onset of pregnancy.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Examination of males and non-pregnant females of the F0 generation revealed that, at doses of 500 mg/kg/day or higher there was a dose-related increase in the liver weight. There was dose-dependent proliferation of smooth endoplasmic reticulum with the associated enlargement of centrilobular hepatocytes and centrilobular sinusoidal dilatation.

OTHER FINDINGS (PARENTAL ANIMALS)
Biochemical studies on females showed a massive elevation of the cytochrome P450 isoenzymes showing pentoxyresorufin dealkylase (P450 II otherwise called P450) activity but little alteration in those showing ethoxyresoruphin O-deethylase (P450 I otherwise called P448) activity. There was also some induction of glutathione S-transferase. In the F0 generation biochemical and morphological studies were only undertaken on control females and females receiving doses of 500 mg/kg/day of BHT so that it is not possible to identify a no-effect limit.
Key result
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was a slight fall in pup number/litter in dams receiving 500 mg/kg/day but this was not statistically significant. There was no evidence for any change in body weight or increase in gross abnormalities between pups born of BHT-treated animals and the pups of control animals in either study. The results give no evidence for a teratogenic effect of BHT even at doses of 1000 mg/kg/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
BODY WEIGHT (OFFSPRING)
Pups of dams receiving the highest dose of BHT (500 mg/kg/day) were lighter than controls.
Food consumption and compound intake (if feeding study):
not specified
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 examination of the livers of the pups at weaning showed a marked induction of pentoxyresorufin delakylase and some induction of glutathione-S-transferase. There was also induction of epoxide hydrolase and of the P450 isoenzymes responsible for benzphetamine N-demethylase activity, enzymes not studied in the F0 generation. The degree of induction of these enzymes declined progressively from weaning onwards.
Urinalysis findings:
not examined
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
ORGAN WEIGHTS (OFFSPRING)
An increase in relative liver weight was observed in weanling pups of dams receiving the overlap dose of 500 mg/kg/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was a slight fall in pup number/litter in dams receiving 500 mg/kg/day but this was not statistically significant. There was no evidence for any change in body weight or increase in gross abnormalities between pups born of BHT-treated animals and the pups of control animals in either study.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
OTHER FINDINGS (OFFSPRING)
Six months after weaning pups treated with the top dose of 250 mg/kg/day showed all the morphological and biochemical changes observed in the F0 generation with the exception of bile duct proliferation. However, examination of pups at earlier times showed marked differences between the F0 and F1 generations. While BHT treatment of dams had no apparent effect on the development of the pups in utero there was a marked suppression of weight gain during lactation and premature development of the adrenal cortex. Biochemical examination of the livers of the pups at weaning showed a marked induction of pentoxyresorufin delakylase and some induction of glutathione-S-transferase. There was also induction of epoxide hydrolase and of the P450 isoenzymes responsible for benzphetamine N-demethylase activity, enzymes not studied in the F0 generation. The degree of induction of these enzymes declined progressively from weaning onwards.

No evidence was obtained in the current study to support the hypothesis that the failure of pups to gain weight was due to direct toxicity from BHT passed in the milk. In this study, litters were reduced at an earlier time. Hence reduction in demand on the mother for milk results in increased pup weight. It is conventional to reduce rat litters to eight because it is the general experience that this is the safe level where there will be no pup loss due to malnutrition. Hence nursing of eight pups to their full potential weight at weaning would appear to be on the limits of the metabolic capacity of a rat. This is supported by the observation that even with control animals there is a slightly greater weight at weaning when litter size is less than eight. A reduction in milk producing capacity would thus be expected to cause a reduction in pup weight which would be markedly ameliorated by reduction in litter size. No such effect would be expected if the effect were due to toxicity by BHT or metabolites to the pups.

It would seem likely that failure of the pups of BHT-treated dams to gain weight during lactation is due to malnourishment not to direct toxicity.
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: all the morphological and biochemical changes observed in the F0 generation with the exception of bile duct proliferation
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
Key result
Reproductive effects observed:
no

In the F0 generation BHT produced the expected hepatic changes in males and in non-pregnant females. These include a dose-related increase in liver weight, induction of cytochrome P450 isoenzymes and other drug metabolising enzymes. In lactating mothers of the F0 generation BHT produced marked liver enlargement and evidence of an adverse effect on the hepatocytes. These effects were dose related. There was no evidence for a teratogenic or foetotoxic effect of BHT. There was, however, a slight reduction in litter size. There was a dose-related drop in the body weight gain of pups nursed by mothers on BHT. This reduction in weight gain was not completely abolished in pups returned to control diet even at 4 weeks after weaning. The reduction of body weight gain in pups is thought to be due to a reduced milk production or quality consequent upon the induction of drug metabolising enzymes in the mother and the energy required to eliminate BHT from the body. In turn this reduced milk production appears to cause some degree of malnutrition in the early weeks of life. Apart from the loss in weight the F1 generation show the same spectrum of treatment-related changes as the F0 generation. The only significant difference observed between the F0 and F1 generation is reduced pup growth during nursing. This may well predispose the rats to develop neoplasia at the end of their lives.

Conclusions:
In the F0 generation BHT produced the expected hepatic changes in males and in non-pregnant females. These include a dose-related increase in liver weight, induction of cytochrome P450 isoenzymes and other drug metabolising enzymes. In lactating mothers of the F0 generation BHT produced marked liver enlargement and evidence of an adverse effect on the hepatocytes. These effects were dose related. There was no evidence for a teratogenic or foetotoxic effect of BHT. There was, however, a slight reduction in litter size. There was a dose-related drop in the body weight gain of pups nursed by mothers on BHT. This reduction in weight gain was not completely abolished in pups returned to control diet even at 4 weeks after weaning. The reduction of body weight gain in pups is thought to be due to a reduced milk production or quality consequent upon the induction of drug metabolising enzymes in the mother and the energy required to eliminate BHT from the body. In turn this reduced milk production appears to cause some degree of malnutrition in the early weeks of life. Apart from the loss in weight the F1 generation show the same spectrum of treatment-related changes as the F0 generation. The only significant difference observed between the F0 and F1 generation is reduced pup growth during nursing. This may well predispose the rats to develop neoplasia at the end of their lives.
Executive summary:

The study was designed to investigate the development of changes in the F0 and F1 generations of rats administered BHT. The following doses were used: 25, 100 and 500 mg/kg/day of BHT in the F0 generation and 25, 100 and 250 mg/kg/day of BHT in the F1 generation. Foetuses were examined at the 20th day of gestation and the pups at weaning, 4 weeks and 6 months after weaning. The livers of these rats were subjected to detailed examination using light and electron microscopy, immunohistochemistry and biochemical analysis. In the F0 generation BHT produced the expected hepatic changes in males and in non-pregnant females. These include a dose-related increase in liver weight, induction of cytochrome P450 isoenzymes and other drug metabolising enzymes. In lactating mothers of the F0 generation BHT produced marked liver enlargement and evidence of an adverse effect on the hepatocytes. These effects were dose related. There was no evidence for a teratogenic or foetotoxic effect of BHT. There was, however, a slight reduction in litter size. There was a dose-related drop in the body weight gain of pups nursed by mothers on BHT. This reduction in weight gain was not completely abolished in pups returned to control diet even at 4 weeks after weaning. The reduction of body weight gain in pups is thought to be due to a reduced milk production or quality consequent upon the induction of drug metabolising enzymes in the mother and the energy required to eliminate BHT from the body. In turn this reduced milk production appears to cause some degree of malnutrition in the early weeks of life. Apart from the loss in weight the F1 generation show the same spectrum of treatment-related changes as the F0 generation. The only significant difference observed between the F0 and F1 generation is reduced pup growth during nursing. This may well predispose the rats to develop neoplasia at the end of their lives.

Endpoint:
two-generation reproductive toxicity
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 toxicity for reproduction of the substance.
Reason / purpose:
reference to same study
Reason / purpose:
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 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.

Details on mating procedure:
Male rats were allowed access to female rats for 3 weeks commencing 5 weeks after first offering the BHT-containing diet.
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
Details on study schedule:
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.
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
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS AND BODY WEIGHTS:
The rats were examined daily and weighed weekly.


Postmortem examinations (parental animals):
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.

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.

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.
Postmortem examinations (offspring):
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). 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.

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.
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.

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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
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:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Groups of rats were killed and autopsied 4 and 22 wk after wean- ing. The body weight of the top dose group 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.
Food consumption and compound intake (if feeding study):
not specified
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 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
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
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:
no effects observed
Histopathological findings:
no effects observed
Description (incidence and severity):
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.
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Apart from a reduced body weight, the pups of dams exposed to BHT developed normally.

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.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
other: Bodyweight (General toxicity)
Organ:
liver
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Reproductive effects observed:
no

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 NOAEL for parental toxicity is 500 mg/kg bw/day for females and 100 mg/kg bw/day for males. The NOAEL for pup body weight and development was 100 mg/kg bw/day. No adverse effects on reproductive performance were observed.
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. The NOAEL for parental toxicity is 500 mg/kg bw/day for females and 100 mg/kg bw/day for males. The NOAEL for pup body weight and development was 100 mg/kg bw/day. No adverse effects on reproductive performance were observed.

Endpoint:
two-generation reproductive toxicity
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 effects on reproduction of the substance.
Reason / purpose:
reference to same study
Reason / purpose:
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:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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)
Details on mating procedure:
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.
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
Parental animals: Observations and examinations:
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.
Postmortem examinations (parental animals):
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:
not specified
Dermal irritation (if dermal study):
not examined
Mortality:
not specified
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.
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.

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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
The gestation rate was comparable among groups.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
other: Bodyweight gain (General toxicity).
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified
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 / viability:
no mortality observed
Description (incidence and severity):
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):
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):
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.
Sexual maturation:
not specified
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).
Histopathological findings:
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.

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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Gestation rate was not affected by the treatment. The Armitage-Cochran test for linear trend in proportions demonstrated that the fraction of litters with ten or more pups decreased significantly with BHT dose (P < 0.001). At weaning, these pups, particularly males, showed a dose-related reduction of body weight being clearly statistically significant at 100 and 500 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: Number of litters of ten or more pups at birth.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
other: Bodyweight gain (General toxicity) and Number of litters at birth.
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Reproductive effects observed:
no
Conclusions:
The NOAEL for maternal toxicity was 100 mg/kg bw/day and the NOAEL for the F1 was 25 mg/kg-bw/day based on body weight gain and number of pups per litter at birth.
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. The NOAEL for maternal toxicity was 100 mg/kg bw/day; body weight gain of the F0 animals was significantly reduced in the high-dose group (500 mg/kg bw/day). Gestation rate was not affected by the treatment. The Armitage-Cochran test for linear trend in proportions demonstrated that the fraction of litters with ten or more pups decreased significantly with BHT dose (P < 0.001). At weaning, these pups, particularly males, showed a dose-related reduction of body weight being clearly statistically significant at 100 and 500 mg/kg bw/day.

Endpoint:
toxicity to reproduction
Remarks:
other: dose ranging experiment for a two generation study
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 toxicity for reproduction of the substance. No data on GLP.
Reason / purpose:
other: The present study is a dose-range finding experiment of the cross referenced test.
Principles of method if other than guideline:
To determine the maximum dietary dose of butylated hydroxytoluene (BHT) tolerated by female rats exposed prior to and throughout pregnancy, and by pups similarly exposed in utero and until weaning. Treatment groups each comprised three males and sixteen females. There were a total of four groups. After acclimatisation, these groups received diets designed as follows: control group, 500 mg/kg bw/day, 750 mg/kg bw/day and 1000 mg/kg bw/day. Male and female rats were weighed on arrival, after allocation to treatment groups and at weekly intervals thereafter. Females continued to be weighed weekly until the pups were weaned. The numbers of live and dead pups in each litter were counted, and the litter weight recorded. The litters were reweighed at weaning. The average food consumption by males and females in each cage was measured at weekly intervals, as was the food consumption of pregnant and nursing dams. All animals were observed daily for clinical or behavioural signs of toxicity, and for symptoms of ill-health. At weaning, dams and pups were also be examined extemally, killed, opened ventrally and subjected to macroscopic internal examination.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Bantin and Kingman Ltd. (Hull, U.K.)
- Age at study initiation: males 11 weeks, females 7 weeks
- Housing: Animals were housed on sterilised sawdust in solid-bottomed NKP polypropylene cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Three weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 h light/12 h dark cycle

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
Diet mixes were prepared by adding an appropriate amount of finely ground BHT to a weighed quantity of powdered CRM diet and mixing in a planetary action mixer carrying a paddle-type blade.
Details on mating procedure:
The males were allowed access to groups of three untreated females at intervals during a four week period to encourage subsequent mating with the treated females. While the male was present with the untreated females, the food of the female rats was removed. At the end of this period the male in each group with the poorest success in mating was discarded. After three weeks, one treated male was allowed access to eight females that have been exposed to the same level of BHT. While the male was present the food of the female rats was removed. When palpation confirmed that a female rat is pregnant, that animal was removed from the group and housed singly, while continuing to be exposed to the same level of BHT as previously. After
confirmation of fertilisation in at least eight females per group, the males, and remaining females was killed.
Duration of treatment / exposure:
Before mating and during mating. Exposure of the pregnant females to BHT was continued through to the birth of the young and until weaning (21 dys after delivery).
Frequency of treatment:
Daily
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Eight females per group
Control animals:
yes, plain diet
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Male and female rats were weighed on arrival, after allocation to treatment groups and at weekly intervals thereafter. Females continued to be weighed weekly until the pups were weaned.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The average food consumption by males and females in each cage was measured at weekly intervals, as was the food consumption of pregnant and nursing dams.



Litter observations:
PARAMETERS EXAMINED
The numbers of live and dead pups in each litter were counted, and the litter weight recorded. The litters were reweighed at weaning.

Postmortem examinations (parental animals):
GROSS NECROPSY
At weaning, dams were also be examined externally, killed, opened ventrally and subjected to macroscopic internal examination.

HISTOPATHOLOGY / ORGAN WEIGHTS
Samples of liver from all dams were be removed to 10% neutral buffered formalin. In addition other tissues were removed, and all tissues subjected to histological examination.
Postmortem examinations (offspring):
GROSS EXAMINATION:
At weaning, pups were also be examined externally, killed, opened ventrally and subjected to macroscopic internal examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
Samples of liver from all weanlings were be removed to 10% neutral buffered formalin. In addition other tissues were removed, and all tissues subjected to histological examination.
Statistics:
Where appropnate, statistical comparisons of data was be made by using Student's t-test, and the level of significance of differences in group means indicated.
Clinical signs:
no effects observed
Description (incidence and severity):
Treatment with BHT caused no serious effects on the general health of the rats in the period under consideration. There was a slight increase in fur discoloration in treated animals and, on some occasions rats treated with BHT appeared sensitive to handling.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The female rats took readily to the BHT containing diet. The food consumption of treated animals was consistently higher than controls from the fourth week of the study onwards. The presence of BHT in the diet had no statistically significant effect on body weight gain although a dose related trend to reduced body weights was visible. Male rats showed a dose related fall in food consumption and in body weight gain in the first week of being offered BHT containing diets. Food consumption and body weight gain subsequently returned to control values although the weight lost in the first week was never regained. Nevertheless the weight loss in the first week does not indicate any toxic effects of BHT on male animals but rather reflects the fact that older rats have a markedly greater aversion to changes in diet palatability than younger animals. The mean achieved intake of BHT was close to the nominal figure in both males and females.

Observation of pregnant rats treated with BHT showed no serious differences between groups treated with BHT and control rats. The slight increase in fur discoloration observed prior to confirmation of pregnancy continued. The weight gain during pregnancy followed the expected pattern except in rats treated with 750 and 1000 mg/kg BHT where there appeared to be some inhibition of the weight gain normally shown in the last weeks of pregnancy. The concentration of BHT in the diet was not adjusted during pregnancy.

The condition of the control dams and dams of rats treated with mid and low doses of BHT remained generally good during lactation. Dams treated with high doses of BHT were, however, markedly lighter han dams in other groups. The pups of dams treated with all doses of BHT developed markedly more slowly than control pups and, in some high dose animals, were cold to the touch in the third week of life. The brown staining observed in dams at earlier times continued and there was patchy fur loss in some animals. lnflammation of the teats were observed in some animals. As expected the food consumption of all groups of dams rose during lactation. This increase was less marked in rats treated with BHT than in control rats and the inhibition of weight gain during pregnancy appeared dose related.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The female rats took readily to the BHT containing diet. The food consumption of treated animals was consistently higher than controls from the fourth week of the study onwards. The presence of BHT in the diet had no statistically significant effect on body weight gain although a dose related trend to reduced body weights was visible. Male rats showed a dose related fall in food consumption and in body weight gain in the first week of being offered BHT containing diets. Food consumption and body weight gain subsequently returned to control values although the weight lost in the first week was never regained. Nevertheless the weight loss in the first week does not indicate any toxic effects of BHT on male animals but rather reflects the fact that older rats have a markedly greater aversion to changes in diet palatability than younger animals. The mean achieved intake of BHT was close to the nominal figure in both males and females.

Observation of pregnant rats treated with BHT showed no serious differences between groups treated with BHT and control rats. The slight increase in fur discoloration observed prior to confirmation of pregnancy continued. The weight gain during pregnancy followed the expected pattern except in rats treated with 750 and 1000 mg/kg BHT where there appeared to be some inhibition of the weight gain normally shown in the last weeks of pregnancy. The concentration of BHT in the diet was not adjusted during pregnancy.

The condition of the control dams and dams of rats treated with mid and low doses of BHT remained generally good during lactation. Dams treated with high doses of BHT were, however, markedly lighter han dams in other groups. The pups of dams treated with all doses of BHT developed markedly more slowly than control pups and, in some high dose animals, were cold to the touch in the third week of life. The brown staining observed in dams at earlier times continued and there was patchy fur loss in some animals. lnflammation of the teats were observed in some animals. As expected the food consumption of all groups of dams rose during lactation. This increase was less marked in rats treated with BHT than in control rats and the inhibition of weight gain during pregnancy appeared dose related.
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:
not examined
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):
ORGAN WEIGHTS (PARENTAL ANIMALS)
Treatment with BHT caused a marked increase in liver weight in all dams treated with BHT. There was little change with dose. It should, however, be noted that the liver weights are almost 10% of body weights and it is our experience that this is the maximum degree of enlargement possible in rats. The liver weight of pups of dams mated with BHT was depressed in line with the general failure to thrive, there was no change in the liver weight of the pups when expressed on a body weight basis.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
GROSS PATHOLOGY (PARENTAL ANIMALS)
Control dams and dams treated with low and mid doses of BHT appeared fit and well three weeks after the birth of the pups. On autopsy heavy deposits of fat were observed on the body wall and around the kidneys and adrenals as is normal in lactating females. Dams treated with high doses of BHT were markedly lighter than other groups but remained active and showed no signs of systemic toxicity. On autopsy little fat was observed around the body wall, kidneys or adrenals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Treatment with BHT did not affect the readiness of male rats to mate with untreated female rats or their success in mating. There was no difference between the litter size or weight or in the average pup weight between litters fathered by male rats treated with BHT and female rats.

Prior to this part of the experiment the male rat in each group with the poorest mating record was discarded. The remaining two male rats were each caged with eight female rats treated with the same amount of BHT on a body weight basis until a total of at least eight of the sixteen females in each group became pregnant. The results showed that successful mating occurred less frequently in rats treated with 1000 mg/kg body weight of BHT than in the other experimental groups.
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Treatment with BHT caused no serious effects on the general health of the rats in the period under consideration. There was a slight increase in fur discoloration in treated animals and, on some occasions rats treated with BHT appeared sensitive to handling.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The female rats took readily to the BHT containing diet. The food consumption of treated animals was consistently higher than controls from the fourth week of the study onwards. The presence of BHT in the diet had no statistically significant effect on body weight gain although a dose related trend to reduced body weights was visible. Male rats showed a dose related fall in food consumption and in body weight gain in the first week of being offered BHT containing diets. Food consumption and body weight gain subsequently returned to control values although the weight lost in the first week was never regained. Nevertheless the weight loss in the first week does not indicate any toxic effects of BHT on male animals but rather reflects the fact that older rats have a markedly greater aversion to changes in diet palatability than younger animals. The mean achieved intake of BHT was close to the nominal figure in both males and females.

Observation of pregnant rats treated with BHT showed no serious differences between groups treated with BHT and control rats. The slight increase in fur discoloration observed prior to confirmation of pregnancy continued. The weight gain during pregnancy followed the expected pattern except in rats treated with 750 and 1000 mg/kg BHT where there appeared to be some inhibition of the weight gain normally shown in the last weeks of pregnancy. The concentration of BHT in the diet was not adjusted during pregnancy.

The condition of the control dams and dams of rats treated with mid and low doses of BHT remained generally good during lactation. Dams treated with high doses of BHT were, however, markedly lighter han dams in other groups. The pups of dams treated with all doses of BHT developed markedly more slowly than control pups and, in some high dose animals, were cold to the touch in the third week of life. The brown staining observed in dams at earlier times continued and there was patchy fur loss in some animals. lnflammation of the teats were observed in some animals. As expected the food consumption of all groups of dams rose during lactation. This increase was less marked in rats treated with BHT than in control rats and the inhibition of weight gain during pregnancy appeared dose related.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Treatment with BHT did not affect the readiness of male rats to mate with untreated female rats or their success in mating. There was no difference between the litter size or weight or in the average pup weight between litters fathered by male rats treated with BHT and female rats.

Prior to this part of the experiment the male rat in each group with the poorest mating record was discarded. The remaining two male rats were each caged with eight female rats treated with the same amount of BHT on a body weight basis until a total of at least eight of the sixteen females in each group became pregnant. The results showed that successful mating occurred less frequently in rats treated with 1000 mg/kg body weight of BHT than in the other experimental groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Treatment with BHT caused a marked increase in liver weight in all dams treated with BHT. There was little change with dose. It should, however, be noted that the liver weights are almost 10% of body weights and it is our experience that this is the maximum degree of enlargement possible in rats. The liver weight of pups of dams mated with BHT was depressed in line with the general failure to thrive, there was no change in the liver weight of the pups when expressed on a body weight basis.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Control dams and dams treated with low and mid doses of BHT appeared fit and well three weeks after the birth of the pups. On autopsy heavy deposits of fat were observed on the body wall and around the kidneys and adrenals as is normal in lactating females. Dams treated with high doses of BHT were markedly lighter than other groups but remained active and showed no signs of systemic toxicity. On autopsy little fat was observed around the body wall, kidneys or adrenals.
Key result
Remarks on result:
other: This is a dose-range finding study, its purpose is not to unequivocally determine effect levels but to determine a range of concentrations to be used in the main study
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Pups from dams treated with the lowest dose of BHT were markedly stunted in their growth but their fur condition was good and they were active and exploratory. Pups from dams treated with the mid and high doses of BHT were severely stunted, showed poor fur condition and were markedly less active than pups from other groups.

Pups from two litters, totalling fourteen animals, from each dose group were maintained on control diet for four weeks after weaning. Pups born to dams mated with 1000 mg/kg/day of BHT were so sickly that four had to be killed for humanitarian reasons within one week of weaning. Four weeks after weaning the pups born to dams receiving BHT-containing diets remained lighter than control pups. Pups born to dams receiving 500 mg/kg of BHT showed no sign other than the check in growth but pups born to dams receiving 750 or 1000 mg/kg of BHT continued to show poor fur condition until termination of the experiment four weeks after weaning.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Pups from dams treated with the lowest dose of BHT were markedly stunted in their growth but their fur condition was good and they were active and exploratory. Pups from dams treated with the mid and high doses of BHT were severely stunted, showed poor fur condition and were markedly less active than pups from other groups.

Pups from two litters, totalling fourteen animals, from each dose group were maintained on control diet for four weeks after weaning. Pups born to dams mated with 1000 mg/kg/day of BHT were so sickly that four had to be killed for humanitarian reasons within one week of weaning. Four weeks after weaning the pups born to dams receiving BHT-containing diets remained lighter than control pups. Pups born to dams receiving 500 mg/kg of BHT showed no sign other than the check in growth but pups born to dams receiving 750 or 1000 mg/kg of BHT continued to show poor fur condition until termination of the experiment four weeks after weaning.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no statistically significant difference between litter number or litter weight between pups born of control rats or pups born from sires and dams treated with BHT although there did appear to be a dose-related trend towards reduced litter size and the average weight of pups of dams treated with 750 mg/kg of BHT was significantly smaller than that of control pups.
Food consumption and compound intake (if feeding study):
not specified
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In no group of animals were any malformations observed although in pups of dams treated with mid and high doses of BHT there was the disproportionate development of the head as compared with the rest of the body which is to be expected when growth is inhibited to the extent found in this study.
Gross pathological findings:
not examined
Histopathological findings:
not specified
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
CLINICAL SIGNS (OFFSPRING)
Pups from dams treated with the lowest dose of BHT were markedly stunted in their growth but their fur condition was good and they were active and exploratory. Pups from dams treated with the mid and high doses of BHT were severely stunted, showed poor fur condition and were markedly less active than pups from other groups.

Pups from two litters, totalling fourteen animals, from each dose group were maintained on control diet for four weeks after weaning. Pups born to dams mated with 1000 mg/kg/day of BHT were so sickly that four had to be killed for humanitarian reasons within one week of weaning. Four weeks after weaning the pups born to dams receiving BHT-containing diets remained lighter than control pups. Pups born to dams receiving 500 mg/kg of BHT showed no sign other than the check in growth but pups born to dams receiving 750 or 1000 mg/kg of BHT continued to show poor fur condition until termination of the experiment four weeks after weaning.

BODY WEIGHT (OFFSPRING)
There was no statistically significant difference between litter number or litter weight between pups born of control rats or pups born from sires and dams treated with BHT although there did appear to be a dose-related trend towards reduced litter size and the average weight of pups of dams treated with 750 mg/kg of BHT was significantly smaller than that of control pups.

GROSS PATHOLOGY (OFFSPRING)
In no group of animals were any malformations observed although in pups of dams treated with mid and high doses of BHT there was the disproportionate development of the head as compared with the rest of the body which is to be expected when growth is inhibited to the extent found in this study.
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
Remarks on result:
other: This is a dose-range finding study, its purpose is not to unequivocally determine effect levels but to determine a range of concentrations to be used in the main study
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (nominal)
System:
other: General toxicity
Organ:
not specified
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
no
Relevant for humans:
not specified

BHT at all doses caused a marked inhibition of the post-natal weight gain of pups. Three weeks after birth the pups of dams receiving the two higher doses of BHT were emaciated and some pups of dams receiving the highest dose were killed on humanitarian grounds. The effect appeared to be associated with poor milk production rather than BHT toxicity firstly because generally pups of smaller than normal litters were significantly heavier than those in normally-sized litters and secondly because the pups showed no increase in liver weight, even on a body weight basis. As the dams remained healthy and, except in the case of dams receiving the highest dose of BHT, were not significantly reduced in weight compared with controls, it was concluded that the postulated reduction in milk production is due to some specific effect of BHT rather than to an overall effect on the dams condition. The livers of the dams were greatly enlarged, being over two-times the weight, on a body weight basis, of non-lactating rats of this age. It is known that liver enlargement in rats treated with BHT is associated with induction of microsomal drug-metabolising enzymes. The livers of control pregnant and lactating rats are also enlarged and this is believed to be due to the increased synthesis of serum lipoproteins needed to support milk formation. It is possible that the livers of BHT-treated dams are unable to meet the simultaneous demands for synthesis of smooth endoplasmic reticulum, for drug metabolising enzymes, as well as rough endoplasmic reticulum, for apolipoprotein synthesis, and that as a result lipid cannot be exported to the mammary glands at the speed needed to maintain pup growth.

 

Conclusions:
The pups of dams treated with 750 or 1000 mg/kg/day of BHT appear permanently damaged and it would be quite inappropriate to use these doses. The reduction of pup weight in rats treated with 500 mg/kg/day of BHT in our study is almost identical to that found by Olsen et al (1986) and we would recommend that this dose be used in the main experiment.
Executive summary:

To determine the maximum dietary dose of butylated hydroxytoluene (BHT) tolerated by female rats exposed prior to and throughout pregnancy, and by pups similarly exposed in utero and until weaning. Treatment groups each comprised three males and sixteen females. There were a total of four groups. After acclimatisation, these groups received diets designed as follows: control group, 500 mg/kg bw/day, 750 mg/kg bw/day and 1000 mg/kg bw/day. Male and female rats were weighed on arrival, after allocation to treatment groups and at weekly intervals thereafter. Females continued to be weighed weekly until the pups were weaned. The numbers of live and dead pups in each litter were counted, and the litter weight recorded. The litters were reweighed at weaning. The average food consumption by males and females in each cage was measured at weekly intervals, as was the food consumption of pregnant and nursing dams. All animals were observed daily for clinical or behavioural signs of toxicity, and for symptoms of ill-health. At weaning, dams and pups were also be examined extemally, killed, opened ventrally and subjected to macroscopic internal examination. The results indicate that administration of BHT at doses of up to 1000 mg/kg body weight/day has no effect on the overall health of either adult male or adult virgin female rats although there was marked hepatomegaly. It was necessary to house together male and female rats treated with 1000 mg/kg/day on greater number of occasions before an acceptable number of pregnancies were achieved than control animals or animals treated with 500 or 750 mg/kg/day of BHT. No similar effect was found when test males were mated to control females. This suggests that, at 1000 mg/kg/day, BHT has an effect on the receptiveness or fertility of female rats. The pregnancies of all groups of rats proceeded normally in rats treated with 500 mg/kg/day of BHT but rats treated with 750 or 1000 mg/kg/day showed a reduced weight gain in the last week of pregnancy. As there was only a small, statistically insignificant reduction in overall litter weight it would appear likely that the effect on maternal weight gain reflected reduced fat deposition or some similar effect. BHT at all doses caused a marked inhibition of the post-natal weight gain of pups. Three weeks after birth the pups of dams receiving the two higher doses of BHT were emaciated and some pups of dams receiving the highest dose were killed on humanitarian grounds. The effect appeared to be associated with poor milk production rather than BHT toxicity firstly because generally pups of smaller than normal litters were significantly heavier than those in normally-sized litters and secondly because the pups showed no increase in liver weight, even on a body weight basis. As the dams remained healthy and, except in the case of dams receiving the highest dose of BHT, were not significantly reduced in weight compared with controls, it was concluded that the postulated reduction in milk production is due to some specific effect of BHT rather than to an overall effect on the dams condition. The livers of the dams were greatly enlarged, being over two-times the weight, on a body weight basis, of non-lactating rats of this age. It is known that liver enlargement in rats treated with BHT is associated with induction of microsomal drug-metabolising enzymes. The livers of control pregnant and lactating rats are also enlarged and this is believed to be due to the increased synthesis of serum lipoproteins needed to support milk formation. It is possible that the livers of BHT-treated dams are unable to meet the simultaneous demands for synthesis of smooth endoplasmic reticulum, for drug metabolising enzymes, as well as rough endoplasmic reticulum, for apolipoprotein synthesis, and that as a result lipid cannot be exported to the mammary glands at the speed needed to maintain pup growth.

Endpoint:
three-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although the study was not conducted according to the recommended guidelines (number of animals per group, reproductive parameters, etc.), it provides some scientific valid information to assess the toxicity to reproduction effects of the substance to be taking into account for weight of evidence.
Principles of method if other than guideline:
Crj:CD-1 mice (F0 and F1generations) received ca. 0, 23, 68, 203 or 608 mg/kg bw/day BHT in the diet during premating, mating, gestation and lactation periods (ca. 11 weeks). The study was designed to evaluate the reproductive, developmental and behavioural effect of BHT in lower dose levels in mice during three generations.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
other: Crj:CD-1
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
The animals in the F0 generation were 5 weeks o fage at start of the study. At 9 weeks of age, each female was paired with one male from the same treatment group for a period of 5 days. The males were removed from the females after 5 days and the females were allowed to produce and rear their pups.

The animals in the F1 generation were mated at 9 weeks of age, avoiding sibling matings.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
From 5 weeks of age of the F0 generation to the weaning of the F2 generation.
Frequency of treatment:
Daily
Details on study schedule:
The pups were removed from their dams at 4 weeks of age and were selected at random to continue treatment. The control sizes of the F1 generation were control 10/sex and treatment group 10/sex/group.
Dose / conc.:
0.015 other: %
Remarks:
Approximately 23 mg/kg-bw/day
Dose / conc.:
0.045 other: %
Remarks:
Approximately 68 mg/kg-bw/day
Dose / conc.:
0.135 other: %
Remarks:
Approximately 203 mg/kg-bw/day
Dose / conc.:
0.405 other: %
Remarks:
Approximately 608 mg/kg-bw/day
No. of animals per sex per dose:
10 animals per sex per group
Control animals:
yes, plain diet
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 generation on postnatal day 0: litter size, litter weight and sex ratio. The pups were weighed on postnatal day 0, 4, 7, 14 and 21 in the lactation period.

The functional and behavioural developmental parameters were measured for the individual pups in the lactation period in F1 and F2 generations.
Statistics:
Litter size, litter weight and the body weight of the pups were assessed with Bonferroni´s multiple comparison test after ANOVA or Kruskal-Wallis test. Sex ratio was assessed with the Chi-square test. The neurobehavioural parameters were assessed with the Mann-Whitney U-test.
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality:
not specified
Description (incidence):
Two dams died during the 2nd week of the lactation period. One dam in the 0.015 % group and the other in the 0.045 % group.
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not examined
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not specified
Key result
Remarks on result:
not determinable
Key result
Critical effects observed:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two dams died during the 2nd week of the lactation period. One dam in the 0.015 % group and the other in the 0.045 % group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no consistent or dose-related effect of BHT on litter weight at birth. The body weight of the pups during the lactation period was significantly different from control in some treatment groups; increased at PND 0,4 and 21 in the 0.015 % group, reduced in PND 14 in the 0.135 % group and reduced in the PND 7,14 and 21 in the 0.405 % group.
Food consumption and compound intake (if feeding study):
not examined
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Description (incidence and severity):
There were a few effects on the neurobehavioural parameters during the lactation period, while surface righting at PND 7 in the 0.015 % group was significantly increased. There was no consistent compound or dose-related effect in open field activity at 3 weeks of age, while ambulation of male mice in the 0.045% group was significantly reduced.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not specified
There was no significant effect of BHT on litter size and litter weight at birth. There was no significant effect of BHT on the sex ratio at birth. The body weight of the pups in the lactation periods was significantly increased in the 0.015% group for all measurements periods. The survival index at PND 21 was control = 100%, 0.015 % BHT= 100%, 0.015 % BHT= 99.1 %, 0.135% BHT=99.1 and 0.405 % BHT =99.1 %.
Key result
Dose descriptor:
NOAEL
Effect level:
608 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
other: Neurobehaviour
Key result
Critical effects observed:
no
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Two dams died during the 2nd week of the lactation period. One dam in the 0.015 % group and the other in the 0.045 % group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no consistent or dose-related effect of BHT on litter weight at birth. The body weight of the pups during the lactation period was significantly different from control in some treatment groups; increased at PND 0,4 and 21 in the 0.015 % group, reduced in PND 14 in the 0.135 % group and reduced in the PND 7,14 and 21 in the 0.405 % group.
Food consumption and compound intake (if feeding study):
not specified
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not specified
Description (incidence and severity):
There were a few effects on the neurobehavioural parameters during the lactation period, while surface righting at PND 7 in the 0.015 % group was significantly increased. There was no consistent compound or dose-related effect in open field activity at 3 weeks of age, while ambulation of male mice in the 0.045% group was significantly reduced.
Developmental immunotoxicity:
not examined
There was no biologically significant adverse effect on reproductive and neurobehavioural parameters found in this study. The body weight of the pups was increased in the 0.015% BHT group at birth and during the lactation period for each generation, while there was no consistent effect in other groups.

In the neurobehavioural parameters in the lactation period, a few parameters were increased in treatment groups, while there was no significant consistent compound- or dose-related effect for each generation.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
608 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Neurobehaviour, reproductive performance
Remarks on result:
other: see any other information on results inc. tables
Key result
Critical effects observed:
no
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no consistent or dose-related effect on litter weight at birth. The bodyweight of the pups in the lactation period was significantly increased at the 0.015 % group for all measurement periods.
Food consumption and compound intake (if feeding study):
not specified
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Histopathological findings:
not examined
Other effects:
not specified
Description (incidence and severity):
There was no consistent or dose-related effect of BHT on litter size or sex ratio at birth. The survival index at PND 21 was control= 100 %; 0.015 %BHT=100%; 0.045 % BHT= 99.1%; 0.135 % BHT=99.1 % and 0.405 % BHT=99.1 %.
The incidence of external malformation was observed in two female pups in the 0.015% group: two pups showed eyes opening at birth and were smaller than other pups from the same litter. The animals of external malformation became similar to sparse fur mice in adult.
Behaviour (functional findings):
not specified
Description (incidence and severity):
There were a few effects o the neurobehavioural parameters in the lactation period, while surface righting at PND 4 in the 0.135 % group and negative geotaxis at PND4 in the 0.405 % group were significantly increased. In open field activity at 3 weeks of age, ambulation was significantly reduced in both the 0.045% male and female groups and 180º turn was reduced in all male treatment groups.
Developmental immunotoxicity:
not examined
The animals with external malformation were examined in the reproductive test and the characteristic was confirmed. The character was confirmed as a genetical mutant and recessive character, the same as that reported by Nakano et al. (1967). Although it cannot be asserted that this mutation was caused by the effect of BHT because it might be spontaneous.
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
608 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental neurotoxicity
other: Litter size, litter weight, sex ratio.
Remarks on result:
other:
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

The dose levels of BHT (up to 608 mg/kg-bw/day) in this study produced no adverse effect on reproductive and neurobehavioural parameters.

Conclusions:
The dose levels of BHT in this study produced no adverse effect on reproductive and neurobehavioural parameters.
Executive summary:

In a three-generation study, Crj:CD-1 mice (F0and F1generations) received ca. 0, 23, 68, 203 or 608 mg/kg bw/day BHT in the diet during premating, mating, gestation and lactation periods (ca. 11 weeks). There were no effects on the number of litters, number of pups, litter size, litter weight and sex ratio in any dose group of F1and F2animals or on neurobehavioural parameters in F1and F2 generation. The body weight of pups was increased at the lowest dose at birth and during lactation period for each generation.

Effect on fertility: via oral route
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
chronic
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

Weight of evidence: Experimental results from developmental studies in rats published in scientific articles and read-across approach from an analogue.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
chronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The analogue CAS No. 119-47-1 (6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol) which shares the same functional group (alkylphenol) with the substance CAS no. 128-37-0 (2,6-di-tert-butyl-p-cresol), also has comparable values for the relevant molecular properties. Therefore, the results obtained with the substance CAS No. 119-47-1 can be used for the read-across approach.

Based on published data, the read-across approach is applied from 6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol and taking into account the molecular weights:

BHT: The NOAEL for maternal toxicity is 60.5 mg/kg bw/day and the NOAEL for developmental toxicity was 243 mg/kg bw/day (the highest dose tested).

Justification for classification or non-classification

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