Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproduction/developmental toxicity screening test (equivalent to OECD 422, GLP)

- Parental and developmental NOAEL: 1000 mg/kg bw/day

NOAEL (P): 1000 mg/kg bw/day (actual dose received) (male/female) (No treatment related effects)

NOAEL (F1): 1000 mg/kg bw/day (actual dose received) (male/female) (No treatment related effects)

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 April 2003 - 2 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to GLP and an equivalent of OECD guideline 421.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: (P) 8 wks
- Weight at study initiation: (P) Males: 226-262 g; Females: 156-193 g
- Housing: Individually, in accordance with the Guide for the Care and Use of Laboratory Animals (1996)
- Diet (e.g. ad libitum): Ad libitum, certified rodent diet
- Water (e.g. ad libitum): Ad libitum, municipal water
- Acclimation period: approx. 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-26.1
- Humidity (%): 30-70
- Air changes (per hr): min. of 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared approximately every 4 weeks at graded concentrations of 25, 125 and 500 mg/ml and adminstered at a dosing volume of 2 ml/kg.

VEHICLE
- Concentration in vehicle: 25, 125 and 500 mg/ml
- Amount of vehicle (if gavage): 2 ml/kg
- Lot/batch no. (if required): 062K0006
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually in shoebox cage
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance in corn oil was analysed by HPLC/UV for homogenicity, stability and concentration.
Duration of treatment / exposure:
- Female: 2 weeks prior to mating, during mating period (up to 2 weeks) and during gestation, lactation and until post-partum day 4
- Male: 2 weeks prior to mating, during mating period (up to 2 weeks) and additionally treated for a total minimum of 35 consecutive days
Frequency of treatment:
Daily
Details on study schedule:
Not relevant, as the study is a screening study (only P exposed to test article)
Remarks:
Doses / Concentrations:
50, 250, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on findings from previous toxicity studies with the test material
- Rationale for animal assignment (if not random): At random
Positive control:
Not relevant
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations: moribundity and mortality, general appearance, overt signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations:
Not measured during cohabitation
Males: prior to treatment initiation and weekly thereafter
Females: GD 0,7, 14 and 20, PD 0 and 4
Fasted body weight collected prior to terminal sacrifice, except in lactating females.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly prior to mating.Not measured during cohabitation. Females: GD 0, 4, 7, 14 and 20, PD 0 and 4. Males: weekly until sacrifice.
Oestrous cyclicity (parental animals):
Not performed
Sperm parameters (parental animals):
Not performed
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, fetal weight (litter weight)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
The following tissues were preserved: vagina, ovaries with oviducts, pituitary gland, cervix, testes, epididymides, seminal vesicles, prostate, coagulating gland and uterus.

Evaluation of histopathology and organ weights was performed for the ovaries, testes and epididymides from animals in the vehicle control and high-dose groups (10/sex/group).
Postmortem examinations (offspring):
Not performed
Statistics:
- Mean and SD
- One-way ANOVA (homogeneity in variance)
- Post-hoc comparisons using Dunnett's test (when main effect differences were found)
- Chi-square test (mating performance and absolute pup survival)
Reproductive indices:
Not calculated
Offspring viability indices:
Not calculated
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
not considered treatment related
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
not considered treatment related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality and no treatment related clinical signs observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No difference in body weight or body weight gain for treated males as compared to the control group. For treated female groups, significant increases in body weight were noted as compared to the control group (low dose: GD7 and 14, mid/high dose: GD 0, 7, 14 and at termination). Body weight gain was only significantly increased during week 1 of precohabitation. These effects were considered to be unrelated to treatment, as they were not observed in males.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment related effects on mating index and length of gestation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No effects on male organ weights were noted. Only the fasted weight was significantly increased in the mid and high dose group females, but this was considered to be caused by the increase in body weight.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment related effects observed in both males and females.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment related effects observed in both males and females.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No treatment related effects
Clinical signs:
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
not considered treatment related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
The post-implantation loss was higher in the mid and high dose groups (not statistically significant). However, a statistically significant increase in the absolute number of stillbirths in the mid and high dose groups was noted. For the mid dose group this was caused by 10 deaths in one litter. The number of litter deaths between PD0-4 was significantly decreased for all treatment groups. The overall number of litter deaths was only significantly decreased for the low dose group. Due to the lack of a dose related response and similar overall deaths across the groups, the effects were considered unrelated to treatment.

BODY WEIGHT (OFFSPRING)
No effects were noted on the mean body weights of litters during PD0-4.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No treatment related effects
Reproductive effects observed:
not specified

RESULTS OF TEST DOSING GROUPS
Control Low (50 mg/kg bw/day) Medium (250 mg/kg bw/day) High (1000 mg/kg bw/day)
ANALYSES
Stability Greater than 7 weeks
Concentration Within 5% of target concentrations
Homogeneity Within 5% of target concentrations
 
PATERNAL TOXIC EFFECTS BY DOSE LEVEL 
Number of animals (M) 12 12 12 12
Mortality X X X X
Clinical signs X X X X
Body weight X X X X
Body weight gain X X X X
Food consumption X X X Significantly increased in week 5
Gross necropsy X X X X
Organ weights changes (relative and absolute) X X X X
Histopathology X X X X
MATERNAL TOXIC EFFECTS BY DOSE LEVEL 
Number of animals (F) 12 12 12 12
Mortality X X X X
Clinical signs X X X X
Body weight X Significantly increased on GD 7 and 14 Significantly increased on GD 0, 7 and 14 and at termination Significantly increased on GD 0, 7 and 14 and at termination
Body weight gain X Significantly increased during week 1 of precohabitation Significantly increased during week 1 of precohabitation (also resulting in increased total gain) Significantly increased during week 1 of precohabitation (also resulting in increased total gain)
Food consumption X Significantly increased in week 1 X Significantly increased on GD 14 and 20
Number pregnant per dose level 11 12 11 12
Number of corpora lutea 148 159 146 176
Pre-implantation loss 6 8 8 10
Number of implantations 142 151 138 166
Post-implantation loss 12 8 30 20
Duration (days) of pregnancy (mean +/- SD) 22.1 +/- 0.7 22.3 +/- 0.62 22.5 +/- 0.82 22.1 +/- 0.29
Number of pups born 130 145 121 (109 after correction for 1 litter, which was excluded due to water deprivation) 154
Number of stillborn fetuses 0 2 13* (increase, but caused by 10 deaths in one litter) 9* (increase)
Number alive on day 0 130 143 108 (96 after correction for 1 litter, which was excluded due to water deprivation) 145
Gross necropsy X X X X
Organ weights changes (relative and absolute) X X Significantly increased fasted weight Significantly increased fasted weight
Histopathology X X X X
FETAL DATA 
Number of litters 11 12 11 12
Mean litter size 11.8 12.1 11.0 12.8
Sex ratio (M/F) 0.9:1 1.4:1 1.1:1 0.8:1
Body weights per litter X X X X
Mean postnatal survival per litter (day 0-4) 10.7 11.6 8.7 11.8
Mean postnatal deaths per litter (day 0-4) 1.1 0.2 1.2 0.3
Absolute numbers of postnatal survival (day 0-4) 118 141 96 (one litter excluded, due to water deprivation) 145
Absolute numbers of postnatal deaths (day 0-4) 12 2* (decrease) 0* (decrease) 4* (decrease)
Total number of deaths 12 4* (decrease) 13 (caused by 10 deaths in 1 litter) 13
X = no effects (as compared to control group)
* = significant difference (as compared to control group)
Conclusions:
Under the conditions of this study, oral (gavage) administration of Phosflex 61B to rats did not result in overt signs of parental toxicity. This also accounts for the offspring. Therefore, the NOAEL for parental and developmental toxicity was established to be 1000 mg/kg bw/day.
Executive summary:

Phosflex 61B was administered in doses of 0, 50, 250 or 1000 mg/kg bw/day to rats. Treatment was done 2 weeks prior to mating, during mating (male/female) and for female during gestation, lactation and post partum up to day 4. Males were treated for at least 35 consecutive days. Mortality, clinical signs, body weights, food consumption were recorded in the parental animals. Also gross necropsy and organ weights and histopathology for the reproductive organs were studied. Reproductive performance was determined as well. For the litters, viability, fetal and litter weights and gross anomalies were recorded.

No mortality or overt signs of parental toxicity were observed in the parental rats. No effect was seen on body weight and food consumption, while reproductive performance was normal. Gross necropsy and organ weight data and histopathology of the reproductive organs revealed no adverse findings.

Mean litter size and mean number of live pups was comparable between the treatment groups. Additionally, no effects on litter weights were observed. Percent post-implantation loss was higher in 250 and 1000 mg/kg groups (not statistically significant). Subsequently, a statistically significant increase in the absolute number of stillbirths in the 250 and 1000 mg/kg groups was noted. However, overall a similar number of pup deaths was observed across all groups. While the incidence was the same, the timing of the event was somewhat different. In the 250 and 1000 mg/kg groups, most of the deaths occurred on day 0, while this was after day 0 in the control group. Overall, pup survival from day 0 to 4 was lower in the 250 mg/kg group (due to 10 deaths in one litter), higher in the 50 mg/kg group and approximately the same as control in the 1000 mg/kg group.

Under the conditions of this study, oral (gavage) administration of Phosflex 61B to rats did not result in overt signs of parental toxicity. This also accounts for the offspring. Therefore, the NOAEL for parental and developmental toxicity was established to be 1000 mg/kg bw/day.

Additional information

No relevant information available

Effects on developmental toxicity

Description of key information

McIrvine 2020: Effects on Embryo‑Fetal Development in the Han Wistar Rat by Oral Gavage Administration (OECD 414, GLP)

The NOAEL for embryofetal survival, growth and development was considered to be 1000 mg/kg/day.

The NOAEL for Maternal toxicity was considered to be 300 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
As per ECHA Decision number: CCH-D-2114449848-30-01/F
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
July 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Chemical name: Reaction mass of p-t-butylphenyldiphenyl phosphate and bis(p-t-butylphenyl)phenyl phosphate, synonyms : tBuTPP- low TPP, Phosflex 71B-HP, Fyrquel EHC plus or Syn-O-Ad 8499

EC number (per REACH) 939-505-4
I
ntended use: Industrial chemical.

Appearance: Clear, transparent liquid.

Storage conditions: At ambient temperature (15 to 25C) in the dark.

Supplier: Sponsor.
Batch number: 18235H0299

Expiry date: 28 August 2020.

Purity: Multi constituent substance, considered to be 100% when within specifiaction
COA attached
Species:
rat
Strain:
other: Han Wistar
Details on test animals or test system and environmental conditions:
Animals:
Strain/Species RccHan™;WIST.
Supplier Envigo RMS UK.
Number of animals ordered 88 females.
Duration of acclimatization Six days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 85 to 91 days old.
Weight range of the animals at the start of the study (Day 0 of gestation) 187 to 224 g.

Animal Care and Husbandry:
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature
and relative humidity Monitored and maintained within the range of 20-24C and 40-70%. There were no deviations from these ranges.

Lighting A rtificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
Cage distributionץ The cages constituting each group were blocked by group and mounted in batteries.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage
Acclimatization up to four animals
During pairing one (stock) male and one female
Gestation one female

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method of preparation
The required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous. The formulation was transferred to the final containers, via syringe whilst magnetically stirring.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2 to 8°C).
Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Formulation Analysis
Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures were determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix as part of another study, Covance Study Number LP22JS.
Stability was confirmed as one day at ambient temperature (15 to 25°C) and 15 days at refrigerated temperature (2 to 8°C).

Achieved concentration: Samples of each of the first and last formulations prepared were analyzed for achieved concentration of the test item.

Analysis: The method of analysis and results are presented in Attachment 13.2.
Details on mating procedure:
Male/female ratio: 1:1 with identified stock males.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation: When positive evidence of mating was detected.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day
See also attachment of Study Design.
Frequency of treatment:
Once Daily
Duration of test:
Day 6 to 19 after mating
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control- Corn oil
Dose / conc.:
100 mg/kg bw/day
Remarks:
Lowest dose
Dose / conc.:
300 mg/kg bw/day
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Highest dose
No. of animals per sex per dose:
20 animals per group
Control animals:
yes
yes, concurrent vehicle
Details on study design:
See attachment
Maternal examinations:
Serial Observations

Mortality: A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases as described in Section 3.7.

Clinical Observations: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing:
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
• Pre-dose observation
• At the end of dosing each group
• One to two hours after completion of dosing
• As late as possible in the working day.

Clinical Signs: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight:
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Food Consumption:
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-7, 8-10, 11-13, 14-16 and 17-19 after mating inclusive.

Thyroid Hormone Analysis:
Blood samples were collected at the following occasion:
Occasion Animals
At termination All surviving adults


Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule: Animals surviving until the end of the scheduled study period were killed on Day 20 after mating.
Sequence: To allow satisfactory inter-group comparison.

Organ Weights: For bilateral organs, left and right organs were weighed separately. Requisite organs were weighed for animals killed at scheduled intervals.
Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
Ovaries and uterine content:
For females surviving to term, the following was recorded:
Uterus Gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals (including those prematurely sacrificed, where possible):
For each ovary/uterine horn, Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

Apparently non pregnant animals The number of uterine implantation sites were checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski, 1964)).
Fetal examinations:
Fetal Examination and Processing:
Examination of all viable fetuses: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex and ano-genital distance of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter Sexed internally and eviscerated.

Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.

Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.
Statistics:
Statistical Analysis
The analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
Litter and fetal weights
Ano-genital distance, average for each litter adjusted for litter average fetal body weight
Organ weights, absolute and adjusted for terminal body weight
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) x 100
Number of corpora lutea

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations - Number of live fetuses) x 100
Number of implantations

All group values and SD were calculated from the individual litter values.

Ano-genital distance were presented both as absolute/unadjusted and adjusted for fetal body weight, using the weight recorded at necropsy
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation, often excessive, was seen for 5/20 animals receiving 100 mg/kg/day and for the majority of animals receiving 300 or 1000 mg/kg/day (17/20 and 18/20 animals respectively). Additionally, chin rubbing was apparent for 8/20 animal receiving 300 mg/kg/day and 14/20 animals receiving 1000 mg/kg/day. These signs are often seen when animals are dosed via the oral gavage route and are generally considered to reflect distaste or slight irritancy of the dosing formulations rather than any systemic effect of treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (animal number 64) treated at 1000 mg/kg/day was dispatched to necropsy on gestation Day 17 due to continued body weight loss from Day 13 and poor clinical condition. Clinical signs included brown staining to the anus, piloerection, red staining to the muzzle. At macroscopic examination the animal was found to have a small cecum, with abnormal contents and a dark liver but, a clear underlying reason for the decline in clinical condition could not be established. The female was pregnant with 11 live young. This death appeared atypical compared with the other animals at this dose level and an association with treatment was considered unlikely but could not be totally discounted
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Females receiving 1000 mg/kg/day showed marginal mean body weight loss of 2 g during the first two days of treatment compared to a mean body weight gain of 7 g in the controls. Thereafter, body weight gain to gestation Day 18 of gestation was similar to control, however body weight gain from Day 19-20 of gestation was low (46% of controls). Overall body weight gain during the treatment period, gestation Days 6-20 was 83% of the control value, and 42% when adjusted for the contribution of the gravid uterus. The differences from control achieved statistical significance (p<0.01).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females receiving 1000 mg/kg/day showed notably low mean food consumption, compared to control, during Day 6-8 of gestation (47% of control food intake for this period). Thereafter, an improvement in food intake was apparent, although food consumption remained significantly lower than control (p<0.01) throughout the remainder of the study.
Females receiving 300 mg/kg/day showed low mean food consumption, compared to control, during Day 6-8 of gestation (71% of control value). Thereafter, an improvement in food intake was apparent, although values remained significantly lower than control until Day 17 of gestation (p<0.01 or p<0.05), by Days 17-20 food consumption was similar to control.
There was no clear effect of treatment on food consumption during gestation for females receiving 100 mg/kg/day.
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):
The following effects were seen and were not considered adverse:

Group mean absolute organ weights of the liver were high at 100, 300 or 1000 mg/kg/day (110%, 131% and 151%, of the control value, respectively). When adjusted for terminal body weight, all of these values remained high (112%, 132% and 161% of the controls, respectively) attaining statistical significance (p<0.01). The increased weight of the liver was considered most likely to reflect enzyme induction, a typical adaptive response to the presence of a xenobiotic.

Group mean adjusted organ weights of the kidneys, thyroids and parathyroids for females receiving 1000 mg/kg/day were high when compared with the weights of these organs from control animals (106% änd 138% of the controls, respectively), both mean organ weights attained statistical significance (p<0.01).

Mean gravid uterine weight was similar to control for females receiving 100, 300 or 1000 mg/kg/day, despite the effect on maternal body weight at 1000 mg/kg/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no findings at macroscopic examination of females considered to be related to treatment with tBuTPP- low TPP.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathology

A test item-related finding of minimal to slight diffuse follicular cell hypertrophy was seen in the thyroid of females treated with 300 or 1000 mg/kg/day with a clear dose-relationship. Increased breakdown of thyroid hormones due to enzyme induction in the liver is a common mechanism of induction for this finding, and the higher liver weights seen in this study may reflect that change. The thyroid glands of rats are reported to be highly sensitive when compared with humans due to physiologic differences in thyroid homeostasis, including higher circulating TSH concentrations, a shorter half-life of T4, and species differences in thyroid transport proteins. Therefore, this finding is considered to be rat specific and therefore of no relevance to man (Greaves, 2012)

Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis

The analysis of serum thyroxine (T4) and triiodothyronine (T3) concentrations at scheduled
termination on Day 20 of gestation revealed that mean serum T3 concentrations at 100, 300 or 1000 mg/kg/day were 94%, 81% and 77% of control values attaining statistical significance, (p<0.05 or p<0.01, respectively), when compared with the control group at 300 or 1000 mg/kg/day. The range of T3 values were 258 – 636 pg/mL in the control group,
247 – 542, 182 – 505 and 184 – 433 pg/mL in the treated groups receiving, 100, 300 or 1000 mg/kg/day, respectively. The individual T3 concentration values for 3 control females, 3 females receiving 100 mg/kg/day, 7 females receiving 300 mg/kg/day and for 11 females receiving 1000 mg/kg/day were below the historical control data range
(289.6 – 886.0 pg/mL).
Mean serum T4 concentrations were 87%, 82% and 65% of control values at 100, 300 and 1000 mg/kg/day, respectively; the difference from control values was statistically significant at 300 and 1000 mg/kg/day (p<0.01). The range of T4 values for each group was 11600 – 29200 pg/mL in the control group, and 9570 – 23000, 8500 – 21600 and 7480 – 20600 pg/mL in the groups treated at 100, 300 or 1000 mg/kg/day, respectively. The individual T4 concentration values for 1 female receiving 100 mg/kg/day, 2 females receiving 300 mg/kg/day and for 8 females receiving 1000 mg/kg/day were below the historical control data range (10729.8 – 34962.0 pg/mL).
The analysis of serum thyroid stimulating hormone (TSH) concentrations at scheduled termination on Day 20 of gestation revealed that the mean TSH level was 111%, 230% and 247% of control values at 100, 300 and 1000 mg/kg/day, attaining statistical significance in rats receiving 300 or 1000 mg/kg/day, when compared with the control group (p<0.01). The range of TSH values for each group was 229 – 4240 pg/mL in the control group, 250 – 4420, 589 – 6210, and 912 – 9440 pg/mL in the groups treated at 100, 300 or 1000 mg/kg/day, respectively. The individual TSH concentration values for 6 females receiving 300 mg/kg/day and 7 females receiving 1000 mg/kg/day were above the historical control data range (172.4 – 4472.6 pg/mL).
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:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
One female (animal No. 26) that received 100 mg/kg/day tBuTPP- low TPP was found to be not pregnant at necropsy.
Litter data, as assessed by corpora lutea count, pre-implantation loss, numbers of implantations, early resorptions and late resorptions, post-implantation loss and number of live young, were similar in all groups. The mean sex ratio in offspring females receiving 100 mg/kg/day was 41.6%M compared to 52.1%M in the controls, with a lack of dose response this is considered unlikely to be related to treatment. There was no indication that maternal treatment with tBuTPP- low TPP had any effect on embryofetal survival.

Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: No additional effects seen
Key result
Abnormalities:
no effects observed
Localisation:
other:
Description (incidence and severity):
No abnormalities detected
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Total litter and fetal weights were considered to be unaffected by maternal treatment with tBuTPP- low TPP.
At 1000 mg/kg/day, mean fetal weights, compared to the controls, were marginally low (male and overall, 96% of control) but only mean fetal weight for females (95% of control) attained statistical significance (p<0.05). All mean values (male, female and overall) were within the HCD range (3.0 – 4.4, 2.7 – 4.1 and 2.9 – 4.3 g). One male mean fetal weight (Litter No. 65) and two overall fetal weights (Litter Nos. 65 and 66) were outside of the respective 98 percentile HCD range, and therefore, the minor difference in fetal weights at 1000 mg/kg/day at the magnitude observed, were considered to be consistent with normal variation.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: No effects detected
Key result
Abnormalities:
no effects observed
Localisation:
other:
Description (incidence and severity):
No effects detected
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
no
Conclusions:
Conclusion
Treatment at 1000 mg/kg/day (the limit dose) withtBuTPP- low TPPwas associated with one death, the etiology of which was unclear, and reductions in body weight gain and food consumption.The NOAEL for maternal toxicity was therefore considered to be 300 mg/kg/day.
The NOAEL for embryofetal survival, growth and development was considered to be 1000 mg/kg/day.  
Executive summary:

SUMMARY

The purpose of this study was to assess the influence of tBuTPP- low TPP on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy in the Han Wistar Rat.

Three groups of 20 females received tBuTPP- low TPP at doses of 100, 300 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus weight and organ weights were recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results

The mean concentrations of formulations sampled from the first and last dose preparations were within 11% of the nominal concentration confirming the accuracy of the formulation procedure and the difference from mean remained within 3%, confirming precise analysis.

The clinical condition of pregnant females receiving 100, 300 or 1000 mg/kg/day was unaffected by treatment withtBuTPP- low TPP.

There were two premature deaths on study. One control female was killed for welfare reasons on gestation Day 12 due to an error during the dosing procedure. One female receiving 1000 mg/kg/day was killed for welfare reasons on gestation Day 17 after showing poor clinical condition and continued body weight loss from Day 13. This death appeared atypical but an association with treatment could not be totally discounted.  

Females receiving 1000 mg/kg/day showed marginal mean body weight loss of 2 g during the first two days of treatment compared to a mean body weight gain of 7 g in the controls, andnotable low food intakeduring the first two days of treatment(47% of control food intake for this period). Thereafter, an improvement in food intake was apparent, although food consumption remainedsignificantly lower than control (p<0.01) throughout the remainder of the study, but lower body weight gain was only apparent on Day 19-20 of gestation(46% of controls).However,overall body weight gain during the treatment period, gestation Days 6-20 was 83% of the control value, and 42% when adjusted for the contribution of the gravid uterus.

Females receiving 300 mg/kg/day showed low food consumption during Day 6-8 of gestation (71% of control value), thereafter, an improvement in food intake was apparent, although values remained significantly lower than control until Day 17 of gestation (p<0.01 or p<0.05), by Days 17-20 food consumption was similar to control. There was no corresponding effect on body weight. There was no effect of treatment on body weight gain and food consumption during gestation for females receiving 100 mg/kg/day.     

For females receiving 300 or 1000 mg/kg/dayserum thyroxine (T4) and triiodothyronine (T3) concentrations were low (81% and 77% for T3 and 82% and 65% of control values for T4, respectively). The concentration of thyroid stimulating hormone (TSH) forfemales receiving 300 or 1000 mg/kg/daywas high at scheduled termination on Day 20 of gestation (230% and 247% of control values). Microscopic examination of the thyroids revealedminimal to slight diffuse follicular cell hypertrophy at 300 or 1000 mg/kg/day, and high mean absolute and adjusted thyroid weights (138% of the controls) were apparent for females receiving 1000 mg/kg/day.

 

Group mean adjusted organ weights of the kidneys for females receiving 1000 mg/kg/day were high when compared with the weights of this organ from control animals (106% of the controls). Group mean absolute organ weights of the liver were high at 100, 300 or 1000 mg/kg/day (110%, 131% and 151%, of the control value, respectively). When adjusted for terminal body weight, all of these values remained high (112%, 132% and 161% of the controls, respectively) attaining statistical significance (p<0.01).

There were no findings at macroscopic examination of females considered to be related to treatment withtBuTPP- low TPP.

Litter data, litter and fetal weights,ano-genital distance and the type, incidence and distribution of major and minor abnormalities and skeletal variants did not indicate any effect of maternal treatment on the survival, growth and morphological development of the fetus at 100, 300 or 1000 mg/kg/day.

At 1000 mg/kg/day, mean fetal weights, compared to the controls, were marginally low (male and overall, 96% of control) but only mean fetal weight for females (95% of control) attained statistical significance (p<0.05). As all mean values (male, female and overall) were within the HCD range (3.0 – 4.4, 2.7 – 4.1 and 2.9 – 4.3 g) this was considered to reflect normal biological variation. There was no effect on fetal weight at 100 or 300 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimich 1 Guidleine study
Additional information

Reproduction/developmental toxicity screening test (equivalent to OECD 422, GLP)

- Parental and developmental NOAEL: 1000 mg/kg bw/day

A NOAEL of 1000 mg/kg bw/day for developmental toxicity was established also in two additional available studies and was based on the absence of adverse effects (Downs 1982) .

The NOAEL was supported by additional studies: one study (Rodwell 1980) was the pilot study for the other (Rodewell 1981), a prenatal developmental toxicity study. Both studies established a NOAEL of 1000 mg/kg bw/day as well, based on the absence of adverse effects up to the highest dose tested.

Justification for classification or non-classification

Based on the NOAEL of 1000 mg/kg bw/day that was established and the criteria outlined in Annex I of CLP Regulation 1272/2008/EC, there is no need to classify this substance for repro- or developmental toxicity.

 

Additional information