2,4-di-tert-butylphenol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a combined 1-Generation Reproduction Toxicity Study / Repeated Dose Toxicity Study was conducted in rats. Dietary administration of DTBP to parental animals prior and through mating, gestation and lactation had no effect on reproductive performance when numbers of animals mated, numbers of litters born, sex ratio and viability of the progeny was considered. However, the total number of pups born and the mean number of pups per dam was lower in parental animals exposed to DTBP at 300 mg/kg bw/day and in those animals, mean pup weight at weaning was also lower than in control animals. Whilst lower pup weight at weaning was also noted for the 150 mg/kg bw/day group this was associated with a higher number of pups per litter and therefore was not an effect of treatment with DTBP. A NOAEL of 150 mg DTBP/kg bw/day was identified in this study.

Link to relevant study records

Reference

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)

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

Specific details on test material used for the study:

Purity at least 97%

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD strain

Sex:

male/female

Details on test animals or test system 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

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

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

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

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

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: 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.

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.

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

Key result

Critical effects observed:

not specified

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

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

no effects observed

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

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

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Sufficient to address requirements.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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 parental animals prior and through mating, gestation and lactation. After weaning, randomly selected F1 progeny pups were exposed to DTBP via the experimental diet for 13 weeks or exposed to the experimental diet for 13 weeks followed by a 4 week treatment free period. The results of 13 week exposure of the F1 generation are reported in the repeat dose toxicity section of the dossier.

The reproductive performance of three groups of rats each comprising 15 males and 15 females (parental generation) treated via diets containing DTBP at concentrations which provided an approximately constant intake of 50, 150 or 300 mg/kg bw/day was assessed. The animals were fed the experimental diet for 4 weeks before mating and throughout mating, gestation and lactation. In addition to general health and well-being observations (clinical symptoms, body weight, food and water intake) reproductive observations included time to mating, total number mated in each group, pregnancy rate, duration of gestation, number of litters born, total number of pups, mean number of pups per dam, sex ratio of pups and viability during lactation.

One male animal of the parental generation at dose level 300 mg/kg bw/day 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 bw/day died after a prolonged gestation period, and at necropsy was noted to have a fetus presented for birth in the breech position. All other parental 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 bw/day showed retarded body weight gain during the pre-mating period, and those treated at 300 mg/kg bw/day showed body weight losses during the first 2 weeks of the study. Animals treated at 50 mg/kg bw/day 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 bw/day was reduced during the pre-mating period but similar throughout gestation and lactation, 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 bw/day were similar to those of the controls.

Reproductive capability of the parental generation animals was unimpaired by the ingestion of DTBP at dose levels up to 300 mg/kg bw/day. Only 1 female (50 mg/kg bw/day) in the study failed to mate within the 14 day pairing period; this was considered to be unrelated to treatment with DTBP as there was 100% pregnancy rate in the 150 and 300 mg/kg bw/day females.

No effect of treatment was noted on duration of gestation, numbers of litters born or sex ratio of the progeny. However, the data suggested that ingestion of 300 mg DTBP/kg bw/day elicited a reduction in the mean number of progeny born (179 pups or 11.9 pups per dam in comparison to 202 pups and 13.5 pups per dam in the control group).

Growth rate of the 300 mg/kg bw/day F1 progeny was reduced during lactation (300 mg/kg bw/day group mean pup weight at weaning was 30.7 g, whilst control mean pup weight was 36.3 g) . The former effect was not apparent at dose levels of 50 and 150 mg/kg bw/day. Although reduced growth rate was also apparent in the progeny of animals treated at 150 mg/kg bw/day, it was associated with a higher average number per litter.

There was no effect of treatment on viability of the F1 generation up to weaning and there were no deaths in any of the experimental groups during the F1 generation feeding phase.

Post-mortem examination of the parental generation animals treated with DTBP at dose levels of up to 300 mg/kg bw/day and the surplus F1 progeny 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 bw/day for 13 weeks did not reveal any gross or microscopic lesions which could be attributed to the ingestion of DTBP.

Dietary administration of DTBP to parental animals prior and through mating, gestation and lactation had no effect on reproductive performance when numbers of animals mated, numbers of litters born, sex ratio and viability of the progeny was considered. However, the total number of pups born and the mean number of pups per dam was lower in parental animals exposed to DTBP at 300 mg/kg bw/day and in those animals, mean pup weight at weaning was also lower than in control animals. Whilst lower pup weight at weaning was also noted for the 150 mg/kg bw/day group this was associated with a higher number of pups per litter and therefore was not an effect of treatment with DTBP.

It may therefore be concluded that the "no effect" level established in this study was 150 mg DTBP/kg bw/day.

Short description of key information:
NOAEL (no-observable adverse effect level) parent animals and pups = 150 mg/kg body weight/day.

Justification for selection of Effect on fertility via oral route:
This study was scored a reliability rating of 2. Due to its age it pre-dates GLP and current guidelines. Nevertheless it investigates the reproductive performance of parenteral generation animals and the viability of the offspring exposed to the test substance for 4 weeks before mating, throughout mating, gestation and lactation.

Effects on developmental toxicity

Description of key information

The maternal and developmental No observed Adverse Effect Level (NOAEL) for 2,4-Di-tert-butylphenol was identified to be 70 mg/kg bw/day in a prenatal developmental toxicity study in the rabbit.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March to September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: supporting information on dose selection

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2,4-Di-tert-butylphenol
- Analytical purity: 99.76 %
- Retest date of the lot/batch: November 2019

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Age at study initiation: 18 and 20 weeks old
- Weight at study initiation: between 2895 and 4245 g
- Housing: On arrival and following randomization females were housed individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20 °C
- Humidity (%): 47 to 76%
- Air changes (per hr): >=10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08.05.22019 or 10.05.2019 To: 07.06.2019

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Formulations were heated to a maximum temperature of 40°C (±5°C) for 60 minutes (+/- 5 minutes) to obtain visual homogeneity. The dosing formulations were prepared at least weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature under controlled conditions (water bath set at 20°C (±5°C)) for at least 30 minutes and dosed within 24 hours after removal from the refrigerator.
Test item dosing formulations were kept at room temperature and protected from light until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity/density of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure (for details see "any other information on materials and methods incl. tables). According to these the test item is only soluble in oily vehicles.
- Concentration in vehicle: 0, 20, 50, 140 mg/L
- Amount of vehicle (if gavage): 0.5 mL/kg bw
- Lot/batch no. (if required): MKCG3257 + MKCH1635

The test substance was administered to the animals once daily by oral gavage 7 days a week from Day 6 to Day 28 post-coitum, inclusive. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic catheter attached to a plastic disposable syringe. A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Formulations were heated to a maximum temperature of 40°C (±5°C) for 60 minutes (+/- 5 minutes) (for exceptions, see Appendix 8) to obtain visual homogeneity. The dosing formulations were prepared at least weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature under controlled conditions (water bath set at 20°C (±5°C) from 18 May 2019 onwards) for at least 30 minutes and dosed within 24 hours after removal from the refrigerator.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20177664) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity.

Details on mating procedure:

Time-mated animals were used.

Duration of treatment / exposure:

gestation day 6 - 28

Frequency of treatment:

daily

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

70 mg/kg bw/day

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the results of a tolerability (see attachment) and a dose range-finding study (see supporting study), the following doses were selected for the prenatal toxicity study in rabbits:
- 10 mg/kg bw
- 25 mg/kg bw
- 70 mg/kg bw

Maternal examinations:

mortality/moribundity: twice daily
clinical signs: once daily
body weights: Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum
food consumption: Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum. For the main study, food consumption was additionally measured over Days 3-6 post-coitum.
Water consumption: was monitored on regular basis throughout the study by visual inspection of the water bottles/containers. No quantitative investigation.
gross necropsy: an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs
organ weights (uterine, liver and kidney)
number of corpora lutea
(gravid) uterine weight
uterine contents

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Live fetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube. Recognizable live fetuses of animals sacrificed before planned necropsy (Female Nos. 37, 58, 86 and 87) were euthanized by decapitation. For recognizable fetuses or normal implantations in development of females sacrificed before planned necropsy or that delivered before the day of scheduled necropsy, a gross external examination was performed (if possible). Recognizable fetuses with abnormalities were fixed in 10% buffered formalin.

Litters of females surviving to scheduled necropsy, were subjected to detailed external, visceral and skeletal examinations, as described in the following sections.
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

Each viable fetus was examined in detail to detect macroscopic visible abnormalities and their weight was determined. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed. One late resorptions with malformations (A030-03) were fixed in 10% buffered formalin.

All fetuses were internally sexed and examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe ref 1. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar.
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.
All carcasses, including the carcasses without heads, were eviscerated, skinned, labeled and fixed in 96% aqueous ethanol for subsequent examination of skeletons.

All eviscerated fetuses, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson.
Subsequently, the skeletal examination was done on all fetuses from all groups.
All specimens were archived in glycerin with bronopol as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and Study Director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Statistics:

All stat. tests were conducted at the 5% signif. level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Num. data collected on scheduled occasions for the listed variables were analyzed as indicated acc. to sex and occasion. Descriptive stat. number, mean and SD (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential stat. were performed acc. to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric: Datasets with at least 3 groups (the designated control and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (% of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (% per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed stat. signif. (p<0.05) intergroup variance, Dunn’s test was used to compare compound-treated to control.

Incidences:
An overall Fisher’s exact test was used to compare all groups at the 5% signif. level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% signif. level if the overall test was significant.
No stat. were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Historical control data:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Reduced faeces production (up to severe) was noted in the majority of the animals across all groups, including controls, with a slightly higher persistence and/or severity at 70 mg/kg/day. Transient diarrhea (on a single day) was observed in one female of the low dose group, one female of the mid dose group and two females of the high dose group.

In the high dose group another female presented with hunched posture at the end of the treatment period. In the mid dose group one female showed piloerection during the last eight days of treatment and another female of this group had a lean appearance at the end of treatment. At the incidence observed and in the absence of dose response, these finding were considered unrelated to treatment with the test item.

Other clinical signs noted during the treatment period included scabs, scars, wounds, erythema, alopecia, watery discharge from the eye, restlessness, missing toes (Female of the mid dose group, which was probably missed during the health inspection of the animals and was only noted during handling for dosing) and salivation. These clinical signs occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):

effects observed, non-treatment-related

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female of the high dose group was euthanized on Day 3 post-coitum, before initiation of dosing, as it presented with clinical signs including abnormal, hunched and flat posture, uncoordinated movements, dropped and tilted head, abnormal gait and quick breathing. At necropsy, she was found to have a broken cranial spine.

In total, four females were euthanized in extremis after dosing had been initiated:
One female of the high dose group, was sacrificed on Day 18 post-coitum as she presented with minimal to absent food consumption and persistent body weight loss (up to 9%) from the initiation of treatment onwards. Clinical signs included severely reduced faeces production for several consecutive days and piloerection on Day 18 post-coitum. No macroscopic findings were noted at necropsy. As the condition of the female was more severe than the health condition of the remainder of high dose females, absent food consumption with concurrent body weight loss is occasionally observed in rabbits, the preterm sacrifice of this female was considered unrelated to treatment.

Another female of the high dose group, was sacrificed on Day 18 post-coitum as severe clinical signs were observed during the morning check on this day, most likely due to an earlier oral misgavage, including flat posture, gasping and paleness of the skin. At necropsy, dark red discolored lungs were observed, together with a dark red fluid in the thoracic cavity. In addition, granular tan contents in the pericardium were noted. On the days prior to this preterm sacrifice, the animal presented with very low or absent food consumption and persistent body weight loss (up to 7%) from initiation of treatment onwards, and with a reduction in faeces production (up to severe) for several consecutive days.

One female of the low and one female of the mid dose group were sacrificed on Day 15 post-coitum as they presented with absent food consumption from the pre-treatment period (Day 3 post-coitum) together with body weight loss from Day 0 post-coitum onwards (5 and 15% , respectively). Severe reduction in faeces production from the initiation of treatment onwards was noted for both females. Additional clinical signs for the animal of the mid dose group included hunched posture, lean appearance, piloerection and red fluid on the manure tray for one to three days prior to sacrifice. This female was found emaciated and had a reddish discoloration of the thymus. No macroscopic findings were noted at necropsy for the animal of the low dose group. As the absence of food consumption and loss of body weight commenced prior to initiation of dosing, the preterm sacrifice of these animals was not attributed to treatment with the test item.

In addition, one female of the control and one female of the low dose group were euthanized on Days 27 and 29 post-coitum, respectively, as they delivered their offspring early/aborted. The control animal presented with hunched posture and absent faeces production on the days prior to sacrifice and reduced faces production (up to moderate) from the initiation of treatment onwards. In addition, severe body weight loss (11%) on Day 27 post-coitum and absent food consumption over Days 21-27 post-coitum was observed. The female of the low dose group was observed with reduced faeces production on several days and with diarrhea for one day. Body weight loss (up to 7%) was observed on between Days 6 to 9 post-coitum and between Days 21 and 29, with recovery in between, and absent food consumption concurrently with the body weight loss. As these early deliveries occurred in the control group and low dose group only, this was considered incidental and unrelated to the treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the high dose group, a statistically significant mean body weight loss of 1% was noted over Days 6-9 post-coitum with subsequent significant reduction in body weight gain over Days 6-15 post-coitum when compared to concurrent control mean. From Day 21 post-coitum onwards, mean body weight gain in the high dose group remained in the same range as controls.

In the mid dose group, absent mean body weight gain was observed over Days 6-9 post-coitum. In addition, body weight gain over Days 12-15 post-coitum was slightly reduced when compared to concurrent control mean, but without reaching statistical significance.

No relevant changes in body weight and body weight gain were noted in the low dose group.

Mean body weight loss corrected for uterus weight was observed for all groups, including controls. All values remained within the same range and were considered unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In the high dose group food consumption (before or after allowance for body weight) was reduced over Days 6-15 post-coitum when compared to concurrent control mean (up to 52% of relative values during the first 3 days of treatment; statistically significant between Days 6-12 only), followed by a recovery from Day 18 post-coitum onwards.
In the low and mid dose group, a slight reduction (not statistically significant) in food consumption (before or after allowance for body weight) compared to concurrent controls was noted between Days 6-12 post-coitum. As the effects were only slight and not statistically significant, these were considered not toxicologically relevant.
Any other statistically significant changes were considered chance findings as no trend was apparent regarding dose and duration of treatment.

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, non-treatment-related

Description (incidence and severity):

Organ weights and organ:body weight ratios of treated animals were considered to be comparable to those of control animals, except for the mean kidney:body weight ratio in the high dose group for which a statistically significantly increase was observed. This slight increase was considered not to be a sign of toxicity in the absence of a dose response and macroscopic findings in the kidney.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Incidental findings among control and treated animals included alopecia, pale discoloration of the liver and reduced size of the gallbladder. In addition, incidental finding among treated animals included cyst(s) on the oviduct, accentuated lobular pattern of the liver, enlarged gallbladder with mucous content, agenesis of the gallbladder, hardened right median liver lobe with grey-white foci, ectopic splenic tissue and grey-white/greenish isolated nodules on the papillary process of the liver. In the absence of a dose-related incidence these were considered of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

A slight (not statistically significant) decrease in late resorption in fetuses at 25 mg/kg/day was observed. In the absence of a dose response this was considered a chance finding and not related to treatment with the test item.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The slight (not statistically significant) decrease in viable fetuses at 25 mg/kg/day, resulted from a slight increase in late resorption. In the absence of a dose response this was considered a chance finding and not related to treatment with the test item.

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

One females of the control group and one female of the low dose group had an early delivery on day 27 and 29, respectively.

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 70 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: overall effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the low dose group, mean fetal female weight were slightly below the lower limit of the historical control range. However, in the absence of a dose response, and all other mean fetal weight being comparable to the control group and remaining within the historical control data, this slightly lower fetal weight observed was considered to be of no toxicologically relevance.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on external morphology observed following treatment up to 70 mg/kg/day.
Two external malformations were observed in this study at scheduled necropsy. I the high dose group one fetus was observed with a short tail with matching skeletal findings (vertebrae posterior to caudal vertebra #9 absent; caudal vertebra #8 and #9 malformed and fused). In the mid dose group one fetus was observed with flexure of both tarsals, which could not be substantiated by skeletal examinations as this was only performed for litters of the control and high dose group. Because the latter malformation was observed in a litter with only one alive fetus and three late resorptions, the mean group incidence of this finding (5.3% per litter) raised above the historical control maximum value of 1.1 % per litter. Nevertheless, both findings were observed previously in historical control fetuses and due to the single occurrence, they were considered chance findings.
One female of the control group delivered her offspring early on Day 27 post-coitum. One of her fetuses was observed with an omphalocele. As this female was part of the control group, this malformation was not related to the test item.
No external variations were observed in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on skeletal morphology following treatment at 70 mg/kg/day.
AIn the mid and high dose group one fetus each were observed a skeletal malformation, as a vertebral anomaly with or without associated rib anomaly was found during skeletal examination. A vertebral anomaly was observed also observed in two control fetuses.
In addition, one fetus in the control group and one fetus in the mid-dose group were observed with a vertebral centra anomaly. Due to the group distribution none of the skeletal findings were considered to be test item-related. No other skeletal malformations were observed
Skeletal variations occurred at an incidence of 71.4%, 69.4%, 66.9 and 84.7% per litter in the control, 10, 25 and 70 mg/kg/day groups, respectively. All noted variations were considered not to be related to the treatment with the test item, as they occurred infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on visceral morphology following treatment up to 70 mg/kg/day.
Visceral malformations occurred in 5 (4), 2 (2), 2 (2) and 3 (2) fetuses (litters) in the control, 10, 25 and 70 mg/kg/day groups, respectively.
At 70 mg/kg/day, one fetus had a thick diaphragm and one fetus had malpositioned testes. In addition, another fetus was observed with a large atrium and an excessive amount of grey-white fluid in the thorax and abdominal cavity.

At 25 mg/kg, a small kidney was observed in one fetus and a malpositioned kidney occurred in another fetus.

At 10 mg/kg/day, Tetralogy of Fallot was observed in one fetus and malpositioned testis in another fetus.

The affected control fetuses either had Tetralogy of Fallot (one fetus), malpositioned kidney, interrupted aortic arch, lung cyst or a solid deposit in the gallbladder in only one fetus each.
The single occurrence and/or group distribution of these malformations does not indicate a test item-relationship and therefore all were considered to be spontaneous in origin.
The visceral variation absent or small gallbladder showed a remarkable group distribution as it was observed in 3.1%, 0.4%, 0.0% and 0.0% of fetuses per litter in the control, 10, 25 and 70 mg/kg/day groups, respectively, yielding statistical significance for the mid and high dose incidences. However, as the control incidence was higher than the historical maximum value, the decreased incidence of this variation was considered unrelated to the test item.
All other variations noted were considered unrelated to treatment as they occurred in the absence of a dose-related trend, infrequently, in control fetuses only, and/or at frequencies that were within the range of available historical control data.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 70 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

		SEX		viable fetuses	dead fetuses	resorptions		Post implantation loss	Implantations sites	Corpora Lutea	Pre implantation loss	Fetal weights [g]	No. of gravid Females
Dose [mg/kg bw/d]		M	F			early	late
0	TOTAL	87	101	188	0	9	2	11	199	216	17	NA	20
	MEAN	4.4	5.1	9.4	0.0	0.5	0.1	0.6	10.0	10.8	0.8	37.7
	S.D.	1.66	2.14	1.88	0.00	0.76	0.45	0.83	2.01	2.09	0.93	4.87
10	TOTAL	88	85	173	1	3.	6.	110	183	200	17	NA	19
	MEAN	4.6	4.5	9.1	0.1	0.3	0.3	0.5	9.6	10.5	0.9	37.1
	S.D.	2.03	2.34	2.92	0.23	0.50	0.67	1.07	3.09	2.52	1.20	6.39
25	TOTAL	71	87	158	0	9.	10.	19	177	204	27	NA	19
	MEAN	3.7	4.6	8.3	0.0	0.5	0.5	1.0	9.3	10.7	1.4	38.1
	S.D.	2.05	2.04	2.98	0.00	0.96	1.02	1.25	2.73	2.49	1.61	5.17
70	TOTAL	87	84	171	0.	12	0	12	183	203	20	NA	18
	MEAN	4.8	4.7	9.5	0.0	0.7	0.0	0.7	10.2	11.3	1.1	36.6
	S.D.	1.47	2.22	1.76	0.00	1.14	0.00	1.14	1.15	1.74	1.45	4.99

Conclusions:

Based on the results in this prenatal developmental toxicity study, the maternal and developmental No observed Adverse Effect Level (NOAEL) for 2,4-Di-tert-butylphenol was established as being 70 mg/kg/day.

Executive summary:

The objectives of this study were to determine the potential of 2,4-Di-tert-butylphenol to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post coitum, inclusive. The dose levels in this study were selected to be 0, 10, 25, 70 mg/kg/day, based on the results of the dose range finder (see RSS supporting study).  Higher doses were not tolerated (see report tolerability study in non-pregnant rabbits, attachment to supporting DRF study).  

No treatment related mortality was observed during this study. Reduced faeces production (up to severe) was observed in the majority of the animals across all groups, including controls, with a slightly higher persistence and/or severity at 70 mg/kg/day.  This slight increase in persistence and/or severity at 70 mg/kg/day could be related to the reduction in food consumption on the first days of treatment (Days 6-12 post coitum) at 70 mg/kg/day, which also resulted in a mean body weight loss of 1% on Day 9 post-coitum and subsequent reduction in body weight gain between Day 9 and 15.  As complete recovery of food consumption was observed from Day 18 post coitum, resulting in a total recovery in body weight gain from Day 21 post coitum onwards, these effects were considered non-adverse.  

No toxicologically significant changes were noted in any of the remaining maternal parameters investigated in this study (i.e. macroscopic examination and organ weights). No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results in this prenatal developmental toxicity study, the maternal and developmental No observed Adverse Effect Level (NOAEL) for 2,4-Di-tert-butylphenol was established as being 70 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

Remarks:

Dose range finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

March to April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline required

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2,4-Di-tert-butylphenol
- Analytical purity: 99.76 %
- Retest date of the lot/batch: November 2019

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Age at study initiation: 19 weeks old
- Weight at study initiation: between 3393 and 4066 g
- Housing: On arrival and following randomization females were housed individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20 °C
- Humidity (%): 41 to 58%
- Air changes (per hr): >=10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Formulations were heated to a maximum temperature of 40°C (±5°C) for 60 minutes (+/- 5 minutes) to obtain visual homogeneity. The dosing formulations were prepared at least weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature under controlled conditions (water bath set at 20°C (±5°C)) for at least 30 minutes and dosed within 24 hours after removal from the refrigerator.
Test item dosing formulations were kept at room temperature and protected from light until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity/density of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure (for details see "any other information on materials and methods incl. tables). According to these the test item is only soluble in oily vehicles.
- Concentration in vehicle: 0, 20, 50, 140 mg/L
- Amount of vehicle (if gavage): 0.5 mL/kg bw
- Lot/batch no. (if required): MKCG3257 + MKCH1635

The test substance was administered to the animals once daily by oral gavage 7 days a week from Day 6 to Day 28 post-coitum, inclusive. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic catheter attached to a plastic disposable syringe. A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Untreated females were mated at the Supplier, and were at Day 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).

Duration of treatment / exposure:

on days 6 to 28 post-coitum

Frequency of treatment:

daily on days 6 to 28 post-coitum

Dose / conc.:

17.5 mg/kg bw/day (actual dose received)

Dose / conc.:

35 mg/kg bw/day (actual dose received)

Dose / conc.:

70 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A non-GLP tolerability study (see attached report in "additional background material") in rabbits was performed in non-pregant New Zealand White rabbits with doses up to 150 mg/kg/bw. In this study, 2/3 animals at 90 mg/kg/day presented with persistent body weight loss (up to 10%) and absent to low food consumption from start of treatment onwards that led to discontinuation of treatment after 5-7 days. Therefore, this dose level was deemed too highfor the dose range finder.

Maternal examinations:

mortality/moribundity: twice daily
clinical signs: once daily
body weights: Animals were individually weighed on Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum
food consumption: Food consumption was quantitatively measured for Days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum. For the main study, food consumption was additionally measured over Days 3-6 post-coitum.
Water consumption: was monitored on regular basis throughout the study by visual inspection of the water bottles/containers. No quantitative investigation.
gross necropsy: an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs
organ weights (uterine, liver and kidney)
number of corpora lutea
(gravid) uterine weight
uterine contents

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

Each viable fetus of animals surviving to planned necropsy will be externally examined indetail and weighed. All live fetuses will be euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube. In case this is not possible due to a malformation, the fetus will be euthanized by interscapular injection of sodium pentobarbital (Euthasol® 20%).
Nonviable fetuses of animals surviving to planned necropsy (the degree of autolysis is minimal or absent) will be examined and weighed. For late resorptions a gross external
examination will be performed (if possible).
Fetuses of animals found dead or sacrificed before planned necropsy will be externally examined in detail and euthanized (if necessary) by decapitation or by sodium pentobarbital
(Euthasol® 20%).
Sex will not be determined. No visceral (internal) or skeletal examination will be performed as default, however in the case of (suspected) malformations, more detailed fetal examinations might be performed.
Malformed late resorptions will be collected and fixed in 10% buffered formalin. Malformed fetuses will be collected and fixed in the most appropriate fixative (based on type of malformation). Fetuses and late resorptions without malformations will be discarded.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Reduced faeces production (up to severe) was observed in all pregnant animals surviving to scheduled necropsy, including controls, with a higher persistence (lasting ≥ 14 days) at 35 and 70 mg/kg/day (only the non-pregnant Animal No. 12 presented with no clinical signs over the entire study period).
Animal No. 7 at 17.5 mg/k/day was observed with transient red fluid on the manure tray for 2 consecutive days (Days 12-13 post-coitum).
Incidental findings (i.e. scabs, scars) occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control and high dose animal (Nos. 4 and 33, respectively) were sacrificed for animal welfare reasons on Day 14 and 15 post-coitum, respectively, as they presented with absent or almost absent food intake from start of treatment, together with piloerection and/or body weight loss (up to 6%). At necropsy, Female No. 4 was found to be emaciated.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Animals at 70 mg/kg/day presented with a mean body weight loss of 1% from start of treatment until Day 9-12 post-coitum, followed by a recovery thereafter. This decrease in body weight gain resulted in a lower mean body weight (3% vs control) over Days 15-21 post-coitum and at the end of the treatment period (on Day 29 post-coitum).
Animals at 35 mg/kg/day presented with absent body weight gain over Days 12-15 post-coitum, with a mean body weight loss of 1% over Days 15-18 post-coitum (mainly attributed to animals Nos. 13, 14 and 18 with BW loss during that period), followed by a slight recovery thereafter. This decrease in body weight resulted in a lower mean body weight (up to 3%) vs control over Days 15-29 post-coitum.
No relevant changes in mean body weight and body weight gain were noted at 17.5 mg/kg/day.

No statistically significant changes were observed in mean body weight gain corrected for gravid uterus. All mean values were within the range of available HCD.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 70 mg/kg/day, mean food consumption was reduced vs control (up to 47% in relative values) from start of treatment until Day 18 post-coitum, followed by a recovery thereafter. This reduction in food intake was accompanied by a 1% mean body weight loss from start of treatment until Day 9-12 post-coitum, followed by a recovery afterwards. The decrease in body weight gain resulted in a slightly lower mean body weight (3% vs control) over Days 15-21 post-coitum and at the end of the treatment period (on Day 29 post-coitum).
At 35 mg/kg/day, mean food consumption was slightly reduced vs control over Days 6-9 and Days 12-18 post-coitum (up to 32% in relative values). A mean body weight loss of 1% was noted at this dose level over Days 15-18 post-coitum (mainly attributed to 3/6 animals with body weight loss during that period), followed by a slight recovery thereafter. This decrease in body weight gain resulted in a slightly lower mean body weight (up to 3% vs control) over Days 18-29 post-coitum.
No relevant changes in mean body weight and body weight gain were observed at a dose level of 17.5 mg/kg/day. Mean food consumption at this dose level was slightly reduced vs control over Days 6-9 post-coitum (which was attributed to 3/5 pregnant animals) with no relevant changes thereafter.

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, non-treatment-related

Description (incidence and severity):

Slightly higher liver weights were noted for Animal Nos 7 and 12 treated at 17.5 mg/kg/day and slightly lower liver weights were noted for all females treated at 70 mg/kg/day. In the absence of a dose response, this was considered as not toxicologically relevant. Slightly higher mean kidney weights were noted at 17.5 and 35 mg/kg/day, however without a dose response. This was most likely attributed to Animal Nos. 7 and 13, at 17.5 and 35 mg/kg/day, respectively, as slightly higher individual weights were noted for these two females.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic observations at necropsy did not reveal any alterations that were considered to be toxicologically relevant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions in any group.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes for the number of post-implantation loss were observed.
However, the number of post-implantation loss was increased at 17.5 mg/kg/day compared to controls, which was mainly attributed to the 7 early resorptions of Female No. 7. In addition, the post-implantation number at 35 mg/kg/day was statistically significantly reduced compared to concurrent controls. The pre-implantation loss (%) was statistically significantly reduced at 17.5 and 35 mg/kg/day compared to concurrent controls. However, the percentage noted in the control group was above the historical control data.

Total litter losses by resorption:

not specified

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

Higher number of early reortptions at 17.5 mg/kg/day mainly attributed to one animal that presented 7 early resorptions. No effects on late resorptions.

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead fetuses in any group.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

One female at 17.5 mg/kg bw was not pregant.

Other effects:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Although no statistically significant, the mean combined fetal weights in all test item-treated groups were below the control mean and lower limit of the historical control data, with no dose-related response.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Mean number of viable fetuses was 9.2, 9.6, 11.2 and 9.0 in the control, 17.5, 35 and 70 mg/kg/day groups, respectively. A lower percentage of viable fetuses per litter was noted at 17.5 mg/kg/day that was mainly attributed to Female No. 7 that presented 7 early resorptions and partly contributed to the higher litter incidence of early resorptions at this dose level. The higher number of viable fetuses (absolute and % per litter) at 35 mg/kg/day was attributed to the absence of post-implantation loss in this group.

Changes in sex ratio:

not examined

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

External examination of the fetuses did not show any test item-related abnormalities.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External examination revealed 2 multiple malformed fetuses, each at 17.5 and 70 mg/kg/day groups:
- One low dose fetus presented with multiple abnormalities (namely: facial cleft, small bilateral eye bulge, carpal flexure, right hindlimb hyperextension and trunk omphalocele).
- One high dose fetus was noted also with multiple abnormalities (namely: carpal flexure, trunk thoracogastroschisis, bilateral open eyes, right forelimb rotated outwards, cleft palate, small lower and upper jaw, cranioschisis in the nasal region and brachydactyly). It is noteworthy that the majority of these malformations were observed at lower incidences or not previously recorded in historical control fetuses.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

see skeletal malformations

Other effects:

no effects observed

Conclusions:

Based on the results of the dose range finder, selected dose levels for the main study were 10, 25 and 70 mg/kg.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

70 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Additional information

A prenatal developmental toxicity study in rabbits was performed to determine the potential of 2,4-Di-tert-butylphenol to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested. The substance was given orally by gavage to time-mated female New Zealand White rabbits from Day 6 to 28 post coitum, inclusive. The dose levels in this study were selected to be 0, 10, 25, 70 mg/kg/day, based on the results of the dose range finder (see RSS supporting study).  Higher doses were not tolerated (see report tolerability study in non-pregnant rabbits, attachment to supporting DRF study).  

No treatment related mortality was observed during this study. Reduced faeces production (up to severe) was observed in the majority of the animals across all groups, including controls, with a slightly higher persistence and/or severity at 70 mg/kg/day.  This slight increase in persistence and/or severity at 70 mg/kg/day could be related to the reduction in food consumption on the first days of treatment (Days 6-12 post coitum) at 70 mg/kg/day, which also resulted in a mean body weight loss of 1% on Day 9 post-coitum and subsequent reduction in body weight gain between Day 9 and 15.  As complete recovery of food consumption was observed from Day 18 post coitum, resulting in a total recovery in body weight gain from Day 21 post coitum onwards, these effects were considered non-adverse.  

No toxicologically significant changes were noted in any of the remaining maternal parameters investigated in this study (i.e. macroscopic examination and organ weights). No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results in this prenatal developmental toxicity study, the maternal and developmental No observed Adverse Effect Level (NOAEL) for 2,4-Di-tert-butylphenol was established as being 70 mg/kg/day.

Justification for classification or non-classification

No classification based on the results from the F0/F1 generation oral (dietary) toxicity study in the rat. No effect on the reproductive performance of the F0 generation or on offspring viability was recorded. The number of progeny born in animals treated at 300 mg/kg bw/day was reduced and neonatal growth in these animals was also reduced. However at this dose level and at 150 mg/kg bw/day palatability of the diet was reduced which caused a decrease in food consumption.

The NOAEL in this study was 150 mg/kg bw/day.