Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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
Cross-reference
Reason / purpose:
other: supporting information on dose selection
Reference
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 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
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

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 70 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: overall effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

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

Effect levels (fetuses)

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

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.