Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity oral: NOAEL = 150 mg/kg/day (Combined 1-generation reproduction toxicity/13 week repeat dose toxicity study)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Remarks:
other: oral 1-generation reproduction toxicity combined with repeated dose toxicity screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 1979 - February 1980
Reliability:
2 (reliable with restrictions)
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Internal HLE protocol: P943/21/4/3/552/d (25.07.79): Three groups of parental generation rats each comprising 15 males and 15 females were fed diets containing 2,4-di-tert-butyl-phenol (DTBP) at dose levels of 50, 150, or 300 mg/kg for 4 weeks before mating and throughout mating, gestation and lactation. Groups of 20 F1 generation progeny of each sex were selected and maintained on the experimental diets for 13 weeks after weaning. From these animals, five male and five female F1 generation progeny from each of the groups were additionally maintained untreated after the 13 week feeding phase, to investigate the nature of any treatment-related changes observed. Additionally, similar groups of animals were fed untreated powdered diet for similar periods to function as control. Extended haematology, blood chemistry and pathology was conducted on the F1 generation progeny animals. Though the study is not a current OECD guideline study, the experiment combines elements of a 1-Generation Reproduction Toxicity Study (OECD 415) and the Repeated Dose 90-day Oral Toxicity Study in Rodents (OECD 408) and has been considered relevant to address these endpoints.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
Purity at least 97%
Species:
rat
Strain:
other: Sprague-Dawley CD strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (U.K.) Ltd., Manston Road, Margate, Kent
- Age at study initiation: (F0) 0 weeks
- Fasting period before study: None
- Housing: by sex in either stainless steel grid cages suspended over cardboard-lined aluminium trays or in solid-bottomed polypropylene cages furnished with autoclaved softwood chips, 5 animals per cage, single room, exclusive to the study
- Diet (e.g. ad libitum): free access with exception of overnight deprivazion prior to blood sampling and urine collection (approx. 16 h)
- Water (e.g. ad libitum): free access with exception of overnight (approx. 16 h) deprivation prior to urine collection
- Acclimation period (F1): none (born under test conditions)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-25°C
- Humidity (%): 40-60%
- Air changes (per hr): 18-20
- Photoperiod (hrs dark / hrs light): 10 hrs/14 hrs
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
(test article incorporated into powdered diet)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly intervals for each dose and sex group
- Mixing appropriate amounts with (Type of food): Rat and mouse Diet No. 1 Expanded and reground, B.P. Nutrition (U.K.) Ltd., Stepfield, Witham, Essex
- Storage temperature of food: not specified, diet samples were stored at +4°C till further analysis
-The weighted amount of test article was ground in a mortar with a small amount of powdered diet to produce a homogenous mixture of the test article concentration. The concentrate was made up to the final weight with powdered diet and then mixed in a Gardener 3C double cone blender for 10 minutes. From study week 6 onwards, the mixing time was extended to 15 minutes in order to achieve greater homogeneity.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test and control diet prepared for the 1st and 6th weeks of the parental generation feeding phase, and the first and last batches prepared for the F1 generation feeding phase were analyzed for their DTBP content. Additionally, stability of the test substance in the diet was tested after one week at 40 °C. All analytical recovery rates were in the range of 87.8% -113.6% of the nominal test substance amount.
Duration of treatment / exposure:
-Test article/diet mixes continuously available to parental generation animals for 28 days prior to mating and throughout mating, gestation and lactation in females and to parental male animals during mating and until conception in females
- Weaned progeny selected were given appropriate diet continuously for 13 weeks.
Frequency of treatment:
-continuously in diet
Remarks:
Doses / Concentrations:
0, 50, 150, 300 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
F1 offspring: 20 animals per sex and dose, randomly selected for 13 weeks after weaning
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: pretest
- Rationale for animal assignment: random
Positive control:
None
Observations and examinations performed and frequency:
PARAMETERS EXAMINED:
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other: organ weights in study animals ( adrenals, brain, heart, kidneys, liver, pituitary, spleen, testes/ovaries, thyroids). Water consumption per cages was recorded daily, food consumption per cage was recorded weekly.

GROSS EXAMINATION OF DEAD PUPS:
- Not conducted as no dead pups found.

OPHTHALMOSCOPY:
- Method: Keeler, direct ophthalmoscope, after pupil dilatation with 1% tropicamide solution ( Mydriacil, Alcon Laboratories, Texas, US)
- Time schedule: prior to 13 week feeding phase (progeny selected for further test) and after 4, 8, 12 weeks of treatment (animals of groups 0 mg/kg and 300 mg/kg, designated for necropsy at 13 weeks)

LABORATORY STUDIES:
- Time schedule: after 0,4,8 and 12 weeks (blood and urine), after 9 weeks (blood), and during final week of treatment-free period from recovery animals (blood)
- Method (blood): orbital sinus puncture under light ether (Diethyl ether, Analar Grade, BDH Chemicals, Poole, Dorset)
- Method (urine): overnight collection (deprivation of food and water) by isolation in stainless steel metabolism cages
- Haematological examinations: erythrocyte count, haemoglobin concentration, packed cell volume (derived index), mean cell haemoglobin (derived index), mean cell haemoglobin concentration (derived index), mean cell volume, total leucocyte count, differential leucocyte count
- Haematological method: blood samples withdrawn into EDTA anti-coagulant, quality control by in-house scheme and by the International American Hospital Supply DADE scheme
- Blood chemistry parameters: glucose, urea, total protein (females only), albumin, albumin/globulin ratio, glutamate-pyruvate transaminase activity, glutamate-oxaloacetate transaminase activity, total bilirubin, alkaline phosphatase activity (females only), sodium ions, potassium ions
- Haematological method: blood samples withdrawn into EDTA anti-coagulant
- Analytical quality control: by in-house scheme and by the International American Hospital Supply DADE scheme
- Urine analysis: quantitative (volume, specific gravity), semi-quantitative (pH, protein, reducing substances, glucose, ketones, bilirubin, urobilinogen), microscopic examination of centrifuged deposits
Sacrifice and pathology:
SACRIFICE
- Animals not selected for further study were sacrificed via intraperitoneal injection of pentobarbitone sodium solution. These animals were subjected to postmortem macroscopic examinations as follows:

GROSS NECROPSY
All major organs and tissues were examined for the presence of gross lesions.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Excess F1 progeny: Gross lesions observed at necropsy were fixed in 10% buffered formalin.
- F1 selected for study: The tissues (adrenals, brain, heart, kidneys, liver, pituitary, spleen, testes/ovaries, thyroids), were prepared for microscopic examination and weighed, respectively. Samples of the following tissues (except for the eyes fixed in Davidson's fluid and bone marrow smear fixed in methanol) were fixed in 10% buffered formalin (for all groups and processed to slides for histological examination in the control and high dose group): adrenals, aorta, bone( rib), bone marrow (sternal), bone marow smear, brain, caecum, colon, duodenum, epididymides, eye, gross lesions, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes (cervical and mesenteric), mammary gland, oesophagus, optic nerve, pancreas, pituitary, salivary gland (submaxillary), sciatic nerve, seminal vesicle, skeletal muscle (quadriceps), skin, spinal cord (high cervical), spleen, stomach, testes/ovaries, thymus (where present), thyroids, tongue, trachea, uterus/prostate, urinary bladder. Microscopic examination of all listed tissues was performed by a veterinary pathologist for control (Group 1) and high dose (Group 4) animals.
Statistics:
- Body weight: analysis of variance, t-test
- Haematology data: analysis of variance, t-test
- Blood chemistry data: Wilcoxon's rank sum test
- Absolute and relative organ weights: anaylsis of variance, t-test
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment related mortalities and no clinical effects (minor clinical observations did not suggest an effect to treatment with DTBP at any dose level, no deaths in any experimental group during feeding phase).
Mortality:
no mortality observed
Description (incidence):
There were no treatment related mortalities and no clinical effects (minor clinical observations did not suggest an effect to treatment with DTBP at any dose level, no deaths in any experimental group during feeding phase).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced growth rate at 300 mg/kg during lactation, reduced growth rate at 150 mg/kg was associated with a higher average litter number. A dose related retardation of body weight gain in all animals but considered secondary due to palability issues.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Dose-dependent reduction in food consumption and body weight gain.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Overall water consumption of all DTBP-treated groups was slightly lower than controls during treatment. Even more marked difference in water consumption during 4 week treatment-free period.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Observed ocular lesions did not suggest an effect of treatment with DTBP at dose level 300 mg/kg.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Animals treated with 300 mg/kg DTBP showed at the beginning reduced haemoglobin, packed cell volume, mean cell volume and white blood cell counts. These effects were considered to reflect the degree of growth retardation observed during lactation period.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Investigation of the blood chemistry in animals treated at 300 mg/kg/d did not reveal any consistent evidence of an effect of DTBP ingestion
Urinalysis findings:
no effects observed
Description (incidence and severity):
No consistent qualitative or quantitative differences in the urinary constitution of control and DTBP treated animals (300 mg/kg).
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver weight significantly higher during feeding phase in male and female animals at 300 mg/kg. After 4 week without treatment, liver weights returned to normal values. These observations are indicative of an adaptive response to DTBP ingestion.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Observed lesions did not suggest an effect of treatment with DTBP at any dose level.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Observed lesions during histopathological evaluation occurred either in isolation or at low frequency, all lesions found were of the same severity, diverse in their nature and observed in the control and DTBP-treated group (300 mg/kg).
Details on results:
VIABILITY
- External appearance: no differences between DTBP-treated progeny and control progeny (daily observation)
- Mortality: no mortality in any experimental groups during the feeding phase and the subsequent treatment-free period, one control female died shortly after week 0 blood sampling (animal was replaced)

CLINICAL SIGNS
- Overall survival rate: substantially higher at 150 mg/kg compared to all other groups (due to no litter loss at this dose level)
- Source dependent clinical changes observed in controls and all treatment groups: patchy hair loss, ocular/nasal secretion, cutaneous dry scab formation, crooked dentition
- Presence of raised rings around the tail, persistent for approx. 2 weeks, were observed in one control animal, four animals at 50 mg/kg, one animal each at 150 mg/kg and 300 mg/kg.
- Observed clinical observations did not suggest an effect of treatment with DTBP at any dose level.

BODY WEIGHT AND FOOD CONSUMPTION
- Mean pup weight at 300 mg/kg: lower compared to control at day 10 and day 21 post partum
- Mean pup weight at 150 mg/kg: significantly lower than controls at day 21 post partum
- Mean pup weight at 50 mg/kg: significantly lower than controls at day 21 post partum
- There was a dose-dependent reduction in food consumption and body weight gain at all dose levels of DTBP employed. The effect in the females (4% at 50 mg/kg and up to significant 13% at 300 mg/kg) was less marked than in the males (significant body weight gain reduction at all DTBP dose levels, 9% reduction at 50 mg/kg and up to 31% reduction at 300 mg/kg).
- Male animals at 150 mg/kg and 300 mg/kg gained weight at greater rate than controls during 4 week treatment-free period.
- Food consumption in males: during treatment reduction of 8% at 50 mg/kg, 12% at 150 mg/kg, and 20% at 300 mg/kg compared to control, during treatment-free period also lesser food consumption than control
- Food consumption in females: during treatment maximum reduction of 6% at 300 mg/kg compared to control, during treatment-free period less food consumption than control (up to 20% difference at 300 mg/kg)
- Food consumption data during treatment-free period was derived from only one cage per group, therefore biological significance of differences between control and DTBP-treated animals is questionable.

WATER CONSUMPTION
- Overall water consumption of all DTBP-treated groups was slightly lower than controls during treatment. Even more marked difference in water consumption during 4 week treatment-free period.
- Water consumption data during treatment-free period was derived from only one cage per group, therefore biological significance of differences between control and DTBP-treated animals is questionable.

TEST SUBSTANCE INTAKE
- Mean dose level males: 50 mg/kg/d at nominal dose level 50 mg/kg/d (range 41-61 mg/kg/d), 149 mg/kg/d at nominal dose level 150 mg/kg/d (range 118-180 mg/kg/d), 296 mg/kg/d at nominal dose level 300 mg/kg/d (range 235-365 mg/kg/d)
- Mean dose level females: 50 mg/kg/d at nominal dose level 50 mg/kg/d (range 40-56 mg/kg/d), 150 mg/kg/d at nominal dose level 150 mg/kg/d (range 116-180 mg/kg/d), 299 mg/kg/d at nominal dose level 300 mg/kg/d (range 239-374 mg/kg/d)

ORGAN WEIGHTS
- Absolute liver weight significantly lower in all male groups at 150 mg/kg and at 300 mg/kg.
- Absolute heart, gonads, adrenals, spleen, and kidneys weight significantly lower in all animals at 300 mg/kg and partially at 150 mg/kg.
- Relative liver weight significantly higher at 300 mg/kg in males 20% and females 40% and at 150 mg/kg in females 22%. Remained significantly higher in females at 150 mg/kg after the 4 week treatment-free period.
- Relative thyroid and spleen weight significantly higher at 300 mg/kg in males.
- Relative pituitary and kidney weight significantly higher at 300 mg/kg in males and at 150 mg/kg in males.
- Relative brain weight significantly and dose-related increased at 300 mg/kg in males/females and at 150 mg/kg in males. Remained significantly higher in all females of DTBP treated groups and males at 300 mg/kg after the 4 week treatment-free period.
- Relative testis weight significantly and dose-related increased at 300 mg/kg in males and at 150 mg/kg in males.
- All other absolute and relative organ weights were not significantly different from the controls.

GROSS PATHOLOGY
- Observed lesions: principally confined to skin, lungs and liver, including focal alopecia (especially at tail base), ischaemic injury to the papillary process of the caudate liver lobe and foci of pulmonary discolouration
- Present in controls, at 50 mg/kg and at 150 mg/kg.
- Remark 1: in excess F1 no gross lesions attributable to DTBP ingestion
- Remark 2: relatively frequent occurrence of similar lesions in all rats from the specified source
- Observed lesions did not suggest an effect of treatment with DTBP at any dose level.

HISTOPATHOLOGY
- Observed lesions: peribronchial and perivascular lymphoid aggregates in the lungs, multifocal pericholangitis in the liver, diffuse congestion and lymphadentitis in the mandibular lymph nodes, focal eosinophilic gastritis underlying the limiting ridge and testicular interstitial oedema
- Observed lesions during histopathological evaluation occurred either in isolation or at low frequency, all lesions found were of the same severity, diverse in their nature and observed in the control and DTBP-treated group (300 mg/kg).

OPTHALMOSCOPY
- Ocular lesions observed: intravenal haemorrhage, lenticular opacity, fibrous corneal adhesions
- Enlargement and opacity of the right eye were found in one female at 150 mg/kg, four animal in the controls and high dose groups showed either eye enlargement or corneal opacity as a result of the orbital sinus puncture procedure.
- Observed ocular lesions did not suggest an effect of treatment with DTBP at dose level 300 mg/kg.

HAEMATOLOGY
- Erythrocyte count: significantly higher at 300 mg/kg after 8 weeks (males)
- Haemoglobin level: significantly lower at 300 mg/kg at treatment start (males/females), after 4 weeks (females), and after final 4 weeks of treatment period (males).Control and treatment animals showed progressive increase in haemoglobin level during first 9 weeks consistent with maturation. Decrease in haemoglobin level during the final 4 weeks more marked in animals at 300 mg/kg.
- Packed cell volume (derived index): significantly lower at 300 mg/kg at treatment start (males/females), after 4 weeks (males/females), after 8 weeks (females) and after final 4 weeks of treatment period (males)
- Mean cell volume: significantly lower at 300 mg/kg at treatment start (males/females), after 4 weeks (males/females), after 8 weeks (males), after final 4 weeks of treatment period (males/females) and after 4 week treatment-free period (females)
- Leucocyte count: significantly lower at 300 mg/kg at treatment start (males/females)

URINE ANALYSIS
- No consistent qualitative or quantitative differences in the urinary constitution of control and DTBP treated animals (300 mg/kg).

BLOOD CHEMISTRY
- Glucose: significantly (slightly) higher at 300 mg/kg (males/females) after 8 weeks
- Blood urea nitrogen level: significantly (slightly) higher at 300 mg/kg (males/females) at treatment start and after 8 weeks (males)
- Total protein (females only): not significantly different to control
- Albumin: not significantly different to control
- Albumin/globulin ratio: not significantly different to control
- Glutamate-pyruvate transaminase activity: significantly (slightly) lower at 300 mg/kg after 8 weeks (males/females)
- Glutamate-oxaloacetate transaminase activity: significantly (slightly) lower at 300 mg/kg after 8 weeks (males/females)
- Total bilirubin: not significantly different to control
- Alkaline phosphatase activity (females only): significantly (slightly) higher at 300 mg/kg after 12 weeks and not significantly (slightly) higher after 4 week treatment-free period
- Sodium ions: not significantly different to control
- Potassium ions: not significantly different to control
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight
Key result
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight
Key result
Critical effects observed:
not specified
Conclusions:
In conclusion, dietary administration of DTBP at a dose level of 300 mg/kg daily for 13 weeks elicited a primary toxic effect on the growth rate of the rats. At a dose level of 150 mg/kg, elicited growth retardation was considered secondary in relation to reduced diet palatability. With the exception of adaptive hepatic changes and palatability effects, the "no-effect" level established in this study was 150 mg DTBP/kg/day; the liver effects were not considered adverse.
Executive summary:

The 1 -generation fertility / Repeated dose toxicity study was carried out in the rat to investigate the oral toxicity of 2,4-di-tert-butyl-phenol (DTBP). F1 progeny was exposed to the test article through gestation and lactation and for 13 weeks after weaning. In addition, the nature of the treatment-related changes observed during the 13 week treatment period were investigated during a subsequent 4 week treatment-free period for some of the animals.

The parental generation rats were fed diets containing 2,4 -di-tert-butyl-phenol (DTBP) at dose levels of 50, 150 or 300 mg/kg/day for 4 weeks before mating and throughout mating, gestation and lactation. Groups of 20 generation F1 progeny of each sex were maintained on the experimental diets for 13 weeks after weaning. Similar groups of animals fed untreated powdered diet for similar periods acted as control group. Five male and 5 female animals from each of the groups were maintained untreated for 4 weeks after the 13 week feeding phase, to investigate the nature of any treatment-related changes observed.

Reproductive capability of the parental generation animals was unimpaired by the ingestion of DTBP at dose levels of up to 300 mg/kg/d. Ingestion of 300 mg DTBP/kg/d elicited a significant reduction in the mean number of progeny born and reduced the growth rate of the F1 progeny during lactation. The former effect was not apparent at dose levels of 50 and 150 mg/kg/d. The reduced growth rate was also apparent in the progeny of animals treated at 150 mg/kg/d and was associated with a higher average litter number.

There were no substance related deaths in any of the experimental groups during the feeding phase. Daily observations of these animals did not reveal any overt signs of toxicity attributable to the ingestion of DTBP. From the onset of treatment of the animals selected for further study, there was a dose-dependent reduction in the food consumption and body weight gain at all dose levels of DTBP employed. The effect in the females was less marked than in the males. At dose levels of 50 and 150 mg/kg/d the severity of the effect on food consumption was of a similar magnitude to that of the effect on body weight, and probably indicated reduced diet palatability. However, at the highest dose level employed (300 mg/kg/d) the retardation of body weight gain was disproportionally high in relation to the effect on food consumption and therefore considered a primary toxic effect. The water consumption of DTBP-treated groups of animals was generally lower than that of the controls during the 13 week feeding phase. No ocular defects were observed in the animals treated with DTBP at 300 mg/kg/d which could be related to the ingestion of the test article. A low incidence of defects was observed in the control and dose level 300 mg/kg/d animals. Ocular lesions observed included intravitreal haemorrhage, lenticular opacity and fibrous corneal adhesions. The nature of the lesions suggested that they were traumatic lesions resulting from the retro-orbital sinus puncture procedure for blood sampling. At the start of the feeding phase, animals treated with 300 mg/kg DTBP showed reduced haemoglobin, packed cell volume, mean cell volume and white blood cell counts. These effects were considered to reflect the degree of growth retardation observed during the lactation period. Investigation of the blood chemistry and urine constituents in animals treated at 300 mg/kg/d did not reveal any consistent evidence of an effect of DTBP ingestion. The relative liver weight of male and female animals at 300 mg/kg/d was significantly higher than that of the controls. A similar but less marked effect was also apparent in female animals treated at 150 mg/kg/d. After 4 weeks without DTBP ingestion, relative liver weights had returned to normal values in animals treated at 300 mg/kg/d. These observations are indicative of a physiological (adaptive) response to the ingestion of DTBP. This is confirmed by the lack of any microscopic changes in the liver. The relative weight of the kidney of males treated at 150 and 300 mg/kg/d, and the relative spleen weight of males treated at 300 mg/kg/d were increased. The biological significance of these observations is questionable in the absence of microscopic changes. Post-mortem and microscopic examination of the tissues and organs of the animals treated with DTBP at 300 mg/kg/d for 13 weeks did not reveal any gross or microscopic lesions which could be attributed to the ingestion of DTBP. Dietary administration of DTBP at a dose level of 300 mg/kg daily for 13 weeks to rats elicited a primary toxic effect on growth rate, and at dose level of 150 mg/kg/d, elicited growth retardation which was secondary to reduced diet palatability.

With the exception of adaptive, hepatic changes and palatability effects, the "no adverse effect" level established in this study was 150 mg DTBP/kg/d, which is considered a no adverse effect level.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

The 1 -generation fertility / Repeated dose toxicity study with 2,4-di-tert-butyl-phenol (DTBP) was carried out in the rat to investigate the fertility in the parental generation (F0) and the effects of sub-chronic exposure in the F1 generation who were exposed to DTBP throughout gestation, lactation and via the diet for 13 weeks after weaning. In addition, the nature of the treatment-related changes observed during the 13 week treatment period were investigated during a subsequent 4 week treatment-free period for sub-groups of F1 generation animals at each dose level.

The parental generation rats were fed diets containing DTBP at concentrations which provided an approximately constant intake of 50, 150 or 300 mg/kg bw/day for 4 weeks before mating and throughout mating, gestation and lactation. Groups of 20 generation F1 progeny of each sex were randomly selected from the weaned offspring of litters which were approximately the same age and maintained on the experimental diets for 13 weeks after weaning. Similar groups of animals fed untreated powdered diet for similar periods acted as control group. Five male and 5 female animals from each of the groups were maintained untreated for 4 weeks after the 13 week DTBP exposure phase, to investigate the reversibility of any treatment-related changes observed.

F0 and F1 animals were observed frequently for clinical reactions to treatment, bodyweights were measured at weekly intervals and the food consumed by each cage of animals was measured weekly. Water consumption for each cage of F0 and F1 animals was measured daily for F0 animals prior to mating and daily for F1 animals during the 13 week exposure period. Ophthalmoscopic examinations and clinical pathology (haematology, clinical chemistry and urinalysis) were performed at approximate 4 week intervals during the 13 week exposure period. At the end of the 13 week exposure period and at the end of the 4 week treatment free period all animals were sacrificed and examined macroscopically. A full selection of tissues were harvested, selected tissues were weighed, and tissues were processed for histological examination in control and 300 mg/kg bw/day animals.

The reproductive capability of the parental generation animals has been described in Section 5.9.3 of the dossier whilst the sub-chronic toxicity findings are described in this summary.

There were no substance related deaths in any of the experimental groups during the DTBP exposure phase. Daily observations of these animals did not reveal any overt signs of toxicity attributable to the ingestion of DTBP. From the onset of treatment of the animals selected for further study, there was a dose-dependent reduction in the food consumption and body weight gain at all dose levels of DTBP employed. The effect in the females was less marked than in the males. At dose levels of 50 and 150 mg/kg bw/day the severity of the effect on food consumption was of a similar magnitude to that of the effect on body weight, and probably indicated reduced diet palatability. However, at the highest dose level (300 mg/kg bw/day) the retardation of body weight gain was disproportionally high in relation to the effect on food consumption and therefore considered a primary toxic effect. The water consumption of DTBP-treated groups of animals was generally lower than that of the controls during the 13 week DTBP exposure phase.

No ocular defects were observed in the animals treated with DTBP at 300 mg/kg bw/day which could be related to the ingestion of the test article. At the start of the DTBP exposure phase, animals treated with 300 mg/kg DTBP showed reduced haemoglobin, packed cell volume, mean cell volume and white blood cell counts. These effects were considered to reflect the degree of growth retardation observed during the lactation period. Investigation of the blood chemistry and urine constituents in animals treated at 300 mg/kg bw/day did not reveal any consistent evidence of an effect of DTBP ingestion. The relative liver weight of male and female animals at 300 mg/kg bw/day was significantly higher than that of the controls. A similar but less marked effect was also apparent in female animals treated at 150 mg/kg bw/day. Post-mortem and microscopic examination of the tissues and organs of the animals treated with DTBP at 300 mg/kg bw/day for 13 weeks did not reveal any gross or microscopic lesions which could be attributed to the ingestion of DTBP.

 After 4 weeks without DTBP ingestion, relative liver weights had returned to normal values in animals treated at 300 mg/kg bw/day. These observations are indicative of a physiological (adaptive) response to the ingestion of DTBP. This is confirmed by the lack of any microscopic changes in the liver. The relative weight of the kidney of males treated at 150 and 300 mg/kg bw/day, and the relative spleen weight of males treated at 300 mg/kg bw/day were increased. The biological significance of these observations is questionable in the absence of microscopic changes.

Dietary administration of DTBP at a dose level of 300 mg/kg daily for 13 weeks to rats elicited a primary toxic effect on growth rate, and at dose level of 150 mg/kg bw/day, elicited growth retardation which was secondary to reduced diet palatability.

With the exception of adaptive, hepatic changes and palatability effects, the "no observed adverse effect" level established in this study was 150 mg DTBP/kg/d, which is considered a NOAEL.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
This study was scored a reliability rating of 2. Due to its age it pre-dates GLP and current guidelines, however in terms of repeat dose toxicity it presents a worst case study as the animals were exposed to the test substance during gestation, lactation and for 13 weeks following weaning.

Justification for classification or non-classification

Repeated dose toxicity oral:

No classification. The primary toxic effect observed in the study was a reduction of growth rate at 300 mg/kg/day. The NOAEL in this study was 150 mg/kg/day.