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
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
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
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
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
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.

Endpoint:
toxicity to reproduction
Adequacy of study:
supporting study
Reliability:
other: Statement
Principles of method if other than guideline:
QSAR OECD toolbox in silico calculation
Reproductive effects observed:
not specified
Executive summary:

Potential estrogenic activity:

QSAR OECD toolbox in silico calculation:

Estrogen receptor binding: non binder, impaired OH or NH2 group (Database version: 3.5.0/3.1.2 8)

Mechanistic in vitro studies according to ToxCast/Tox21:

Only 1/12 estrogen receptor agonist related tests is positive indicating no consistent effect.

"ATG_ERa_TRANS_up Active

"OT_ERa_EREGFP_0120 Inactive

"OT_ERa_EREGFP_0480 Inactive

"OT_ER_ERaERa_0480 Inactive

"OT_ER_ERaERa_1440 Inactive

"OT_ER_ERaERb_0480 Inactive

"OT_ER_ERaERb_1440 Inactive

"OT_ER_ERbERb_0480 Inactive

"OT_ER_ERbERb_1440 Inactive

"Tox21_ERa_BLA_Agonist_ratio Inactive

"Tox21_ERa_LUC_BG1_Agonist Inactive

"Tox21_ERa_LUC_BG1_Antagonist Inactive

Negative in “ER Gene (Reporter Gene Assay)” according to EADB dtatbase. Nishihara. 2000. cited in EADB databes (dated 16.2.2015)

No endocrine activity in E-Screen; no transcriptional activation of estrogen receptor (ER); Jobling. 1995. EHP, 103: 582-587.

No endocrine activity in E-Screen; Soto. 1995. EHP, 103 (sup.7): 113-122.

Slight estrogen activity at high concentrations. Ca. 2 fold increase in ER dependent Luciferase activity at 50 and 100 µmol/l; 2-fold increase is slight compared to other compounds (E2 more than 10-fold); no dose response observed; observations at high concentrations not relevant to humans. Overall, artificial test system with limited relevance to human toxicity. Wada. 2004. J Dent Res, 83(3): 222-226.

Potential anti-estrogenic activity:

Mechanistic in vitro studies according to ToxCast/Tox21:

No estrogen receptor antagonistic activity in any test.

"Tox21_ERa_BLA_Antagonist_ratio Inactive

"Tox21_ERa_BLA_Antagonist_viability Inactive

Mechanistic in vivo study:

Weight DECREASE in Uterus + ovaries in uterotrophic assay with premature female rats. The test is not conducted according to actual OECD TG 440; the uterotrophic assay is an “in vivo assay providing data about a single endocrine mechanism, i.e. oestrogenicity.” (OECD TG 440). Since INCREASE in uterus weight is the outcome with estrogenic compounds, the observed decrease with BHT is difficult to interpret. The study does not indicate estrogenic activity and is outside of the applicability domain of the test system. Overall, no conclusion possible on this exploratory study with limited documentation and observations outside the applicability domain of the test. Pop. 2013. Farmacia, 61: 202-211

Conclusion on mechanistic estrogen/anti-estrogen studies: inconclusive data and overall no evidence on any consistent activity.

Potential anti-androgenic activity:

Mechanistic in vitro studies according to ToxCast/Tox21:

No androgen receptor agonist or antagonistic activity in any test.

"ATG_AR_TRANS_up Inactive

"OT_AR_ARELUC_AG_1440 Inactive

"OT_AR_ARSRC1_0480 Inactive

"OT_AR_ARSRC1_0960 Inactive

"Tox21_AR_BLA_Agonist_ratio Inactive

"Tox21_AR_BLA_Antagonist_ratio Inactive

"Tox21_AR_BLA_Antagonist_viability Inactive

"Tox21_AR_LUC_MDAKB2_Agonist Inactive

"Tox21_AR_LUC_MDAKB2_Antagonist Inactive

Mechanistic in vitro studies:

Partial inhibition of testosterone activity in transfected prostate carcinoma cells; EC50: 5.7 µM; inhibition of androgen receptor (AR) promoted luciferase activity at µM concentrations. Exploratory study with very limited documentation and questionable statistical significance (Fig 8 no error bars, control data from separate experiment). Schrader. 2000. Tox Sci, 53: 278-288

Conclusion on mechanistic anti-androgenic study: inconclusive data in a single study with insufficient documentation.

Potential endocrine activity based on apical comprehensive and guideline studies:

Comprehensive and guideline studies are available. No effect on reproductive organs in repeated dose and cancer studies. No consistent effect on fertility. Hyperactivity of the thyroid is frequently reported in repeated dose toxicity studies. This observation is considered secondary as a result of increased liver weight and activity; consequently, these observations are considered to be not correlated to endocrine activity.

Conclusion: the available studies do not indicate any endocrine disruption or endocrine activity.

BHT Evaluations by different committees:

Recent assessments by national and international committees and agencies indicate that BHT has no relevant endocrine activity:

EFSA defined an ADI in 2012. EFSA Journal 2012. 10(3):2588. (http://www.efsa.europa.eu/sites/default/files/scientific_output/files/main_documents/2588.pdf)

German BfR addressed potential endocrine activity of BHT in 2011 and concluded that BHT has no (no or at best very low) endocrine activity. BfR 2011. (http://www.bfr.bund.de/cm/343/bfr_bewertet_untersuchungen_zu_hormonaehnlichen_wirkungen_von_in_natuerlichen_mineralwaessern_vorkommenden_substanzen.pdf )

German MAK commission defined an occupational exposure level (MAK-value). MAK documentation 2004. (http://onlinelibrary.wiley.com/doi/10.1002/3527600418.mb12837e0023/pdf)

OECD SIDS 2002. Siam 14; 2002

ACGIH defined an occupational exposure level (TLV-TWA). ACGIH 2001.

JECFA defined ADI. Report No. 44 (1995)

EFSA 2012: “Overall, the Panel concluded that the present database justifies revision of the SCF ADI of 0-0.05 mg/kg bw/day. Based on the NOAEL of 25 mg/kg bw/day derived from both new 2-generation studies and an uncertainty factor of 100, the Panel established an ADI of 0.25 mg/kg bw for BHT.”

German MAK commission 2004: “In particular the systemic effect on the liver is decisive for deriving the MAK value… If the MAK value of 20 mg/m3 (corresponding to 2.9 mg/kg body weight and day) is observed, prenatal toxicity is not expected to occur. BHT”

Overall, and considering recent reviews by EFSA, MAK and the German BfR, there are no consistent data which indicate that BHT has endocrine activity in vitro or in vivo.

Endpoint:
multi-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable, well-documented publication, which meets basic scientific principles
Principles of method if other than guideline:
other: multigeneration study (F0, F1a, F1b, F2a, F2b)
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
other: BHT contained in the diet
Details on mating procedure:
M/F ratio per cage: 1: 1; Length of cohabitation: 2 oestrous cyles; Proof of pregnancy: daily (copulatory plug or bloody vagina); male was removed after evidence of copulation or after 2 oestrous cycles. If after 2 oestrous cycles, the male failed to copulate, a second male was caged with the female for 2 oestrous cycles; each female was given a maximum of 2 males for 2 oestrous cycles; calculation of mating index and fertility index
Duration of treatment / exposure:
F0 rats were fed their respective diet until 100 days of age (79 days on test) and were maintained on experimental diet for 42 wk; F1b rats were fed the experimental diet until 100 days of age and were maintained on experimental diet for 42 wk
Frequency of treatment:
daily
Details on study schedule:
F0 parental rats were not mated until 100 days of age; after weaning of the F1a generation, the parental females (F0) were given a 10-day rest period and were mated again to obtain the F1b litters. After weaning of this second litter, the parental rats were maintained on experimental diet to permit clinical studies and were sacrificed after 42 wk. 16 females and 8 male (21-day-old) F1b rats were selected randomly to represent the second parental generation. The rats were fed the experimental diet until 100 days of age and mated in the same procedure as described for the F0 rats, to yield 2 litters F2a and F2b. The second litters (F2b) were selected as the parental animals for the third generation (data F3 not shown)
Remarks:
Doses / Concentrations:
0, 300, 1000, 3000 ppm in the diet (about 0, 20, 67, 200 mg/kg body weight and day), adjustment of the fraction of fat in the diet to 20% with lard
Basis:

No. of animals per sex per dose:
16 males and 16 females per dose group
Control animals:
other: yes, plain diet (control I)... (see attached file)
Positive control:
none
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes, BODY WEIGHT: Yes,
Litter observations:
evaluated for physical abnormalities (up to day 21 after birth), number of viable and stillborn members of each litter; survival o at 1 and 5 day after birth
Postmortem examinations (offspring):
GROSS NECROPSY, HISTOPATHOLOGY / ORGAN WEIGTHS
Statistics:
analysis of variance followed by Student's t-test
Reproductive indices:
yes
Offspring viability indices:
yes
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Dose descriptor:
dose level:
Effect level:
3 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on fertility
Reproductive effects observed:
not specified

Parental generation:

Animal behaviour and appearance: no adverse reactions

Body weight gain reduced after 10 wk in male rats, approx. 10% reduction in body weight at 3000 ppm BHT; this effect was equal in both generation, no effect on groths at 1000 ppm

Cholesterol level: at 28 wk, a significant elevation in males and females at 3000 ppm, no effects in the 300, 1000 ppm groups

Clinical studies. no effects

Mortality: no effects

Organ weights: enlargement of the liver in males and females of the 3000 ppm BHT group (approx. 20 % increase rel. liver weight)

Gross and microscopic pathology: both gross and microscopic pathological studies revealed no tissue changes which could be attributed to ingestion of BHT for 42 wk by either generation

Mating performance: no interference with desire or ability to copulate from any level of BHT

Fertility index: no effect on fertility or ability to conceive

Parturition index: no effect on sustaining the pregnancies to parturition

Gestation time: no effects

Lactation index: no interference with lactation and nursing of the offspring

Offspring:

Body weight: reduced at 3000 ppm feeding level ; no effect on weaning weight was detected at 300 and 1000 ppm BHT

Mortality: no effect

Lactation index: indicated normal survival until weaning on day 21

Teratogenic effects: no effect

Executive summary:

In a multigeneration study with Sprague Dawley rats, fed 0, 300, 1000, 3000 ppm BHT (about 0, 20, 67, 200 mg/kg bw and day) in the diet, no effects on reproductive parameters were found. Data are reported on two parent generations and two litters of offspring for each generation. No teratogenic effects have been observed in any of the 4 litters at any feeding level. The only significant effects observed were a reduction in growth rate of parents and offspring, an elevation of serum cholesterol and an increase in the relative liver weight of parents at 3000 ppm feeding level (Frawley 1965).

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable, well-documented publication, which meets basic scientific principles
Principles of method if other than guideline:
other: one-generation study
GLP compliance:
not specified
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
other: BHT contained in diet
Details on mating procedure:
M/F ratio per cage: 1: 1; animals were mated over a 14-day period
Duration of treatment / exposure:
F0 begin of treatment 13 weeks before treatment, BHT was feed throughout the entire study;
examination of the offspring up to the age of 21 days
Frequency of treatment:
daily
Details on study schedule:
when 19 weeks old, the animals were mated over a 14 day period; after completed mating the F0 males were omitted from the study; the presence of plug was checked every evening and morning; 24-hours after birth the litter size was reduced to 8, and equal sex distribution was achieved
Remarks:
Doses / Concentrations:
0, 500 mg/kg bw and day
Basis:

No. of animals per sex per dose:
F0: 46 males and females per dose group
Control animals:
yes, plain diet
Dose descriptor:
dose level:
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant differences in duration of pregnancy and litter size compared to control
Reproductive effects observed:
not specified

F0 generation 500 mg/kg bw dose group: reduced body weight compared to control; no significant differences in duration of pregnancy (22.6 days vs. control, 22.9 days) and litter size (10.0 vs. control 9.8) compared to control.

F1 generation 500 mg/kg bw dose group: body weight gain reduced during lactation; delayed development (e.g. eruption of theeth, opening of eyes and reflexes) during lactation

Executive summary:

In Wistar rats treaded with BHT (500 mg/kg body weight and day), no significant differences in duration of pregnancy (22.6 days vs. control, 22.9 days) and litter size (10.0 vs. control 9.8) were found compared to control (Meyer 1980).

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable documented publication, which meets basic sientific principles.
Principles of method if other than guideline:
other: one-generation study
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
other: BHT contained in diet
Duration of treatment / exposure:
starting 14 days before mating, during breeding, the period of gestation, and the period of lactation (21 days); after weaning dietary exposure of the offspring to BHT continued throughout the remainder of the experiment (up to day 90)
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 0.125%, 0.25%, 0.5% BHT in the diet (about 83, 167, 333 mg/kg bw and day)
Basis:

No. of animals per sex per dose:
at least 13 females
Control animals:
yes, plain diet
Dose descriptor:
dose level:
Effect level:
ca. 333 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reduced litter size, and no effects on the lenght of gestation were found in treated animals compared to the negative control
Reproductive effects observed:
not specified

No reduced litter size, and no effects on the lenght of gestation were found in treated animals compared to the negative control.

From 167 mg/kg body weight (F1): increased postnatal mortality (postnatal days 1 -30)

333 mg/kg bw (F1): reduced body weight gain; delayed development before weaning (180° rotation; opening of the eyes, swimming behaviour; behaviour in the open field); no effect after weaning

Executive summary:

No reduced litter size, and no effects on the lenght of gestation were found in treated animals compared to the negative control (Vorhees 1981).

Endpoint:
toxicity to reproduction
Remarks:
other: Sperm Abnormality Test
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: not reliable due to the application mode (i.p. in mouse) and other methodical deficiencies
Principles of method if other than guideline:
Method: other: Sperm Abnormality Test
GLP compliance:
not specified
Species:
mouse
Strain:
other: hybrid mouse, genotype (C57BL/6 x C3H/He)
Sex:
male
Route of administration:
intraperitoneal
Duration of treatment / exposure:
5 days (daily injections)
Frequency of treatment:
5 days (daily injections)
Remarks:
Doses / Concentrations:
125, 250, 500 or 1000 mg/kg bw day
Basis:

No. of animals per sex per dose:
4 per dose group
Reproductive effects observed:
not specified
Low non-dose related incidence up to 3.5% (controls up to
1%) of abnormal spermatozoa (maximum effect at 125 mg/kg bw day). study with methodical deficiencies, MAK (2004) discussed the study as not reliable

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: non-GLP study, study data do not give sufficient experimental details
Principles of method if other than guideline:
other: multi mating of the F0 generation for 18 months
GLP compliance:
not specified
Species:
mouse
Strain:
other: outbred albino strain, eyeless mice were from ananophthamic strain obtained from the Genetic Department, University of Adelaide
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
other: BHT contained in the diet together with 10% and 20% lard
Duration of treatment / exposure:
F0 generation for 18 months
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 0.1, 0.5% BHT in the diet (about 0, 143, 714 mg/kg body weight and day
Basis:
nominal in diet
No. of animals per sex per dose:
outbred albino and strain with anophthalmia: 144 females total
Control animals:
yes, plain diet
Reproductive effects observed:
not specified

Dietary BHT (0.1% to 0.5%) did not cause anophthalmia in the offspring of albino mice. In the high-dose group, the lengh of gestation was increased and mean pup weight, mean total weight of the litters, and the mean number of live pups were decreased compared to controls. At a concentration of 0.1%, BHT had no adverse effects on the above parameter.

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: non-GLP study, study data do not give sufficient experimental details (e.g. number of animals per dose group not indicated)
Principles of method if other than guideline:
other: one generation study with emphasis of behavioral tests (F1 generation)
GLP compliance:
not specified
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
other: BHT contained in diet
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 0.5% in the diet (about 714 mg/kg body weight and day)
Basis:

No. of animals per sex per dose:
males and females, no other data
Control animals:
yes, plain diet
Reproductive effects observed:
not specified

Dose group: 714 mg/kg body weight, F1 mice:

effects on behaviour (sleep, agression) and learning, learning deficits; no data on prenatal toxicity parameter or maternal toxicity

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: study data do not give sufficient experimental details
Principles of method if other than guideline:
other: one-generation study
GLP compliance:
not specified
Species:
mouse
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
other: BHT contained in the diet
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0.875, 1.75, 2.5% BHT in diet
Basis:

Reproductive effects observed:
not specified

A significant adverse effect on body weight in both F0 and F1 animals was shown in all dose groups. Dose-dependent differences in the evaluated parameter were found in the offspring of BHT exposed adults. Significant differences in litter size, pup weight, and litter weight were particularly evident.

Endpoint:
reproductive toxicity, other
Remarks:
“Statement on potential endocrine disruptor activity of 2,6-di-tert-butyl-p-cresol (BHT; CAS 128-37-0)” prepared as a response to the initial ground for concern reported by ANSES
Type of information:
other: Review
Adequacy of study:
supporting study
Study period:
2016
Reliability:
other: Review
Principles of method if other than guideline:
Terms of Reference
This analysis is based on uncertainty expressed by ANSES on potential endocrine disruptive activity of BHT in the following documents and during a meeting in Paris held on July 19:
a) “Justification Document for the Selection of a CoRAP Substance”, dated 22.03.2016 and
b) “Analysis of the most appropriate risk management option (ROMA)” dated March 2016.

Contents of the statement:

Executive Summary

Terms of Reference

Initial Ground for Concern Expressed by ANSES

Similar Substances / Grouping Possibilities

Endocrine Disruptor and Reprotoxic Effects

Thyroid Gland

OECD Conceptual Framework

EFSA and MAK Concluded in Recent Reviews that Thyroid Function is Affected in Rodents as a Secondary Consequence of Enzyme Induction in the Liver.

Application of the Draft EU Commission Criteria for Endocrine Disruptors

Human Relevance

Adrenal Gland

References (see attached whole statement)

Annex 1: In vivo Studies on BHT within the OECD Conceptual Framework for Testing and Assessment of Endocrine Disruptors (see attached whole statement)

Figures: (see attached whole statement)

.

Terms of Reference

This analysis is based on uncertainty expressed by ANSES on potential endocrine disruptive activity of BHT in the following documents and during a meeting in Paris held on July 19:

a) “Justification Document for the Selection of a CoRAP Substance”, dated 22.03.2016 and

b) “Analysis of the most appropriate risk management option (ROMA)” dated March 2016.

Initial Ground for Concern Expressed by ANSES:

Similar Substances / Grouping Possibilities

The ANSES Justification (2016a) and Analysis (2016b) documents mention in different chapters butylhydroxyanisole (BHA) as a chemically similar compound which is taken into account as an initial reason for concern on BHT.

Figure: see whole statement attached to the IUCLID file.

Based on chemical and toxicological considerations a read across between the two substances is not appropriate. The phenol hydroxyl group is sterically hindered in BHT. According to the OECD Toolbox the chemical reactivity and profiling is different for both substances; e.g. BHT is not considered as an Estrogen Receptor Binder based on “impaired OH or NH2 group” (Schlecker 2016a), whereas BHA is allocated as a “moderate binder, OH group” (Schlecker 2016b).

There are comprehensive evaluations for both compounds available indicating that absorption and metabolism are different (e.g. BHA is metabolised via oxidative O-demethylation, whereas BHT is oxidized on the tert-butyl groups in man) and both compounds differ with respect to toxicologically relevant observations and target organs (EFSA 2011, 2012).

Based on the substantial differences between the two substances I do concur with ANSES conclusion that BHT share common uses with BHA “but the cross-reading with the BHA is not justified” (ANSES Justification. 2016).

Endocrine Disruptor and Reprotoxic Effects

The following information is cited from the ANSES Justification document in the chapter “Endocrine disruptor and reprotoxic effects”:

• “Study on Enhancing the Endocrine Disrupter Priority List with a Focus on Low Production Volume Chemicals, Revised Report to DG Environment (Hersholm, Danemark : DHI Water and Environment, 2007), http://ec.europa.eu/environment/endocrine/documents/final_report_2007.pdf.The european commission on endocrine disruptor listed BHA as a priority substance Cat 1 based on the fact of interfering with hormonal function.” (ANSES 2016a)

In this list in total 575 chemical substances were screened and evaluated as to their potential endocrine disrupting (ED) effects and a preliminary priority list was established at the end of 2006. BHT is not included in the list but BHA. As mentioned above based on substantial differences between the two substances I do concur with ANSES conclusion that cross-reading between BHT and BHA is not justified.

• “PNUE and OCDE, 2,6-di-tert-butyl-p-cresol (BHT) Screening Information Data Set: Initial Assessment Report (Paris: PNUE, 2002), http://www.inchem.org/documents/sids/sids/128370.pdf. A long-term exposure to high doses of BHT is toxic to mice and rats, causing problems to thyroid and other organs (the liver, the kidney, the lungs and the blood)” (ANSES 2016a)

The cited OECD SIDS documentation did not identify thyroid as the primary target of BHT and defined observations on liver and thyroid as the base for the NOAEL. Doses above 25 mg/kg bw/day BHT resulted in enlargement of liver, induction of several liver enzymes and thyroid hyperactivity (OECD SIDS 2002).

The recent EFSA evaluation on BHT came to a similar conclusion and recognized that the observations on thyroid hyperactivity are secondary effects caused by the liver enzyme-inducing properties of BHT. “The demonstrated effects on hepatic enzyme induction and consequent thyroid hyperactivity in the mid- and high-dose groups, together with the tumour data from the Olsen study, suggested a NOAEL of 25 mg/kg bw/day.” (EFSA 2012).

The thyroid hyperactivity observed in rats is considered as an indirect effect and a result of liver enzyme induction, consequently, not relevant for identification of potential endocrine disruptors according to the criteria proposed by the EU-Commission (2016): “The criteria also underline that endocrine-related adverse effects which are only indirectly triggered by a non-endocrine-related toxicity are not adverse effects that are relevant for the identification of a substance as an endocrine disruptor”. See chapter on thyroid below for a detailed discussion.

In addition, the thyroid observations in rats after exposure to BHT are considered as limited to not relevant for humans (Falcó 2016 and see chapter on human relevance below for a detailed discussion). Which is in agreement with the EFSA conclusion: “The hyperactivity of the thyroid caused by substances with liver enzyme-inducing properties is more or less a reaction specific to rodents since rats, as compared with humans, are especially susceptible to a disturbance of the thyroid-pituitary regulation caused by an increased metabolization of thyroxine (T4) in the liver.” (EFSA 2012).

• “TEDX list of potential Endocrine Disruptor : Hughes et al 2000. Estrogenic alkylphenols induce cell death by inhibiting testis endoplasmic reticulum Ca(2+) pumps, Biochem Biophys Res Commun. 2000 Nov 2;277(3):568-74.” (ANSES 2016a)

The Endocrine Disruption Exchange (TEDX) is a United States environmental organization that purports to list chemicals with the potential to affect the endocrine system. The TEDX website states that “endocrine effects include not only direct effects on traditional endocrine glands, their hormones and receptors (such as estrogens, anti-androgens, and thyroid hormones), but also all other hormones and signaling cascades that affect the body’s systems and processes, including reproductive function and fetal development, the nervous system and behavior, the immune and metabolic systems, gene expression, the liver, bones, and many other organs, glands and tissues.” The TEDX database presents the chemicals for which at least one peer-reviewed study that reports findings on one or more of the above has been published.

The TEDX website does not provide a definition for endocrine disruptors and based on the information available it is obvious that the inclusion of a compound in the list differs from the approach used by the WHO or EU-Commission (2016) to identify endocrine disruptors. In this respect, there are some obvious and fundamental limitations in the database since observations on e.g. “.., gene expression, the liver, bones, and many other organs, glands and tissues.” result in inclusion of a compound in the list.

One study by Hughes et al (2000) is cited as relevant for the listing of BHT. The authors investigated in vitro the activity of different compounds on non-muscle Ca2+ pump (SERCA Ca2+ ATPase), one of the most abundant Ca2+ transporters in eukaryotic cells which removes cytoplasmic Ca2+ from cells. The authors used homogenates from whole testis for their investigation and reported IC50 values for inhibition of Ca2+ ATPase (0.6 µM) and Ca2+ Uptake (13 µM). The authors did not discuss this observation as a direct endocrine related observation but used testes homogenates because “SERCA 2 and 3 Ca2+ pumps are relatively abundant in rat testis microsomal membranes” (Hughes et al 2000). Taking into account that the observations were reported at very high concentrations, not relevant for the in vivo human situation, and no effect on testes are reported in comprehensive in vivo studies these data are considered to be not relevant for human risk assessment or for the identification of potential endocrine disruptors according to WHO or EU-Commission criteria.

• “Some limited data suggest that high doses of BHT can simulate estrogen (Wada, H. et al., 20041), sexual primary female hormone, as well as preventing the expression of male sex hormones, which would result in adverse effects on reproduction (Schrader, TJ et GM Cooke, 20002).” (ANSES 2016a)

The in vitro study by Wada et al. (2004) investigated endocrine-receptor dependent activity in a luciferase reporter-gene assay in human 293T cells. The authors reported weak estrogen like activity (ca. 2 fold increase in luciferase activity reported for BHT and >10-fold increase for the positive control 17β-estradiol). These slight observations were reported without dose response at very high concentrations (50 and 100 µM), and are thus not relevant for the in vivo human situation.

Figure: see whole statement attached to the IUCLID file.

Figure 2c copied from Wada et al 2004.

A literature and database search revealed additional exploratory information which demonstrates no estrogenic activity of BHT.

No estrogen-like activities were observed in two independent E-screen studies in which cell growth of estrogen receptor (ER) positive MCF 7 was investigated (Jobling et al. 1995, Soto et al. 1995).

BHT data are available in the US EPA’s Aggregated Computational Toxicology Online Resource (ACToR 2016). Data included in the database come from a) Rapid, automated (or in vitro high-throughput) chemical screening data generated by the EPA’s Toxicity Forecaster (ToxCast) project and the federal Toxicity Testing in the 21st century (Tox21) collaboration; b) Chemical exposure data and prediction models (ExpoCastDB); c) High quality chemical structures and annotations (DSSTox); c) Physchem Properties Database (PhysChemDB).

BHT is investigated in 15 ER-related in vitro tests. BHT is considered inactive in 14 tests and only one test which investigated ER-dependent m-RNA expression in recombinant HepG2 cells showed some activity at a BHT concentration of 21 µM; a concentration close to the limit concentration to define inactivity (≥ 50µM). Overall, the ToxCast Model Predictions indicate “0” (inactive) for “ER-antagonist” and “ER-agonist” (ACToR 2016).

Figure: see whole statement attached to the IUCLID file.

Copied from the ACToR database dashboard (16.09.2016)

(https://actor.epa.gov/edsp21/)

In addition, as mentioned above, a QSAR OECD toolbox in silico calculation was performed for BHT (Schlecker 2016a). The toolbox indicated “non binder, impaired OH or NH2 group” for the endpoint “Estrogen receptor binding”.

Overall, taking into account all available data it can be concluded that BHT has no estrogenic or anti-estrogenic activity. Slight observations are reported in two tests without dose response at unrealistically high concentration. These data are not consistent with the majority of tests that report no activity on estrogen-related endpoints.

The second study mentioned by ANSES investigated potential androgenic activity. Schrader and Cooke (2000) investigated androgen-receptor dependent activity in a luciferase reporter-gene assay in PC-3 LUCAR1 cells in the presence and absence of 5α-Dihydrotestosterone (DHT). Androgen Receptor Activation was seen for BHT with an EC50 value of 5.7 µM. Whereas BHT was inactive in in the same test in the absence of DHT. This observation at very high concentrations is considered to be not relevant for the human in vivo situation and should not be considered as reliable information to identify endocrine disruptors according to the WHO or EU-Commission criteria.

BHT was investigated in 8 test systems for androgen-related endpoints in the above mentioned US EPA ACToR database. No androgen receptor agonist or antagonistic activity is reported in any test. The ToxCast Model Predictions indicate “0” (inactive) for “AR-antagonist” and “AR-agonist” (ACToR 2016).

Figure: see whole statement attached to the IUCLID file.

Copied from the ACToR database dashboard (16.09.2016)

(https://actor.epa.gov/edsp21/)

Overall, taking into account all available data it can be concluded that BHT has no androgenic or anti-androgenic activity. The observation reported by Schrader and Cooke (2000) was observed at an unrealistically high concentration and is not consistent with other in vitro test reported in the US EPA ACToR database.

Thyroid Gland

In the Justification document ANSES (2016a) identified the thyroid as the main organ of concern in the ED context “Preliminary indication of information that may need to be requested to clarify the concern… There is a concern for thyroid effects. Induction of hepatic enzyme (CYP 450 cytochrome) has been shown in rodent. It has been proposed (but not proven) that this enzymatic induction would lead to increased thyroïd hormons catabolism. An effect on thyroid physiology in male Wistar rats including an acceleration of iodine cycle is proven. Therefore, the relevance to human of the thyroïd effects needs to be further evaluated.” (ANSES 2016a).

According to the OECD document 150 “Guidance Document on Standardised test Guidances for evaluating Chemicals for Endocrine disruption” (2012) all available data should be considered during an evaluation. The conceptual framework consists of 5 levels:

Level 1: Existing Data and Non-Test Information

Level 2: In vitro assays providing data about selected endocrine mechanism(s) / pathways(s) (Mammalian and non mammalian methods)

Level 3: In vivo assays providing data about selected endocrine mechanism(s) / pathway(s)1

Level 4: In vivo assays providing data on adverse effects on endocrine relevant endpoints 2 Level 5: In vivo assays providing more comprehensive data on adverse effects on endocrine relevant endpoints over more extensive parts of the life cycle of the organism

OECD Conceptual Framework

Level 2 in vitro Studies

BHT was investigated for thyroid-related endpoints in three mechanistic in vitro tests in the above mentioned US EPA ACToR database:

Figure: see whole statement attached to the IUCLID file.

Copied from the ACToR database dashboard (16.09.2016)

(https://actor.epa.gov/edsp21/)

Only one test was positive which investigated GH3 rat pituitary cells transformed with Thyroid Responsive Element in front of a luciferase gene. Compound dependent loss of luciferase was investigated in this antagonist test. The reported AC50 value of 41.4 µM was based on an effect at a single high concentration datapoint, very close to the limit concentration of 50 µM to distinguish between active and inactive compounds. Additional, detailed information on the BHT data in this test (see Figure below) indicate that loss of activity was observed at a concentration substantially above the cytotoxicity limit of 10 µM defined for this test. Since the read out is a loss of activity, it might be assumed that the read out might be due to cytotoxic effects of BHT at the highest concentration investigated.

Figure: see whole statement attached to the IUCLID file.

Copied from the ACToR database dashboard (16.09.2016)

(https://actor.epa.gov/edsp21/)

Overall, based on mechanistic in vitro studies there is no clear evidence that BHT has any direct activity on the thyroid receptor.

Level 4 and 5 in vivo Studies

There are multiple in vivo studies available that investigate BHT toxicity in rats and mice with studies ranging from sub-chronic toxicity studies to multi-generation reproduction studies. EFSA evaluated these studies and concluded that the observed thyroid effects in rodents are secondary based on the liver enzyme induction (EFSA 2012; bold added retrospectively by the author of this statement).

“The demonstrated effects on hepatic enzyme induction and consequent thyroid hyperactivity in the mid- and high-dose groups, together with the tumour data from the Olsen study, suggested a NOAEL of 25 mg/kg bw/day.”

“The hyperactivity of the thyroid caused by substances with liver enzyme-inducing properties is more or less a reaction specific to rodents since rats, as compared with humans, are especially susceptible to a disturbance of the thyroid-pituitary regulation caused by an increased metabolization of thyroxine (T4) in the liver.”

ANSES indicated in their justification (ANSES 2016a) that the mechanism defined by EFSA, liver enzyme induction by BHT and consequently thyroid hyperactivity, has “been proposed (but not proven)”.

In order to gain insight in the robustness of the database which supports the mechanism defined by EFSA and other expert committees the following chapters will discuss concurrent liver and thyroid effects in individual in vivo studies which reported data on thyroid gland.

BHT was investigated in different generation reproduction toxicity studies (see Annex 1 for tabulated information).

In an early generation study with limited documentation Frawley et al. (1965) dosed F0, F1a, F1b, F2a and F2b animals with 20, 76 and 200 mg/kg/day in groups of 16 male and female rats/dose. In parents and offspring body weight gain was reduced and liver weight and serum cholesterol increased at 67 mg/kg/day. No effect on thyroid weight or histopathology was reported in this study.

In another generation study by Olsen et al. (1986), groups of 40-60 male and female rats were fed a semi-synthetic diet containing BHT (approx. 0, 25, 100 and 250 mg/kg-bw/day). The study was terminated when offspring were 144 weeks of age. Histopathologic examinations indicated dose-related increases in the numbers of hepatocellular carcinomas in male rats and an increase in hepatocellular adenomas in both male and female rats. No effect on thyroid was reported in this study.

A follow up study by Prince (1994) investigated in more detail hepatic changes in the development of hepatocellular carcinomas. The dosing regimen of the study and the strain of rat used were similar to those in the generation study described by Olsen et al. (1986). F1 animals were dosed for up to 22 months; with interim sacrifice at day 20 of gestation, post natal day (PND) 20, post natal week (PNW) 4, and post natal months (PNM) 6, 11, 16 and 22.

Evidence of thyroid hyper-activity, characterized by reduction of follicular size, absence or reduction of colloid, irregularities in the follicular outline, hyperaemia and increase in the number of follicular cells was noted starting at 11 months at 100 mg/kg/day (mild changes affecting 75-82% of the rats) and at 250 mg/kg/day (marked changes affecting 100% of the rats). Serum thyroxin levels in treated rats did not differ from controls. There were no thyroid related observations in the lowest dose group, 25 mg/kg/day.

Liver effects were observed at all dose levels in this study, starting at the lowest dose of 25 mg/kg/day. At 22 months, there was a higher incidence of eosinophilic and basophilic foci in the high-dose group and there was also a significant increase in the number of rats with hepatic nodules in the high-dose group (6/19 animals compared with none in the other groups). Dose dependent increase in liver weight was observed at all time points with statistical significance mainly at 250 mg/kg/day. Starting at the lowest dose, a dose-related incidence of enlargement and eosinophilia of the centrilobular hepatocytes was observed at the scheduled sacrifices, starting at 6 months. Liver enzyme induction was investigated in detail with the following results:

- Eethoxyresorufin-O-deethylase activity (CYP1A) was statistically increased on PND 20 in all dose groups and on PNW 4 in the 100 and 250 mg/kg/day dose groups. The increase at termination was not statistically significant.

- Pentoxyresorufin-O-depentylase activity (PROD CYP2B) was dose-dependent increased in all dose-groups with statistical significance at 100 and 250 mg/kg/day at all time points. The increases in PROD activity were large, 10-25 fold in the mid-dose, and 20-80 fold in the high-dose groups.

- Epoxide hydrolase activity was increased at all dose groups at all time points with statistical significance starting for some time points in the lowest dose tested.

- Glutathione-S-transferase activity was increase at PND20 at 250 mg/kg/day and later time points at 100 and 250 mg/kg/day.

The data on this study were summarized in a comprehensive review on BHT by the German expert committee “Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area” (MAK Commission, 2012) in a table as follows:

Figure: see whole statement attached to the IUCLID file.

Copied from MAK Commission (2012)

Overall, the comprehensive generation study by Prince (1994) reveals liver weight increase and liver enzyme induction as the primary effect of BHT. Thyroid hyper-activity is observed only at doses where liver weight and multiple liver enzymes functions were substantially increased.

In addition there are repeated dose toxicity studies available which report data on thyroid and liver (see Annex 1 for tabulated information on studies)

Søndergaard and Olsen (1982) reported a 90 day subchronic study. In this study male rats were fed BHT in the diet with doses of approx. 25 and 250 mg/kg/day. Liver and thyroid weights were increased at termination in the high dose group and thyroid weights were also increased at the lower dose level. BHT did not change T3 and T4 blood level at both dose levels. The authors investigated also thyroid weight, but not liver weight, after BHT dosing with 250 mg/kg/day for 30 days in different diets and reported an increase in thyroid weight for all diets used. In addition the iodine uptake was investigated for the control and high dose (250 mg/kg/day) group, but not for the lower dose group, after dosing for 8, 26, 62 or 90 days. A marked increase in the uptake of 125I was observed at all time periods. The authors investigated the biological half-life of thyroxin after 13 and 75 days treatment in the high dose group only, 250 mg/kg/day, and reported an increase for the earlier time point but not for the later time point.

MAK Commission (2012) concluded on the observed increase in relative thyroid weight in this study at 25 mg/kg/day as follows: “In a 90-day study in Wistar rats, increased relative thyroid weights were reported even at about 25 mg BHT/kg body weight and day (500 ppm in the diet) (Søndergaard and Olsen 1982; see Table 3); they were however not confirmed at this low dose level in the other studies carried out with Wistar rats (CEFIC-EBMA 1994; Hirose et al. 1981; Olsen et al. 1986). The findings of Søndergaard and Olsen (1982) are therefore not used for the present assessment.” (MAK Commission 2012).

Taken together, this mechanistic study investigated thyroid related effects as T3 and T4 blood level, iodine uptake and half-life at 250 mg/kg/day. Since at this dose marked concurrent relative liver weight increase was reported (control: 2.8 [unit not given]; 250 mg/kg/day group: 4.3; 54% increase in relative weight), this study is in agreement with the EFSA conclusion that thyroid effects in rodents are secondary as a consequence of liver enzyme induction.

Based on this study ANSES indicated in the CoRAP justification (ANSES 2016a) “An effect on thyroid physiology in male Wistar rats including an acceleration of iodine cycle is proven.” (Bold added retrospectively by the author of this statement).

Søndergaard and Olsen (1982) investigated the half-life according to “Materials and Methods” by collecting blood samples of the high dose group after 16, 20, 40, 44 and 60 h and report the following limited information in the result section:

“(D) Biological half life of thyroxine

The biological half life of thyroxine was increased (P < 0.01) after 13 days (controls 15.6 ± 3.2 h, dosed group 21.5 ± 6.5 h) but not after 75 days of treatment. The average half life was approx.. 18 h.” Søndergaard and Olsen (1982).

This study has severe limitations. The time points investigated are not considered appropriate to derive a half-life around 18 h, no kinetic data are given to judge the kinetic curve or slope over time and the standard deviation of the dosed group covers the control and average half-lifes. The overall data in this study are not consistent, since thyroid weight is increased, no change in T3 and T4 blood level are observed but an increased thyroxine half-life was reported and the authors themselves concluded in the discussion section: “This observed delayed metabolism cannot, however, explain the thyroid changes induced by BHT. The toxicological importance of this finding is disputable”. Søndergaard and Olsen (1982). In addition the observation is reported only for the high dose group with severe liver induction.

Overall, based on the inappropriate study design and insufficient documentation the increase in thyroxine half-life reported in this study should be considered as unreliable and not as proven evidence that BHT accelerates iodine cycle.

In addition, early and limited repeated dose toxicity studies indicate no effect on the thyroid.

Hirose et al (1981) treated 36-57 rats/sex/dose with 125 and 500 mg/kg/day BHT for 104 weeks. Body weight gain was reduced and liver weight was increased in both dose groups. No morphological changes were reported in the liver. No effect on thyroid was reported in this study.

In another study by Hiraga et al (1978) 5-15 rats were dosed for 104 week with 2.5, 10 or 160 mg/kg/day. Mortality in females and liver weight increase was reported in the high dose group. No consistent or dose dependent effect on thyroid weight or histopathology was reported.

An early limited study reported an NOAEL of 75 mg/kg/day based on reduced body weight and increased liver weight after exposure of rats to 7.5, 23, 75 225 and 450 mg/kg/day for 76 weeks. No effect on thyroid tumor induction mentioned; not clear if other thyroid parameters were measured in this study (Williams et al. 1990).

A chronic 105 week feeding study (NCI 1979) dosed 20-50 rats via feed with 225 or 450 mg/kg/day and reported thyroid c-cell hyperplasia but no tumor induction. The authors did not report liver weight data. C-(parafollicular) cells are concerned with the production of calcitonin which influences blood levels of calcium and phosphorus, and bone cell metabolism, whereas the thyroid follicular cells secrete the metabolically active iodothyronines. Nodular and/or diffuse hyperplasia of C-cells is frequenly observed with advancing age in rats and is considered to be a response to long-term hypercalcemia and not directly related to thyroid hormones (Carpen and Martin. 1989). In the same study (NCI 1979) 20-50 mice were also dosed via diet (approx. 450 and 900 mg/kg/day) for 108 weeks. No effect was reported on thyroid up to the high dose tested and reduced body weight gain, liver proliferation and necrosis in males at both doses.

Overall assessment according to the OECD conceptual framework and taking into account the available in vitro and in vivo data it is conclusive to conclude that BHT has no direct effect on the thyroid and the reported thyroid hyperactivity in rodents is a consequence of hepatic enzyme induction.

EFSA and MAK Concluded in Recent Reviews that Thyroid Function is Affected in Rodents as a Secondary Consequence of Enzyme Induction in the Liver.

As mentioned above this conclusion is consistent with EFSA (2012):

“The demonstrated effects on hepatic enzyme induction and consequent thyroid hyperactivity in the mid- and high-dose groups, together with the tumour data from the Olsen study, suggested a NOAEL of 25 mg/kg bw/day.” (Bold added retrospectively by the author of this statement)

This conclusion is also in consistent with the MAK expert committee conclusion on BHT (MAK Commission 2012):

“As a consequence of the enzyme induction in the liver, the thyroid function in rats is affected from about 100 mg/kg body weight in the form of hyperactivity (CEFIC-EBMA 1994; see Table 4). In a 90-day study in Wistar rats, increased relative thyroid weights were reported even at about 25 mg BHT/kg body weight and day (500 ppm in the diet) (Søndergaard and Olsen 1982; see Table 3); they were however not confirmed at this low dose level in the other studies carried out with Wistar rats (CEFIC-EBMA 1994; Hirose et al. 1981; Olsen et al. 1986). The findings of Søndergaard and Olsen (1982) are therefore not used for the present assessment. The hyperactivity of the thyroid caused by substances with liver enzyme-inducing properties is more or less a reaction specific to rodents since rats, as compared with humans, are especially susceptible to a disturbance of the thyroid-pituitary regulation caused by an increased metabolization of thyroxine (T4) in the liver.” (Bold added retrospectively by the author of this statement)

Application of the Draft EU Commission Criteria for Endocrine Disruptors

The EU-Commission documentation on the proposed scientific criteria for endocrine disruptors (2016) gives some guidance how to identify endocrine disruptors:

- Indirectly triggered endocrine-related adverse effects should not be considered relevant for the identification of endocrine disruptors:

“The criteria also underline that endocrine-related adverse effects which are only indirectly triggered by a non-endocrine-related toxicity are not adverse effects that are relevant for the identification of a substance as an endocrine disruptor. This clarification is necessary since, as the result of any generalized toxicity, there may be reactions of the endocrine system which would be the consequence rather than the cause of the specific adverse effect observed.” (EU-Commission. 2016; bold added retrospectively by the author of this statement)

- Reasonable evidence ("biological plausibility") should be considered to determine causality.

As mentioned above ANSES indicated in their justification (ANSES 2016a) that the mechanism defined by EFSA, liver enzyme induction by BHT and consequently thyroid hyperactivity is recognized but indicated further: “Although there is some evidence suggesting that BHT might act on thyroid homeostatis through increased TH hepatic catabolism, in the current knowledge, there is no direct proof that this mechanism is true” (bold added retrospectively by the author of this statement) and states that this mechanism has “been proposed (but not proven)”.

The EU-Commission documentation on the proposed scientific criteria for endocrine disruptors (2016) indicates in the chapter “How to determine causality”:

The 2002 definition has at its heart the link between the mode of action and the adverse effect, (the phrase "and consequently" in the definition). The question remains about the extent to which this link should be clearly established – the degree to which a strict causality should be required. In 2013, the European Food Safety Authority concluded that there has to be "a reasonable evidence base for a biologically plausible causal relationship between the [endocrine mode of action] and the adverse effects seen in intact organism studies", i.e. a "reasonable evidence base" to determine causality. The alternative would have been a more rigid approach to causality (asking, for example, for "conclusive" evidence of the connection). The Commission considers that in practice, it will be very difficult to demonstrate "conclusive evidence" of causality. Therefore, the Commission intends to follow a concept of a reasonable evidence ("biological plausibility") to determine causality.” EU-Commission (2016, bold added retrospectively by the author of this statement)

Overall conclusion: BHT should not be considered as an endocrine disruptor based on the experimental data discussed above and the comprehensive evaluations by international (EFSA 2012) and national (MAK Commission 2012) expert committee there is at least reasonable evidence ("biological plausibility") to determine causality between hepatic enzyme induction and consequent thyroid hyperactivity.

Human Relevance

Experimental data are available in monkey which indicates that the liver is the most sensitive organ. The data on monkeys are summarized by the MAK Commission as follows:

“The liver was the most sensitive organ in primates. Groups of two or three young rhesus monkeys were orally administered 0, 50 and 500 mg BHT/kg body weight in corn oil daily for 28 days. The relative liver weights were not altered in either dose group. In the second week, the activity of nitroanisol demethylase in the liver was slightly, but not significantly (6.6 ± 0.4 μmol/hour and mg protein) increased versus the control (5.0 ± 0.1 μmol/hour and mg protein) in the two animals of the low exposure group. The levels no longer differed in the fourth week. When 500 mg/kg body weight and day was given, the hepatocytes showed a moderate proliferation of the endoplasmic reticulum, a slight increase of lipid droplets in the cytoplasm and a fragmentation of the nucleus in some cells. The nitroanisol demethylase activity, which was determined as a parameter of an enzyme induction in the liver, was slightly increased at the higher dosage and the glucose-6-phosphatase activity was reduced at the higher dosage (Allen and Engblom 1972). The cholesterol concentration was significantly reduced in the plasma of both exposure groups (Branen et al. 1973). Because of the high variability of the cholesterol values and the small number of animals used, the validity of this finding is questionable. The histopathological examinations of all other organs revealed no indications of effects. A NOAEL of 50 mg/kg body weight and day was specified (Allen and Engblom 1972; Table 3). The relevance of the slight changes observed at 50 mg/kg body weight is questionable especially as such a small number of animals was used.” (MAK Commission. 2012, bold added retrospectively by the author of this statement)

The MAK Commission discussed human relevance of the observed thyroid effects with BHT in rodent studies as follows:

“The hyperactivity of the thyroid caused by substances with liver enzyme-inducing properties is more or less a reaction specific to rodents since rats, as compared with humans, are especially susceptible to a disturbance of the thyroid-pituitary regulation caused by an increased metabolization of thyroxine (T4) in the liver.” (MAK Commission. 2012, bold added retrospectively by the author of this statement)

Human relevance of observations in experimental animals should be taken into account and a substance with adverse effects in rodents which are not relevant for humans should not be considered a human endocrine disruptor (EU-Commission 2016b, Solecki 2016).

EFSA (2012) concluded on the human relevance of the BHT effects on the thyroid observed in rodent studies:

“Although rodents and humans share a common physiology in regard to the thyroid-pituitary feedback system, a number of factors contribute to the greater sensitivity of the rat to long-term perturbation of the pituitary thyroid axis which predisposes it to a higher incidence of proliferative lesions in response to chronic TSH stimulation than human thyroid.

Both humans and rodents have nonspecific low affinity protein carriers of thyroid hormone (e.g., albumin). However, in humans, other primates, and dogs there is a high affinity binding protein, thyroxine-binding globulin (TBG), which binds T4 (and T3 to a lesser degree); this protein is not present in rodents, birds, amphibians and fish, and has a 1000-fold greater binding affinity than nonspecific low affinity protein carriers.

Although qualitatively the rat is an indicator of a potential human thyroid cancer hazard, humans appear to be quantitatively less sensitive than rodents to developing cancer from perturbations in thyroid-pituitary status. Given that the rodent is a sensitive model for measuring the carcinogenic influences of TSH and that humans appear to be less responsive, effects on rodents would represent a conservative indicator of potential risk for humans. Rodent cancer studies typically include doses that lead to toxicity, including perturbation in thyroid-pituitary function over a lifetime. The relevance of the experimental conditions to anticipated human exposure scenarios (i.e., dose, frequency and time) should be considered. In addition, chemically-induced effects that are produced by short-term disruption in thyroid-pituitary function appear to be reversible, when the stimulus is removed.” (EFSA 2012, bold added retrospectively by the author of this statement)

Enhanced thyroxine metabolism, due to the stimulation of liver phase 2 metabolism (i.e. UDP-glucuronyltransferases and/or sulfotransferases), leads to a compensatory increase in serum TSH levels, leading to increased thyroid gland weight due to thyroid follicular cell hypertrophy and hyperplasia. The chronic stimulation of the rodent thyroid gland is known to result in thyroid follicular cell hyperplasia and subsequently in the formation of thyroid follicular cell adenomas and carcinomas (Capen, 2001; Curran and DeGroot, 1991; Hard, 1998; Hill et al., 1998; Meek et al., 2003). However, while this mode of action is well established for certain non-genotoxic rodent thyroid gland carcinogens in rats, quantitative species differences are known to exist between rodents and humans (Capen, 2001; Hard, 1998; Hill et al., 1998; Meek et al., 2003). For example, rats have higher constitutive serum TSH levels and shorter half-lives for both T4 and T3; i.e.the T4 half-life in rats is 12-24 hours while in humans the half-life is 5-9 days (Capen, 1992). The longer half-lives of T4 and T3 in humans are due to the presence of a high-affinity serum thyroxine-binding globulin, this protein being absent in adult rats. Although T4 can be bound to serum transthyretin and albumin in rats, these two proteins have a much lower affinity for T4 than human thyroxine-binding globulin (Capen, 2001; Hard, 1998; Hill et al., 1998; Meek et al., 2003), resulting in a large proportion of T4 being unbound in the rat (Capen, 2001). While inducers of hepatic xenobiotic metabolism increase TSH levels and thyroid weight in rodents, studies in humans with Phenobarbital, rifampicin and other enzyme inducers have demonstrated that although T4 levels may be affected, serum TSH levels are not increased (Capen, 2001; Curran and DeGroot, 1991; Meek et al., 2003). These drugs are used in humans without significant effects on the thyroid (Falcó 2016). For example Phenobarbital is used for almost a century therapeutically as a sedative, hypnotic and anti-epileptic agent, with many patients receiving high daily doses of over extended periods. A number of epidemiological investigations of patients receiving pharmacologically active doses of Phenobarbital have not demonstrated any significant increases in the incidence of thyroid gland tumors (IARC, 2001; Meek et al., 2003; Olsen et al., 1989; Whysner et al., 1996).

Overall, human relevance of the observations in experimental animals should be taken into account and a substance with adverse effects in rodents which are not relevant for humans should not be considered a human endocrine disruptor according to the EU-Commission proposal on scientific criteria for endocrine disruptors (2016). There is general agreement that rats are substantially more sensitive to agents that disturb thyroid function compared to humans. This applies also to the thyroid effects observed with BHT in rodent studies and, consequently, the relevance of these effects is limited for humans (EFSA 2012, MAK 2012) and BHT should not be regarded as an endocrine disruptor to humans.

Adrenal Gland

During the Meeting between the REACH registrants and ANSES on July 19, ANSES expressed some uncertainty concerning potential effects of BHT on the adrenal gland.

The in vivo studies which investigated adrenal gland are summarized in Annex 1. Two old studies with major deficiencies (Johnson & Hewgill, 1961 used only 3 rats/sex/dose and indicated ‘genetic effects that led to some variatons in litters’; Sporn & Schöbesch, 1961 used a diet with about 20% casein and only poorly reported the results; this publication is only available in Romanian) showed weight increases in the adrenals of rats after feeding of about 100 or 200 mg BHT/kg bw/day for 6 to 8 weeks.

No effects in the adrenals were seen more reliable and more recent studies as e.g. Prince 1994, Hirose et al., 1981, Hiraga et al., 1978, Williams et al. 1990 and NCI 1979.

According to the EU-Comission documentation (2016b) a weight of evidence should be performed for complex databases to assess quality, reliability, reproducibility and consistency of the scientific evidence. In particular the following aspects should be considered: “both positive and negative results shall be considered”, “Relevance of the study designs” and “The quality and consistency of the data” (EU-Comission 2016b).

Taking into account all available data the adrenal is not considered as a target for BHT.

Executive summary:

There is no evidence from mechanistic in vitro studies that BHT has relevant or consistent activity on endocrine related endpoints. Comprehensive in vivo studies, including generation reproduction toxicity studies, demonstrate that the liver is the primary target with increased liver weight and phase 1 and phase 2 liver enzyme activity. The observed thyroid hyperactivity in rodents is a consequence of BHT dependent liver enzyme activation. This secondary mechanism on the thyroid is confirmed in recent comprehensive evaluations by EFSA (2012) and the German expert committee “Permanent Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area” (MAK Commission, 2012).

Applying the recent EU-Commission proposal on scientific criteria for endocrine disruptors (2016) BHT should not be considered as an endocrine disruptor. There is at least “reasonable evidence” ("biological plausibility") demonstrating that liver induction is the primary effect of BHT leading to the secondary effect thyroid activation in rodents. Consequently, the respective non-endocrine-related liver toxicity should not be considered as an adverse effect that is relevant for the identification of a substance as an endocrine disruptor.

In addition, human relevance of the observations in experimental animals should be taken into account and a substance with adverse effects in rodents which are not relevant for humans should not be considered a human endocrine disruptor according to the EU-Commission proposal on scientific criteria for endocrine disruptors (2016). There is general agreement that rats are substantially more sensitive to agents that disturb thyroid function compared to humans. This applies also to the secondary thyroid effects observed with BHT in studies with rats. Consequently, the relevance of these effects is limited for humans (EFSA 2012, MAK 2012) and BHT should not be regarded as an endocrine disruptor to humans.

Overall, BHT should not be considered as an endocrine disruptor. There are comprehensive studies available covering OECD conceptual framework levels 1 to 5 and no additional studies are necessary to conclude on the mechanism of action that result in thyroid toxicity in rodents.

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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Principles of method if other than guideline:
BHT dissolved in corn oil was administered to pregnant Sprague-Dawley rats by oral gavage during organogenesis period (from 7th to 17th day of gestation) at daily doses of 100, 200, 300, 400 mg/kg. Teratogenic effects were scrutinized at day 20 of gestation.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
From 7th to 17th day of gestation
Frequency of treatment:
Daily
Duration of test:
Teratogenic effects were scrutinized at day 20 of gestation.
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
400 mg/kg bw/day
No. of animals per sex per dose:
No data
Control animals:
not specified
Key result
Remarks on result:
not determinable
Remarks:
Only abstract available
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Remarks on result:
not determinable
Remarks:
Only abstract available
Key result
Abnormalities:
no effects observed
Localisation:
other: External and visceral observations
Key result
Developmental effects observed:
no

Pregnant performances and fetal developments were not affected by treatment of BHT and no significant differences were detected in maternal body weight gains and food intakes. Treated groups showed a dose related increase in relative organ weight of liver at high doses. BHT did not induce significant fetal abnormalities in external and visceral observations. On skeletal examination, significant increases in incidences of variations such as sternebral retardation at 300 mg/kg was observed without dose dependence.

Conclusions:
Pregnant performances and fetal developments were not affected by treatment of BHT and no significant differences were detected in maternal body weight gains and food intakes. Treated groups showed a dose related increase in relative organ weight of liver at high doses. BHT did not induce significant fetal abnormalities in external and visceral observations. On skeletal examination, significant increases in incidences of variations such as sternebral retardation at 300 mg/kg was observed without dose dependence.
Executive summary:

BHT dissolved in corn oil was administered to pregnant Sprague-Dawley rats by oral gavage during organogenesis period (from 7th to 17th day of gestation) at daily doses of 100, 200, 300, 400 mg/kg. Teratogenic effects were scrutinized at day 20 of gestation.

Pregnant performances and fetal developments were not affected by treatment of BHT and no significant differences were detected in maternal body weight gains and food intakes. Treated groups showed a dose related increase in relative organ weight of liver at high doses. BHT did not induce significant fetal abnormalities in external and visceral observations. On skeletal examination, significant increases in incidences of variations such as sternebral retardation at 300 mg/kg was observed without dose dependence.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Only one dose was used and only a limited number of parameters studied.
Principles of method if other than guideline:
Butylated hydroxytoluene (BHT) 500 mg/kg body wt/day was given in the diet to F0 rats from 6 weeks of age to weaning of the F1-generation (growth period to age 19 week, gestation period and lactation period) and subsequent to F1-animals until 21 days of age. Body weight of F0 animals was recorded, and the influence on the F1-generation was investigated applying crossfostering. Mortality, litter size, weight gain and some developmental events as well as tests for auditory and visual function and locomotor coordination in the F l-generation were recorded
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-week old
- Housing: Stainless steelwire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1 ºC
- Humidity (%): 60 ± 5%
- Air changes (per hr): 8 air changes per hour
- Photoperiod (hrs dark / hrs light): electric light from 9 pm to 9 am
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
When 19 weeks old, the animals were mated over a 14-day period, 1 female per male per cage. The presence of plug was checked every evening (designated day 0) and morning (designated day 1). After completed mating the F0 males were omitted from the study.
Duration of treatment / exposure:
Butylated hydroxytoluene (BHT) 500 mg/kg body wt/day was given in the diet to F0 rats from 6 weeks of age to weaning of the F1-generation (growth period to age 19 week, gestation period and lactation period) and subsequent to F1-animals until 21 days of age.
Frequency of treatment:
Daily
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
F0: 46 animals per sex per dose
Control animals:
yes, plain diet
Maternal examinations:
BODY WEIGHT: Yes
- Time schedule for examinations: Body weight and weight gain were recorded every 14 days except during gestation where a 7-day interval was employed.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food consumption was measured over 1-7, 7-14 and 14-21 day periods.
Fetal examinations:
Dates of birth of F1 rats were recorded as well as litter size and sex ratio. The duration of the gestation period was estimated. Twenty-four hours after birth the litter size was reduced to 8, and equal sex distribution was achieved. Weight of the litter was recorded both before and after standardization, and half of the litter were crossfostered to obtain the groups defined in the study.

The F1 animals were weighed on days 1, 7, 14 and 21 and observed daily. The days of appearance of the following events were noted in each pup: pinna unfolding (3-4 days of age), tooth eruption (10-11 days), startle response to an auditory stimulus (13-15 days) and opening of the eyes (13-15 days).

The following parameters were used on the development of neuromuscular function and orientation responses: static righting reflex (3-4 days of age), cliff avoidance (7-9 days) and righting reflex (16-18 days). Olfactory orientation (11-13 days), visual orientation ("choice of jumping") (21-22 days) and rotarod (22-24 days).

The F1 animals were autopsied at 25 days of age, and the brains were fixed in 4% buffered formalin. A paramedian incision was performed, and 5 μm sections were stained with hematoxylin and eosin. The number of dead cells in the paramedian section of cerebelum were estimated from 1 male and 1 female per litter.
Statistics:
Student´s t-test was performed on body weight F0, weight gain F0, litter size, sex ratio, body weight F1.

Wilcoxons ranks test was applied on special developmental tests and on the estimated number of dead cells in the paramedian section of cerebellum.

In addition the Quick Test-Chi-square was used for the rotarod.
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):
Body weight and weight gain were significantly reduced in dosed F0 animals, both males and females, as compared to controls. In the first 7 days of the gestation period, the dosed females had a significantly slower weight gain. This reduction of weight gain persisted in the rest of the pregnancy period although not statistically significant.
Food consumption and compound intake (if feeding study):
no effects 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:
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 specified
Neuropathological findings:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Description (incidence and severity):
The average duration of pregnancy was 22.0 days in the control group and 22.6 days in the dosed.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The average duration of pregnancy was 22.0 days in the control group and 22.6 days in the dosed.
Changes in number of pregnant:
not specified
Other effects:
no effects observed
Details on maternal toxic effects:
Details on maternal toxic effects:
Body weight and weight gain were significantly reduced in dosed F0 animals, both males and females, as compared to controls. In the first 7 days of the gestation period, the dosed females had a significantly slower weight gain. This reduction of weight gain persisted in the rest of the pregnancy period although not statistically significant.

Key result
Dose descriptor:
LOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Average body weight and weight gain for F1 offspring exhibit a marked statistically significant effect on young rats, nursed by dosed mothers (P < 0.001).
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Average body weight and weight gain for F1 offspring exhibit a marked statistically significant effect on young rats, nursed by dosed mothers (P < 0.001).
The average birth weight was 5.9 for both groups
Reduction in number of live offspring:
not specified
Changes in sex ratio:
effects observed, treatment-related
Description (incidence and severity):
The sex ratio showed a slightly lower proportion of females/litter in the dosed group compared to the control
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The average litter size was 10.0 and 9.8 for the control and dosed group, respectively
Changes in postnatal survival:
not specified
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The developmental parameters indicated in general a delayed development in the 2 groups suckled by dosed mothers parallel to reduced body weight and weight gain. In addition a comparison between the 2 groups with non-dosed mothers indicate a slightly, relatively slower development in the dosed pups. F1 animals nursed by dosed mothers appeared clinically to have a hyperactive behaviour when handled in the first week of the lactation period
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
The average duration of pregnancy was 22.9 days in the control group and 22.6 days in the dosed. The average birth weight was 5.9 for both groups. The average litter size was 10.0 and 9.8 for the control and dosed group, respectively. The sex ratio showed a slightly lower proportion of females/litter in the dosed group compared to the control. Average body weight and weight gain for F1 offspring exhibit a marked statistically significant effect on young rats, nursed by dosed mothers (P < 0.001). The developmental parameters(Pinna unfolding, static righting reflex, cliff avoidance, tooth eruption, olfactory orientation, startle response, eye opening. righting reflex, visual orientation) indicated in general a delayed development in the 2 groups suckled by dosed mothers parallel to reduced body weight and weight gain. In addition a comparison between the 2 groups with non-dosed mothers indicate a slightly, relatively slower development in the dosed pups. F1 animals nursed by dosed mothers appeared clinically to have a hyperactive behaviour when handled in the first week of the lactation period.

Histological examination of the brain revealed a slightly higher incidence of average number of dead cells (pychnosis, karyorrhexis and hyperchromatosis of the nucleus membrane) in the paramedian section of cerebellum, especially in the molecular layer of cortex and in the white matter, in the F1 animals nursed by dosed mothers. Four litters were euthanised when 3 weeks old showing tremor, wobbling movements and an under-nourished appearance. Histological investigation of animals from these litters above revealed a marked higher proportion of dead cells in cerebellum, both in the molecular and granular layer of cortex and the white matter. In addition the pia mater appeared thickened.
Key result
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Developmental events and histological examination of brain
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

The results indicate that 500 mg BHT/kg body wt/day in the diet caused a significant adverse effect on the body weight of the F0 generation. It might be expected that this growth reduction in F0 would have caused a lower birth weight of pups born of dosed mothers. However, no effect of BHT was found on the reproduction data including birth weight. The difference found in sex ratio is considered to be a coincidence, and in addition a previous study did not reveal such an effect. The pararneters for the F1 generation show a marked adverse effect on the young rats nursed by dosed mothers, and this indicates a predominant lactation effect. The above results could be a consequence of a toxic effect on the mother, a direct effect of BHT on the pups or both. In a previous experiment

at the institute a toxic effect of BHT on the kidney has been found in female rats. In addition a pilot experiment has demonstrated, that

BHT is excreted in high concentrations in rat-milk. Thus both theories could be supported. A toxic effect on the dosed mothers, e.g. leading to a decreased food consumption could be investigated in a pairfeeding experiment.

Although this experiment predominantly demonstrates a marked lactation effect, a prenatal influence of BHT can not be excluded, as group control mothers with dosed youngs exhibit a delayed development especially in the first week of the lactation period. This could be a direct effect of BHT on the fetus. In an experiment with [14C] BHT, it has been demonstrated that BHT crosses the placental barrier. Group dosed mothers and control youngs exhibit in general the most pronounced toxic effect of BHT. The fact that BHT has been found-to cross the placental barrier could explain the difference between groups, as a possible induction of a higher

metabolic capability in- the in-utero dosed young could have happened. A possible effect of the applied crossfostering procedure could be elucidated by using a procedure, where only half of every litter are crossfostered. The four litters with tremor and wobbling movements and a high proportion of dead cells in cerebellum could be a result of extreme undernourishment. Whether this effect is due to a lacking of suckling ability by the youngs or an insufficient lactation by the dosed mother is not clear. From the literature it is known that under-nourished youngs exhibit an effect on the development of the cerebellum. In addition hyperactivity found in youngs, nursed by dosed mothers, could be interpreted in the same way.

Conclusions:
The treatment resulted in a decreased weight gain for both F0 and F1. A parallel adverse effect on different developmental parameters was found in the F1 generation. The effect on F1 offspring occurred mainly in the lactation period, caused either by a decreased nutrition due to BHT´s influence on the lactation of the F0 females, BHT´s influence on the suckling ability of the F1 rats or both in combination.
Executive summary:

Butylated hydroxytoluene (BHT) 500 mg/kg body wt/day was given in the diet to F0 rats from 6 weeks of age to weaning of the F1-generation (growth period to age 19 week, gestation period and lactation period) and subsequent to F1-animals until 21 days of age. Body weight of F0 animals was recorded, and the influence on the F1-generation was investigated applying crossfostering. Mortality, litter size, weight gain and some developmental events as well as tests for auditory and visual function and locomotor coordination in the F l-generation were recorded. The applied dose of BHT exerted a significant adverse effect on body weight in both F0 and F1 animals and on several developmental parameters in F1 animals. The effects arose during the lactation period.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
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.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Strain:
Wistar
Key result
Dose descriptor:
NOAEL
Effect level:
60.5 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
mortality
maternal abnormalities
Remarks on result:
other: Based on a read-across from an analogue for which NOAEL for material toxicity= 93.5 mg/kg.bw/day
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
not specified
Description (incidence and severity):
Hair fluffing, diarrhoea, food consumption, weight gain, mortality.
Key result
Dose descriptor:
NOAEL
Effect level:
243 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental toxicity.
Remarks on result:
other: Based on a read-across from an analogue subtance for which NOAEL (developmental toxicity)=375 mg/kg-bw/day
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
Read-across from 6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol. Taking into account the molecular weights:

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).
Executive summary:

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).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Test method similar to OECD 414.
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
2,2´-methylenebis (4-methyl-6-tert-butylphenol) was given orally to pregnant Wistar rats by stomach intubation at the dose levels of 93.5, 187 or 375 mg/kg bw during days 7 to 17 of pregnancy and the effects of the compound on dams and fetal developments were examined.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Route of administration:
oral: gavage
Details on exposure:
- Vehicle: yes
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
From day 7 to 17 of pregnancy
Frequency of treatment:
Daily
Dose / conc.:
93.5 mg/kg bw/day
Dose / conc.:
187 mg/kg bw/day
Dose / conc.:
375 mg/kg bw/day
No. of animals per sex per dose:
20 to 24 pregnant rats for each group
Control animals:
yes, concurrent vehicle
Maternal examinations:
Body weight and mortality
Ovaries and uterine content:
All pregnant rats on the 20th day of pregnancy were killed under ether anesthesia. Through an incision to remove the fetuses, the number of corpora lutea, number of implantations, the number of fetal deaths, the number of fetal survival, sex ratio, fetuses body weight and number of death implantations (early, late) were examined.

Fetal examinations:
External malformations. 1/3 of fetal survival was examined for internal organ abnormalities and the remaining 2 / 3 were examined for skeletal abnormalities.
Statistics:
Experimental results are evaluated as a unit matrix, test, t-test or rank sum test and the rate of 5% and 1% risk compared with the level of the control group.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the dams at the two higher doses of 187 and 375 mg/kg, toxic signs such as hair fluffing and diarrhoea were observed, and their body weight gain and food consumption were suppressed. Two dams, which showed marked diarrhoea in the highest dose group, died.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
In the dams at the two higher doses of 187 and 375 mg/kg, toxic signs such as hair fluffing and diarrhoea were observed, and their body weight gain and food consumption were suppressed. Two dams, which showed marked diarrhoea in the highest dose group, died.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the dams at the two higher doses of 187 and 375 mg/kg, toxic signs such as hair fluffing and diarrhoea were observed, and their body weight gain and food consumption were suppressed. Two dams, which showed marked diarrhoea in the highest dose group, died.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the dams at the two higher doses of 187 and 375 mg/kg, toxic signs such as hair fluffing and diarrhoea were observed, and their body weight gain and food consumption were suppressed. Two dams, which showed marked diarrhoea in the highest dose group, died.
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 specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Description (incidence and severity):
Slight increase in fetal death was found in the highest dose group.
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In the dams at the two higher doses of 187 and 375 mg/kg, toxic signs such as hair fluffing and diarrhoea were observed, and their body weight gain and food consumption were suppressed. Two dams, which showed marked diarrhoea in the highest dose group, died.
Key result
Dose descriptor:
NOAEL
Effect level:
93.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
not specified
Description (incidence and severity):
In the dams at the two higher doses of 187 and 375 mg/kg, toxic signs such as hair fluffing and diarrhoea were observed, and their body weight gain and food consumption were suppressed. Two dams, which showed marked diarrhoea in the highest dose group, died.
Fetal body weight changes:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
A slight increase in fetal death was found in the highest dose group.
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
not specified
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no evidence of fetal malformation attributable to treatment with the compound in any of the dose groups treated, although a slight increase in fetal death was found in the highest dose group.
Key result
Dose descriptor:
NOAEL
Effect level:
375 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Developmental toxicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
It is concluded that 2,2'-methylenebis (4-methyl-6-tert--butylphenol) has a weak lethal effect on fetal development but not a teratogenic effect in the rat.
Executive summary:

2,2´-methylenebis (4-methyl-6-tert-butylphenol) was given orally to pregnant Wistar rats by stomach intubation at the dose levels of 93.5, 187 or 375 mg/kg bw during days 7 to 17 of pregnancy and the effects of the compound on dams and fetal developments were examined. In the dams at the two higher doses of 187 and 375 mg/kg, toxic signs such as hair fluffing and diarrhoea were observed, and their body weight gain and food consumption were suppressed. Two dams, which showed marked diarrhoea in the highest dose group, died. However, there was no evidence of fetal malformation attributable to treatment with the compound in any of the dose groups treated, although a slight increase in fetal death was found in the highest dose group. It is concluded that 2,2'-methylenebis (4-methyl-6-tert-butylphenol) has a weak lethal effect on fetal development but not a teratogenic effect in the rat. This compound consists essentially of two molecules of BHT.

 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable documented publication, which meets basic sientific principles.
Principles of method if other than guideline:
Developmental toxicity study
GLP compliance:
not specified
Species:
hamster
Strain:
other: golden hamster
Route of administration:
oral: gavage
Vehicle:
not specified
Details on mating procedure:
M/F ratio per cage: 1:1
Duration of treatment / exposure:
beginnig on day 6 and continuing daliy through day 10 of gestatio
Frequency of treatment:
daily, beginnig on day 6 and continuing daliy through day 10 of gestation
Duration of test:
up to day 14 of gestation
No. of animals per sex per dose:
at least 21
Control animals:
yes
Dose descriptor:
NOAEL
Effect level:
280 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

The administration of up to 280 mg/kg body weight of the test substance to pregnant hamsters for 5 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occuring spontaneously in the sham-treated controls.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable documented publication which meets basic scientific principles, but study with limitations concerning the small number of animals used and the mixted exposure with BHA and BHT
Principles of method if other than guideline:
One-generation study
GLP compliance:
not specified
Species:
monkey
Strain:
other: rhesus monkey
Route of administration:
oral: feed
Vehicle:
other: a mixture of BHA and BHT was incorporated in the diet
Details on mating procedure:
Animals were fed with BHT for one year prior breeding and for an additional year including a 165-day gestation period
males received for one year the experimental diet (with BHA and BHT) were introduced into the cage of the females at appropriate time for breeding; they remainted with the females for at least 48 hours during a given breeding period. Pregnancy was determined by postconception bleeding and by rectal palpation at 45 days following breeding
Duration of treatment / exposure:
2 years
Frequency of treatment:
daily
Duration of test:
2 years exposure period + up to 2 years post-exposure period
No. of animals per sex per dose:
6 females per dose group
Control animals:
yes, plain diet
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

First year exposure to BHTand BHA (period before breeding): no abnormalities were recorded in the treated animals; no differences in blood chemistry were seen compared to control; treated animals maintained normal menstrual cycles; body weight and food consumption were unaffected

Second year exposure (including pre- and postnatal period): control (6/6), treatment group (5/6) gave birth to normal infants; hematology of treatment group was similar to control; body weight, body lengh, head circumference of pups from the treatment group were equal to control pups; pups of the treatment group and control did not disclose any behavioral abnormalities

Post-treatment period (2 years): dams and pups of the treatment group did not show any abnormalities

Executive summary:

Female rhesus monkeys treated with BHA and BHT (50 mg BHT/kg bw + 50 mg/kg bw) in the diet for two years, showed no advers effects. In addition pups from treated dams showed no addverse effects, too (Allen 1976).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Pregnant diabetic and normal rats were fed either a standard diet or a diet enriched with 1% of the antioxidant butylated hydroxytoluene (BHT). The rats were mated overnight. Throughout pregnancy (i.e., on gestational days 0, 6, 10, 16, and 20), the animals were weighed and had their serum glucose levels determined. At a gestational age of 20 days, the pregnant rats were killed, the fetuses and placentas were quickly dissected out, and the viable fetuses were weighed and examined for gross malformations.
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
oral: feed
Vehicle:
other: commercial pelleted rat diet (R 36, Lactamin AB, Stockholm, Sweden)
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
The rats were mated overnight, and the morning when sperm were found in the vaginal smear was denoted gestational day 0.
Duration of treatment / exposure:
The BHT treatment (1% added to diet) started 4 weeks before mating and continued throughout pregnancy. At a gestational age of 20 days, the pregnant rats were killed.
Frequency of treatment:
daily
Duration of test:
Approx. 48 days
No. of animals per sex per dose:
Normal rats:
7 rats: normal rats fed with control diet
10 rats: feed with BHT (1% in diet)
4 rats: fed with ether treated diet (ether was used as solvent for BHT)

Diabetic rats:
9 rats: diabetic rats fed with control diet
13 rats: feed with BHT (1% in diet)
4 rats: fed with ether treated diet (ether was used as solvent for BHT)
Control animals:
yes
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified

Fetal outcome on gestational day 20 in normal and manifestly diabetic rats with a control diet (N and MD), a BHT-supplemented diet (NB and MDB), or an ether-treated diet (NE and MDE)

 

 Number

of litters

 Number of

implantations

 Number of

resorptions

Number of

malformations 

Resorptions of

implantations per

litter (%)

 Malformations of viable fetuses

per litter (%)

Implantations per litter

 Resorptions per litter

Malformations per litter 

 N

 13  149  9  0  6 ± 3  0  11.5 ± 0.4  0.7 ± 0.4  0

NB

 17  181  16  0  10 ± 3  0 10.6 ± 0.6  0.9 ± 0.3  0

 NE

 6  76  11  2  14 ± 8  5.5 ±5  12.7 ± 1.4  1.8 ± 1.1  0.3 ±0.3

 MD

 14  136  30  22  23 ± 5 19 ± 6 9.7 ± 0.7 2.1 ± 0.5  1.6 ± 0.5

 MDB

 19  199  57  4  28 ± 5 2.3 ± l 10.5 ± 0.4  2.6 ± 0.6 0.2 ± 0.1

 MDE

 4  53  17  9 32 ± 13 21 ±9 13.2 ± 1.3 4.2 ± 1.7 2.2 ± 1.0
Executive summary:

Pregnant diabetic and normal rats were fed either a standard diet or a diet enriched with 1% of the antioxidant butylated hydroxytoluene (BHT). The rats were mated overnight. Throughout pregnancy (i.e., on gestational days 0, 6, 10, 16, and 20), the animals were weighed and had their serum glucose levels determined. At a gestational age of 20 days, the pregnant rats were killed, the fetuses and placentas were quickly dissected out, and the viable fetuses were weighed and examined for gross malformations.

No malformations were found in the normal rats treated with BHT.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable, documented study report, which meets basic scientific principles
Principles of method if other than guideline:
Method: other: test on maternal toxicity and teratogenicity
GLP compliance:
not specified
Species:
mouse
Strain:
other: JCL-ICR
Route of administration:
oral: gavage
Vehicle:
olive oil
Duration of treatment / exposure:
day 7 to 13 of gestation
Frequency of treatment:
once a day
Duration of test:
animals were killed on day 18 of gestation
No. of animals per sex per dose:
26-30 females per group
Control animals:
other: yes, concurrent vehicle and concurrent untreated
Details on study design:
Sex: female
Duration of test: until the 18th day of gestation
Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Abnormalities:
no effects observed
Developmental effects observed:
no
RS-Freetext:
800 mg/kg bw day:
MATERNAL PARAMETER: increased spleen weight; decreased kidney weight (compared to the untreated control animals)
REPRODUCTIVE PARAMETERS: no effects
FETAL PARAMETER: no effects
240 mg/kg bw day:
MATERNAL PARAMETER: no effects
REPRODUCTIVE PARAMETERS: no effects
FETAL PARAMETER: no effects
70 mg/kg bw day:
MATERNAL PARAMETER: no effects
REPRODUCTIVE PARAMETERS: no effects
FETAL PARAMETER: no effects
Executive summary:

Due to the findings of this study, the author concluded, that BHT was not teratogenic to JCL-ICR mice. Dams treated with 800 mg/kg bw and day showed an increased spleen weight and recuced kidney weight (Hiraga 1978).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable, documented study report, which meets basic scientific principles
Principles of method if other than guideline:
Method: other: test on maternal toxicity and teratogenicity
GLP compliance:
not specified
Species:
mouse
Strain:
other: JCL-ICR
Route of administration:
oral: gavage
Vehicle:
olive oil
Duration of treatment / exposure:
day 9 of gestation
Frequency of treatment:
single administration
Duration of test:
animals were killed on day 18 of gestation
No. of animals per sex per dose:
19-20 females per group
Control animals:
yes, concurrent no treatment
Details on study design:
Sex: female
Duration of test: until the 18th day of gestation
Dose descriptor:
LOAEL
Effect level:
1 200 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
1 200 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
RS-Freetext:
1800 mg/kg bw:
MATERNAL PARAMETER: 5/20 died (11th day 3; 14th day 1 and
15th day 1), increased lung and spleen weights
REPRODUCTIVE PARAMETERS: no effects
FETAL PARAMETER: delay of progression of ossification
1200 mg/kg bw:
MATERNAL PARAMETER: 2/20 died (11th day 1 and 15th day 1),
increased lung weight
REPRODUCTIVE PARAMETERS: no effects
FETAL PARAMETER: delay of progression of ossification
Executive summary:

The single administration of maternally severely toxic BHT doses of 1200 or 1800 mg/kg body weight (in the range of lethality) on day 9 of gestation resulted in a slight retardation of ossification (Hiraga 1978).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable documented publication, which meets basic sientific principles
Principles of method if other than guideline:
other: test of teratogenicity
GLP compliance:
not specified
Species:
mouse
Strain:
CD-1
Route of administration:
oral: gavage
Duration of treatment / exposure:
day of gestation: 6-15
Frequency of treatment:
beginning on day 6 and continuing daily through day 15 of gestation
Duration of test:
up to day 17 of gestation
No. of animals per sex per dose:
20 females per dose group
Control animals:
yes
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

The administration of up to 180 mg/kg body weight of the test substance to pregnant mice for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test group did not differ from the number occuring spontaneously in the sham-treated controls.

Executive summary:

The administration of up to 180 mg/kg body weight of the test substance to pregnant CD-1 mice for 10 consecutive days revealed no developemtal toxicity and teratogenicity (FDA 1972).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable documented publication, which meets basic sientific principles.
Principles of method if other than guideline:
other: test of teratogenicity
GLP compliance:
not specified
Species:
rat
Strain:
Wistar
Route of administration:
oral: gavage
Duration of treatment / exposure:
day of gestation: 6-15
Frequency of treatment:
beginning on day 6 and continuing daliy through day 15 of gestation
Duration of test:
up to day 20 of gestation
No. of animals per sex per dose:
at least 23 females per dose group
Control animals:
yes
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

The administration of up to 225 mg/kg body weight of the test substance to pregnant rats for 10 consecutive days had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test group did not differ from the number occuring spontaneously in the sham-treated controls.

Executive summary:

No embryo toxic effects were recorded in this study with Wistar rats up to BHT doses of 225 mg/kg body weight and day (FDA 1972).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable documented publication, which meets basic sientific principles.
Principles of method if other than guideline:
other: one-generation study
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Route of administration:
oral: feed
Vehicle:
other: BHT contained in the diet
Duration of treatment / exposure:
starting 14 days before mating, during breeding, the period of gestation, and the period of lactation (21 days); after weaning dietary exposure of the offspring to BHT continued throughout the remainder of the experiment (up to day 90)
Frequency of treatment:
daily
No. of animals per sex per dose:
at least 13 females
Control animals:
yes, plain diet
Dose descriptor:
LOAEL
Effect level:
167 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

No reduced litter size, and no effects on the lenght of gestation were found in treated animals compared to the negative control.

From 167 mg/kg body weight (F1): increased postnatal mortality (postnatal days 1 -30)

333 mg/kg bw (F1): reduced body weight gain; delayed development before weaning (180° rotation; opening of the eyes, swimming behaviour; behaviour in the open field); no effect after weaning

Executive summary:

In this study with Sprague-Dawley rats an increased postnatal mortality was observed at concentrations of 167 mg/kg body weight and higher. In addition a delayed development of the F1 generation, before weaning, were seen at 333 mg/kg body weight; but after weaning no developmental effects were seen (Vorhees 1981).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Method with major limitations, e.g. limited number of animals ; effect of high lard intake unclear; insufficient documentation
Principles of method if other than guideline:
Method: other: reproductive study
GLP compliance:
no
Species:
rat
Strain:
other: Norway Hooded
Route of administration:
oral: feed
Duration of treatment / exposure:
5 months
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: male/female
Duration of test: 6 months
Abnormalities:
not specified
Developmental effects observed:
not specified
RS-Freetext:
No differences between teratment groups regarding number of
pups born and weaned and on the total weight of litter at 21
days. Anophthalmia in 3 of 30 litters; number of
anophthalmic young to the total number in these three
litters: 1/11, 1/11, 8/11. With the small number it was not
possible to trace the cause of this effect to either genetic
or dietary factors. In a separate experiment, the mating of
anophthalmic male and female rats failed to produce
anophthalmic young even at a BHT concentration of 0.5% in
the diet. Anophthalmia was not observed in any other
experimental animals used by the authors.
Executive summary:

The findings of this inadequately documented long-term study, in which anophthalmia was observed in 3 of 30 litters after BHT administration to male and female hooded Norway rats were assessed as not reliable (MAK 2004).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details
Principles of method if other than guideline:
other: developmental toxicity study
GLP compliance:
not specified
Species:
rat
Strain:
other: Porton SPF
Route of administration:
oral: gavage
Vehicle:
arachis oil
Duration of treatment / exposure:
7 weeks before mating up to 20th day of gestation
Frequency of treatment:
daily administration for 7 wk before pairing; continuing during pregnancy; treatment commenced when rats were 3 or 10 wk old
Duration of test:
up to 20th day of gestation
No. of animals per sex per dose:
20 females
Control animals:
yes
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified
500 mg/kg body weight: dams and foetuses: no effects
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details
Principles of method if other than guideline:
other: developmental toxicity study
GLP compliance:
not specified
Species:
rat
Strain:
other: Tuck albino or Benger hooded
Route of administration:
oral: gavage
Vehicle:
arachis oil
Duration of treatment / exposure:
10 weeks before mating up to 20th day of gestation
Frequency of treatment:
daily administration for 70 days before pairing, continuing through pregnancy
Duration of test:
up to 20th day of gestation
No. of animals per sex per dose:
5-8 females
Control animals:
yes
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified
750 mg/kg body weight: dams and foetuses: no effects
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details
Principles of method if other than guideline:
other: developmental toxicity study
GLP compliance:
not specified
Species:
rat
Strain:
other: Tuck albino
Route of administration:
oral: gavage
Vehicle:
arachis oil
Duration of treatment / exposure:
1st to 20th day of gestation
Frequency of treatment:
daily administration on day 1-20 of pregnancy
Duration of test:
up to 20th day of gestation, a group of rats were allowed to continue to parturition, the offspring being examined at weaning (3 wk old)
No. of animals per sex per dose:
5 females
Control animals:
yes
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified
750 mg/kg body weight: dams and foetuses: no effects
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details
Principles of method if other than guideline:
other: developmental toxicity study
GLP compliance:
not specified
Species:
rat
Strain:
other: Tuck albino, Carworth albino
Route of administration:
oral: gavage
Vehicle:
arachis oil
Duration of treatment / exposure:
once on gestation day 9, 11 or 13
Frequency of treatment:
single administration on day 9, 11 or 13 of pregnancy
Duration of test:
up to 20th day of gestation
Control animals:
yes
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified
1000 mg/kg body weight: dams and foetuses: no effects
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details
Principles of method if other than guideline:
other: developmental toxicity study
GLP compliance:
not specified
Species:
mouse
Strain:
other: ICI, SPF
Route of administration:
oral: gavage
Vehicle:
arachis oil
Duration of treatment / exposure:
7 wk before mating up to 18th day of gestation
Frequency of treatment:
daily
No. of animals per sex per dose:
9-20 females
Control animals:
yes
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified

250 mg/kg body weight: dams and foetuses: no effects

from 300 mg/kg body weight: dams: increased mortality; reduced number of corpora lutea; foetuses: reduced number of foetuses

500 mg/kg body weight: dams: reduced mating index

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details
Principles of method if other than guideline:
other: developmental toxicity study
GLP compliance:
not specified
Species:
mouse
Strain:
other: Evans No. 1
Route of administration:
oral: gavage
Vehicle:
arachis oil
Duration of treatment / exposure:
32-46 days before mating up to 18th day of gestation
Frequency of treatment:
daily
Duration of test:
up to 18 day of gestation
No. of animals per sex per dose:
3-7 females
Control animals:
yes
Abnormalities:
not specified
Developmental effects observed:
not specified

750 mg/ kg body weight: dams and foetuses: no effects

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details
Principles of method if other than guideline:
other: developmental toxicity study
GLP compliance:
not specified
Species:
mouse
Strain:
other: Evans No. 1
Route of administration:
oral: gavage
Vehicle:
arachis oil
Duration of treatment / exposure:
1st to 18th day of gestation
Frequency of treatment:
daily
Duration of test:
up to 18 day of gestation
No. of animals per sex per dose:
10 females
Control animals:
yes
Abnormalities:
not specified
Developmental effects observed:
not specified

750 mg/kg body weight: dams and foetuses: no effects

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details
Principles of method if other than guideline:
other: developmental toxicity study
GLP compliance:
not specified
Species:
mouse
Strain:
other: Evans No. 1
Route of administration:
oral: gavage
Vehicle:
arachis oil
Duration of treatment / exposure:
1st - 18th day of gestation
Frequency of treatment:
single administration on a specific day of pregnancy (day 1-18)
Duration of test:
up to 18th day of gestation
No. of animals per sex per dose:
83 females
Control animals:
yes
Abnormalities:
not specified
Developmental effects observed:
not specified

1000 mg/kg body weight: dams and foetuses: no effects

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: study with restrictions; low numbers of animals evaluated, no of dams: 2, F1: 4 per dose group
Principles of method if other than guideline:
Method: other: see remark field
GLP compliance:
not specified
Species:
mouse
Strain:
CD-1
Route of administration:
oral: feed
Duration of treatment / exposure:
days 17 to 20 of pregnancy
Frequency of treatment:
daily
No. of animals per sex per dose:
F0: 2 dams, F1 animals: 4 (2 from each dam)
Details on study design:
Sex: female
Duration of test: approx. 2-3 days
Abnormalities:
not specified
Developmental effects observed:
not specified
RS-Freetext:
The retinas of BHT-fed control animals demonstrated sporadic moprphologic changes in the form of circular configurations composed to ganglion cells, arcades of nuclear and plexiform layers.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: non-GLP study, study data do not give sufficient experimental details (e.g. number of animals per dose group not indicated)
Principles of method if other than guideline:
other: one generation study with emphasis of behavioral tests (F1 generation)
GLP compliance:
not specified
Species:
mouse
Strain:
Swiss Webster
Route of administration:
oral: feed
Vehicle:
other: BHT contained in diet
Frequency of treatment:
daily
No. of animals per sex per dose:
males and females, no other data
Control animals:
yes, plain diet
Abnormalities:
not specified
Developmental effects observed:
not specified

Dose group: 714 mg/kg body weight, F1 mice:

effects on behaviour (sleep, agression) and learning, learning deficits; no data on prenatal toxicity parameter or maternal toxicity

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details
Principles of method if other than guideline:
other: one-generation study
GLP compliance:
not specified
Species:
mouse
Route of administration:
oral: feed
Vehicle:
other: BHT contained in the diet
Frequency of treatment:
daily
Abnormalities:
not specified
Developmental effects observed:
not specified

A significant adverse effect on body weight in both F0 and F1 animals was shown in all dose groups. Dose-dependent differences in the evaluated parameter were found in the offspring of BHT exposed adults. Significant differences in litter size, pup weight, and litter weight were particularly evident.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: study with considerable restrictions by the very variable fertility of the rabbit strain used and the unusuable findings (maternal mortality and resorptions) occuring in the control group
Principles of method if other than guideline:
other: teratogenicity study
GLP compliance:
not specified
Species:
rabbit
Strain:
other: Dutch-belted
Route of administration:
oral: gavage
Duration of treatment / exposure:
beginning on day 6 and continuing daily through day 18
Frequency of treatment:
beginning on day 6 and continuing daily through day 18
Duration of test:
up to day 29 of gestation
No. of animals per sex per dose:
at least 12 females
Control animals:
yes
Abnormalities:
not specified
Developmental effects observed:
not specified

In this prenatal toxicity study, rabbits were administered BHT by gavage from days 6 to 18 of gestation in doses of 3.2, 14.9, 69.1 or 320 mg/kg body weight and day. An increased incidence of resorptions was observed even in the lowest dose group of 3.2 mg/kg body weight; increased maternal mortality and a reduced number of live foetuses were observed from 14.9 mg/kg body weight and from 69.1 mg/kg body weight, respectively. The authors conclude that BHT has adverse effects on the survival of dams and foetuses, which does not however strictly correlate with the dose. There was no increased incidence of skeletal or viceral anormalities. Since the results are not clearly related to the dose, the findings cannot be evaluated conclusively according to MAK (2004).

MAK 2004 evaluated the individual animal data of this study and found indications that for dams with live foetuses the number of foetuses per dam and the foetal body weights were not substantially altered with an increase in dose. An increase in resorptions, in particulartotal resorptions, was observed from 69.1 mg/kg body weight and day. The validity of this study is considerably restricted by the very variable fertility of the strain used and the unusual findings (maternal mortality and resorptions) occuring in the control group.

               BHT dose (mg/kg body weight and day)
   0  3.2  14.9  69.1  320
mating (n)*  12 16  16  22  20
pregnancies (n)*  12 12  15  19  18 
animals that diet (n)§  1
corpora lutea per dam (n)§  11.27 + 2.33  11.75 + 3.73  11.38 + 2.60  12.25 + 2.08  12.69 + 3.44
implantations per dam (n)§  8.27 + 2.28(11 animals) 7.75 + 2.67 (12 animals) 9.92 + 1.71(13 animals)   9.44 + 2.17(16 animals) 10.06 + 3.45 (16 animals)
resorption sites of all damsin total (n)*  11 32  23  86  81 
live dams with completeresorptions or stillbirths (n)§  2/11 3/12  2/12  6/16   6/15
abortions (n)*  0
live foetuses per pregnantdams (n)*  6.0 5.08  7.17  3.67  4.13 
live foetuses per dams withlive offspring (n)§  7.33 + 3.08(9 animals) 6.78 + 2.33(9 animals)   8.55 + 1.33(10 animals)  6.88 + 2.03(8 animals) 6.89 + 2.89(9 animals) 
 Foetal weights (g)  39.8 39.7  37.3  35.4  34.6 
 * data of authors               
 § MAK (2004) calculations based on the individual animal data               
Executive summary:

Study with considerable restrictions; the authors indicated adverse effects in rabbit dams and foetuses after administration of BHT at gestation day six to eighteen (FDA 1974). A reevaluation done by MAK (2004), which based on the individual animal data indicated that for dams with live foetuses the number of foetuses per dam and the foetal body weights were not subtantially altered with an increase in the dose. An increase in resorption, in particular total resorptions, was observed from 61.9 mg/kg body weight and day. However, MAK considered, that the validity of the study is considerably restricted by the very varable fertility of the strain used and the unususal findings occured in the control group (MAK 2004).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details
Principles of method if other than guideline:
other: developmental toxicity study
GLP compliance:
not specified
Species:
rat
Strain:
other: no data
Route of administration:
oral: feed
No. of animals per sex per dose:
no data
Abnormalities:
not specified
Developmental effects observed:
not specified

Up to 0.3% BHT in diet (210 mg/kg bw and day): no effects

At 1.55% BHT in diet (1033 mg/kg bw and day): dams: drastic loss of weight (no other details); foetuses: foetal mortality (no assessable litters).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details
Principles of method if other than guideline:
other: embryotoxicity study (unspecified)
GLP compliance:
not specified
Species:
rat
Strain:
not specified
Route of administration:
oral: unspecified
Vehicle:
not specified
Details on mating procedure:
no data
Duration of treatment / exposure:
no data
Frequency of treatment:
no data
Duration of test:
no data
No. of animals per sex per dose:
no data
Control animals:
not specified
Abnormalities:
not specified
Developmental effects observed:
not specified

Ionol caused embryotrophic effects when administered orally to rats during pregnancy. However, the published data are insufficient for risk assessment.

Executive summary:

Ionol caused embryotrophic effects when administered orally to rats during pregnancy (Schumskaya 1973). However, the published data are insufficient for risk assessment.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: not assignable, publication which does not give sufficient experimental details, e.g. specification of BHT doses not clear
Principles of method if other than guideline:
other: developmental toxicity study
GLP compliance:
no
Species:
rat
Strain:
other: Walter Reed-Carworth Farms strain
Route of administration:
oral: feed
Vehicle:
other: BHT contained in the diet
Duration of treatment / exposure:
days 1 to 22 of pregnancy
Frequency of treatment:
daily
Duration of test:
After positive mating female rats were randomly distributed into treatment and control groups; 22 days after positive mating, the pregnant rats were killed.
No. of animals per sex per dose:
11 females per group
Control animals:
yes, plain diet
Abnormalities:
not specified
Developmental effects observed:
not specified

No adverse effects of BHT on dams and fetuses were seen; however the spontaneous rate of resorptions was reduced in dams treated with BHT compared to control

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.