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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 1979 - February 1980
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
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. Five male and 5 female F1 generation progeny from each of the groups were 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Purity at least 97%

Test animals

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: (P) approx. 11.5 wks
- Weight at study initiation: (P) Males: 317-362 g; Females: 205-249 g
- 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 (except for mating 1 female:1 male and gestation 1 female), single room, exclusive to the study
- Diet (e.g. ad libitum): free access with exception of overnight (approx. 16 h) deprivation prior to blood sampling and urine collection
- Water (e.g. ad libitum): free access with exception of overnight (approx. 16 h) deprivation prior to urine collection
- Acclimation period: approx. 2.5 weeks

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

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Remarks:
test article incorporated into powdered diet
Details on 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 the 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.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until pregnancy occurred to a maximum of 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing no replacement of first male. Only one female (no. 82 - 50 mg/kg) did not mate.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually in polypropylene cages during gestation



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 was established in females.
-Weaned progeny selected were given appropriate diet continuously for 13 weeks.
Frequency of treatment:
-continuously in diet
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150, 300 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
-(F0) 15 animals per sex and dose
-(F1) 20 animals per sex and dose, randomly selected
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: pretest
- Rationale for animal assignment: random
Positive control:
None

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
- Observations: ill-health, overt toxicity

BODY WEIGHT:
- Time schedule for examinations: weekly, females during gestation in 3-day interval

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION :
- Time schedule for examinations: daily



Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
- The number, sex, litter weight by sex and presence of any external morphological defects was determined 1,4,10 and 21 days postpartum. Animals for further studies were randomly selected (20 males and 20 females per dose level), excess animals were killed.

PARAMETERS EXAMINED:
The following parameters were examined in 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:
- 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
- Urine analysis: quantitative (volume, specific gravity), semi-quantitative (pH, protein, reducing substances, glucose, ketones, bilirubin, urobilinogen), microscopic examination of centrifuged deposits
Postmortem examinations (parental animals):
SACRIFICE
All surviving animals (male/female) were killed 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 WEIGHTS:
Gross lesions observed at necropsy were fixed in 10% buffered formalin. No organ weights were recorded.
Postmortem examinations (offspring):
SACRIFICE
The F1 offspring not selected as parental animals was 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
- Mean litter number at birth, mean pup weights, mean litter weights (corrected): Wilcoxon's rank sum 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
Reproductive indices:
- reproductive performance, litter number, mean number per dam, sex ratio of litter

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No changes in condition or behaviour which suggested an effect of DTBP treatment at any dose level.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain retarded at 150 mg/kg and body weight loss at 300 mg/kg, body weight growth rate at 50 mg/kg was similar to control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Body weight gain retarded at 150 mg/kg and body weight loss at 300 mg/kg, body weight growth rate at 50 mg/kg was similar to control.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Reproductive capability unimpaired, but reduction of mean number of progeny born at 300 mg/kg.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Generalised deterioration in clinical condition in 1 animal (male, 300 mg/kg), necropsy on day 39 revealed internal changes including pallor of all internal organs, discolouration of the liver, kidneys and lymph nodes and prominent splenic white pulp, no cause of clinical deterioration could be deduced from these findings.
- Mortality in one animal (female, 50 mg/kg) on 24th day after pregnancy, post-mortem examination revealed 14 fetuses in utero and one fetus in presented in difficult position for birth, gestation period of this animal lasted approx. 2.5 days longer than average for the study, it was concluded that death was due to complications in parturition.
- Minor and non- specific changes in condition among all groups, commonly observed in rats from the specific source of supply.
- Blood-stained vaginal discharge in many females observed, considered to signify onset of parturition.
- No changed in condition or behaviour which suggested an effect of DTBP treatment at any dose level.

BODY WEIGHT (PARENTAL ANIMALS)
During first 2 weeks of treatment animals in group 300 mg/kg lost weight and animals in group 150 mg/kg generally showed reduced weight gain in comparison with controls. Statistically significant and dose-related reduction in the overall body weight gain in both, males and females, at 150 mg/kg and 300 mg/kg. But weight gain in females during gestation and lactation were comparable to controls. No differences in weight gain of group 50 mg/kg compared to controls.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Overall food consumption at 150 mg/kg was 11 % (males) and 6% (females) and at 300 mg/kg 24% (males) and 13% (females) lower than in the controls. Reduced food consumption was observed in males and females during 4 week pre-mating period (more pronounced in the early part of pre-mating period) and for males also during the succeeding 3 weeks until necropsy. Food consumption of females, 150 mg/kg and 300 mg/kg, was similar to the controls throughout gestation and lactation. No differences in food consumption of group 50 mg/kg compared to controls.

WATER CONSUMPTION (PARENTAL ANIMALS)
Overall water consumption was higher than controls at 300 mg/kg with 26% (males) and 15 % (females) and at 150 mg/kg with 10% (males) and 12% (females) higher than controls. Animals at 50 mg/kg consumed 9% (males) and 13% (females) less water than controls.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
- Mean dose level during pre-mating and mating period: 49 mg/kg (males, at dose level 50 mg/kg, weeks 1-7, range 42-53 mg/kg), 50 mg/kg (females, at dose level 50 mg/kg, weeks 1-4, range 43-53 mg/kg), 150 mg/kg (males, at dose level 150 mg/kg, weeks 1-7, range 120-176 mg/kg), 146 mg/kg (females, at dose level 150 mg/kg, weeks 1-4, range 123-156 mg/kg), 297 mg/kg (males, at dose level 300 mg/kg, weeks 1-7, range 176-357 mg/kg), 303 mg/kg (females, at dose level 300 mg/kg, weeks 1-4, range 196-352 mg/kg)
- Mean dose level during gestation: 53 mg/kg (females, at dose level 50 mg/kg, range 49-59 mg/kg), 158 mg/kg (females, at dose level 150 mg/kg, range 141-167 mg/kg), 326 mg/kg (females, at dose level 300 mg/kg, range 281-365 mg/kg)
- Mean dose level during lactation: 61 mg/kg (females, at dose level 50 mg/kg, range 50-72 mg/kg), 196 mg/kg (females, at dose level 150 mg/kg, range 161-223 mg/kg), 377 mg/kg (females, at dose level 300 mg/kg, range 297-528 mg/kg)
- Remark: calculated intakes probably unreliable due to scattering of food.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating: one mating failure in a female animal at 50 mg/kg, general mating time course at all treatment levels similar to controls, all animals mated within 10 days of pairing.
- Gestation: mean duration of gestation was either 21 or 22 days in all groups (including control).
- Litter production: litter was produced by all female animals in the control, at 150 mg/kg and 300 mg/kg. At 50 mg/kg 13 of 15 females produced litter, one animal failes to mate and the other one died during parturition.
- Mean litter number: 13.5 progeny/dam in controls, 11.9 progeny/dam at 300 mg/kg (significantly lower than control) 14.5 progeny/dam at 50 mg/kg, 13.9 progeny/dam at 150 mg/kg
- Total litter loss during lactation period: 5 control females, 5 females at 50 mg/kg, and 2 females at 300 mg/kg (Neonatal mortality was usually associated with evidence of maternal neglect, such as no milk in the stomach and non-removal of placenta or foetal membranes.)

GROSS PATHOLOGY (PARENTAL ANIMALS)
- Observed lesions: pulmonary sub-pleural grey foci, alopecia, hydronephrosis, single incident of swollen ureters with yellow foci at the junction with the urinary bladder
- Remarks: no gross lesion in the female animal (50 mg/kg) that died due to complications during parturition; the male animal (300 mg/kg) killed, showed several gross lesions including pallor of all internal organs, green kidneys, yellow liver with red punctate foci throughout, prominent splenic white pulp, dark mesentric and yellow mandibular lymph nodes
- Observed lesions and their distribution between treatment groups did not suggest an effect of treatment with DTBP at any dose level.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: body weight

Target system / organ toxicity (P0)

Key result
Critical effects observed:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
Observed clinical observations did not suggest an effect of treatment with DTBP at any dose level.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No differences in external appearance and no deaths in any experimental groups during the F1 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.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Effects on absolute weight of adrenals, brain, heart, kidneys, liver, pituitary, spleen, testes/ovaries, thyroids.
Gross pathological findings:
no effects observed
Description (incidence and severity):
In excess F1 animals no gross lesions attributable to DTBP ingestion.
Histopathological findings:
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).
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
- External appearance: no differences between DTBP-treated progeny and control progeny (daily observation)
- Mortality: no mortality in any experimental groups during the F1 feeding phase and the subsequent treatment-free period (one control female died shortly after week 0 blood sampling and was replaced)

CLINICAL SIGNS (OFFSPRING)
- 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 (OFFSPRING)
- Mean pup weight at 300 mg/kg: lower compared to control at day 10 and day 21 post partum
- Mean litter weights at 300 mg/kg: lower compared to control at day 10 and significantly lower on day 21 (15%) post partum
- Mean pup weight at 150 mg/kg: significantly lower than controls at day 21 post partum
- Mean litter weights at 150 mg/kg: higher than controls during lactation, significantly higher at days 1, 4 and 10 post partum
- Mean pup weight at 50 mg/kg: significantly lower than controls at day 21 post partum
- Mean litter weights at 50 mg/kg: lower than controls at day 21 post partum
- Group mean body weight of progeny from all DTBP-treated groups was significantly lower than controls at onset of treatment.
- 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 (OFFSPRING)
- 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)
- Remark: 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 (OFFSPRING)
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 (OFFSPRING)
- 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 (OFFSPRING)
- 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 weights 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 weights significantly higher at 300 mg/kg in males.
- Relative pituitary and kidney weights 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 weights 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 (OFFSPRING)
- 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 (OFFSPRING)
- 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 (OFFSPRING)
- 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 (OFFSPRING)
- 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 (OFFSPRING)
- No consistent qualitative or quantitative differences in the urinary constitution of control and DTBP treated animals (300 mg/kg).

BLOOD CHEMISTRY (OFFSPRING)
- 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

OTHER FINDINGS (OFFSPRING)
- Sex ratio: no differences during weaning

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
not specified

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, F0 generation animals treated with DTBP at 300 mg/kg bw and 150 mg/kg bw showed reduced food consumption and body weight gain during the pre-mating period. Reproductive capability was unimpaired, but ingestion of 300 mg/kg DTBP elicited a reduction in the mean number of progeny born and reduced the growth rate during lactation. Post weaning, the F1 generation rats showed a dose dependent reduction in food consumption and body weight gain, the effect at 150 mg/kg bw was related to reduced 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.
Executive summary:

A combined 1-Generation Reproduction Toxicity Study / Repeated Dose Toxicity Study was conducted in rats. This study was carried out to investigate the oral toxicity of 2,4-di-tert-butyl-phenol (DTBP) to prenatal animals prior and through mating, gestation and lactation. F1 progeny was maintained on the experimental diet for 13 weeks after weaning. Recovery was investigated in a number of F1 animals throughout an additional subsequent 4 week treatment-free period.

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/day for 4 weeks before mating and throughout mating, gestation and lactation. Groups of 20 F1 generation 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 F1 generation progeny from each of the groups were maintained untreated after the 13 week feeding phase, to investigate the nature of any treatment-related changes observed.

One male animal of the parental generation at dose level 300 mg/kg/d was killed and necropsied on day 39, following a generalised deterioration in condition. A cause of clinical deterioration could not be deduced from the findings at necropsy. A pregnant animal at dose level 50 mg/kg/d died after a prolonged gestation period, and at necropsy was noted to have a fetus presented for birth in the breech position. All other P generation animals survived the scheduled treatment period, and showed no overt signs of toxicity attributable to the ingestion of DTBP.

Parental generation animals treated with DTBP at 150 mg/kg/d showed retarded body weight gain during the pre-mating period, and those treated at 300 mg/kg showed body weight losses during the first 2 weeks of the study. Animals treated at 50 mg/kg/d showed a growth rate similar to that of the controls, as did all DTBP-treated pregnant females during gestation and lactation. The food consumption of parental generation animals treated at 150 or 300 mg/kg/d was reduced during the pre-mating period and water consumption was markedly increased. In conjunction with the reduced weight gain, these data are suggestive of a reduction in the palatability of the diets. The food and water consumption of animals treated at 50 mg/kg/d were similar to those of the controls. Reproductive capability of the parental generation animals was unimpaired by the ingestion of DTBP at dose levels of up to 300 mg/kg/d. However, the data suggested that ingestion of 300 mg DTBP/kg/d elicited a 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. Although reduced growth rate was also apparent in the progeny of animals treated at 150 mg/kg/d, it was associated with a higher average litter number. Post-mortem examination of the parental generation animals and the surplus F1 progeny did not reveal any gross lesions attributable to the ingestion of DTBP at any of the dose levels employed. There were no deaths in any of the experimental groups during the F1 generation 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 F1 generation 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. Therefore, a primary toxic effect on growth rate may have been produced by the ingestion of DTBP at 300 mg/kg/d. The water consumption of DTBP-treated groups of animals was generally lower than that of the controls during the 13 week F1 generation feeding phase. No ocular defects were observed in the F1 generation 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 F1 generation treatment period, 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 was 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 high in relation to those of the controls. The biological significance of these observations is questionable in the absence of microscopic changes in the architecture of these organs. Post-mortem examination of the parental generation animals treated with DTBP at dose levels of up to 300 mg/kg/d did not reveal any treatment related gross lesions. Post-mortem and microscopic examination of the tissues and organs of F1 generation 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 the F1 generation rats probably 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.