Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

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 October 2005 - 2 May 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted by a recognised facility in compliance with GLP to a recent regulatory test guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
This study was conducted in accordance with the protocol, except for the following deviations.

On one occasion, the humidity in the animal room was 74%, which was outside the protocol specified range of 30 to 70%.

On Day 7, the postdose observations for two mpIPTPP males (animal numbers 281 and 282) were conducted one minute prior to the protocol-specified window of 60-90 minutes postdose.

On Day 10, the postdose observations for seven control males (animal numbers 209,211,212,213,214,215, and 217), two Reofos 35 males (animal numbers 218 and 219), one Reofos 65 male (animal number 236), seven Reofos 65 washed males (animal numbers 248, 249, 250, 251, 252, 253, and 255), and three mpIPTPP males (animal numbers 271, 273, and 274) were conducted one to four minutes prior to the protocol-specified window of 60-90 minutes postdose.

On Day 10, the postdose observation for one Reofos 65 washed female (animal number 337) was conducted 7 minutes past the protocol-specified window of 60-90 minutes postdose.

On Day 16, test article administration for one control male (animal number 201) was conducted 1 minute prior the protocol-specified window of 1 hour from the Day 1 dose.

On Day 20, the postdose observations for six control males (animal numbers 201, 202,205,207,208, and 211), and one control female (animal number 288) were conducted 1 minute past the protocol-specified window of 60-90 minutes postdose.

On Day 22, the postdose observations for one Reofos 35 male (animal number 227) and one Reofos 65 female (animal number 314) were conducted 9 and 2 minutes, respectively, beyond the protocol-specified window of 60-90 minutes postdose.

On Day 23, the postdose observations for six Reofos 120 females (animal numbers 341,342,344,345,346, and 348) were conducted 1 to 2 minutes prior to the protocol-specified window of 60-90 minutes postdose.

On Day 35, the postdose observation for one recovery control male (animal number 214) was conducted 24 minutes past the protocol-specified window of 60-90 minutes postdose.

On Day 42, test article administration for all animals was conducted from 1 hour and 23 minutes to 1 hour and 42 minutes past the protocol-specified window of 1 hour from the Day 1 dose.

A terminal body weight for one mpIPTPP male (animal number 271) was not collected at necropsy.

The cranial cavity was not examined at necropsy for all main study females.

The uterus for one control recovery female (animal number 297) was stained in ammonium sulfide solution for detection of implantation sites, and saved in 10% neutral buffered formalin.

In the opinion of the Study Director, these minor deviations did not affect the quality or integrity of the study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Reofos 35
- Physical state: Clear liquid
- Substance type: isopropylated triphenyl phosphate mixture
- Lot/batch No.: 890000305578
- Expiration date of the lot/batch: July 7, 2005
- Storage condition of test material: Room temperature.

- Name of test material (as cited in study report): Reofos 65
- Physical state: Clear liquid
- Substance type: isopropylated triphenyl phosphate mixture
- Lot/batch No.: 20013572
- Expiration date of the lot/batch: July 7, 2005
- Storage condition of test material: Room temperature.

- Name of test material (as cited in study report): Reofos 65 Washed
- Physical state: Clear liquid
- Substance type: isopropylated triphenyl phosphate mixture
- Lot/batch No.: 65/1
- Expiration date of the lot/batch: July 7, 2005
- Storage condition of test material: Room temperature.

- Name of test material (as cited in study report): Reofos 120
- Physical state: Clear liquid
- Substance type: isopropylated triphenyl phosphate mixture
- Lot/batch No.: 890000305577
- Expiration date of the lot/batch: July 7, 2005
- Storage condition of test material: Room temperature.

- Name of test material (as cited in study report): mpIPTPP
- Physical state: Pale-yellow to yellow-brown liquid
- Substance type: isopropylated triphenyl phosphate mixture
- Lot/batch No.: 64/11
- Expiration date of the lot/batch: July 7, 2005
- Storage condition of test material: Room temperature.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina.
- Age at study initiation: 12 weeks
- Weight at study initiation: No data. Changes in body weight are listed in the results tables, which can be found under 'Attached backgound documents.'
- Fasting period before study:
- Housing: In stainless steel, wire-mesh type cages, except during pairing, near partrition, and during lactation. During pairing, animals were cohabited, one male and one female within the same treatment group. On approximately Day 20 of gestation, females were individually housed in plastic cages containing wood chip bedding.
- Diet (e.g. ad libitum): meal Lab Diet Certified Rodent Diet #5002, PMI Nutrition International, Inc.); ad libitum.
- Water (e.g. ad libitum): tap water; ad libitum.
- Acclimation period: 3 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 65-76°F
- Humidity (%): 30 to 74%
- Air changes (per hr): No data.
- Photoperiod (hrs dark / hrs light): approximately 12 hours light per day.

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The basal diet will be meal Lab Diet Certified Rodent Diet #5002, PMI Nutrition International, Inc.
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food: No data.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil - no justification given.
- Concentration in vehicle: Control animals administered 400 mg/kg/day and a dose volume of 5 mL/kg of the vehicle.
- Amount of vehicle (if gavage): No data - The test aricles were diluted with the vehicle in order to achieve the desired dose volume.
- Lot/batch no. (if required): TH1937, TH1938, TM0158 and TO0383.
- Purity: No data: The Study Director is not aware of any potential contaminants likely to be present in the certified diet that would interfere with the results of this study. Therefore, no analyses other than those routinely performed by the feed supplier will be conducted.
-Preparation: To prepare the vehicle, the required amount of corn oil was dispensed into amber glass containers prior to handling the test articles. The vehicle was dispensed weekly and stored refrigerated.
Details on mating procedure:
- M/F ratio per cage: 1 Male to 1 Female
- Length of cohabitation: After evidence of mating was observed, the female was returned to an individual cage for the remainder of the study. The maximum pairing period was 14 days.
- Proof of pregnancy: Evidence of a copulatory plug in the vagina and/or sperm in the vaginal lavage referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing, any females with no confirmed evidence of mating were returned to individual cages until scheduled euthanasia.
- After successful mating each pregnant female was caged: The female was returned to an individual cage for the remainder of the study.
- Any other deviations from standard protocol: On one occasion, the humidity in the animal room was 74%, which was outside the
protocol specified range of 30 to 70%. In addition, several postdose observations and test article administrations were administered late (between 1 minute and 1 hour and 42 minutes late). A terminal body weight for one mpIPTPP male (animal number 271) was not
collected at necropsy. The cranial cavity was not examined at necropsy for all main study females. The uterus for one control recovery female (animal number 297) was stained inammonium sulfide solution for detection of implantation sites, and saved in 10% neutral buffered formalin. All of these minor deviations were considered by the Study Director not to affect the quality or integrity of the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Homogeneity: Duplicate samples (5 mL/sample) from the vehicle and each test article formulation prepared for Week 1 were collected from the top, middle, and bottom of the mixing container using a syringe, while stirrng, and placed in amber glass bottles. The samples were delivered to MPI Analytical at room temperature for homogeneity analysis of the test article formulations.
- Stability: Samples (5 mL/sample) from each test aricle formulation from Week 1 were collected from the container with a syrnge, while stirrng, and placed in amber glass bottles. The samples were stored refrgerated for up to 14 days, and delivercd to MPI Analytical on wet ice after 7
and 14 days for analysis of the stability of the test articles in the vehicle.
- Concentration: Samples (5 mL/sample) from each test article formulation were collected from the preparations for Weeks 2 through 4 and 8 of study. Samples collected for homogeneity analysis were also used to verify the Week 1 concentrations. The samples were collected from the container, while stirring, with a syrnge, placed into amber glass bottles, and stored at room temperature or refrgerated until analyzed for test article concentrations.
- Analyses: All analytical work was conducted by MPI Analytical, a division ofMPI Research, using a Sponsor-supplied analytical method validated under MPI Research Study Number 1038-005.
- Reserve Sample and Test Article Disposition: A reserve sample from each batch of test article used in this study was taken and archived.The remaining test article will be returned to a Sponsor-designated location after completion of the study.
Duration of treatment / exposure:
The main study males were treated for at least 42 consecutive days, while the main study females were treated for up to 54 days, depending on
reproductive performance.
Frequency of treatment:
The test and control articles will be administered once per day at approximately the same time (± one hour from the Day 1 start of dosing) each day.
Details on study schedule:
After 14 days of treatment, the main study males were randomly cohabited, one male to one female, with the corresponding females in the treatment and control group. Each female was housed in the cage of a male during mating. Positive evidence of copulation was established by daily inspection for a copulatory plug in the vagina and/or sperm in the vaginal lavage.
The day on which positive evidence of copulation was observed was considered Day 0 of gestation. After evidence of mating was observed, the female was returned to an individual cage for the remainder of the study. The maximum pairing period was 14 days, at the end of which any females with no confirmed evidence of mating were returned to individual cages until scheduled euthanasia.

After two weeks post-administration of vehicle or test article, recovery males and females were paired (1: 1) for two weeks or until evidence of mating was observed, as described above.

Parturition and F 1 Litter Observations

Beginning on GD 19, main study and recovery females were examined twice daily for signs of partrition. The mated females were allowed to give birth (F1). The duration of gestation was calculated, and any difficulties occurring at parturition were recorded. The day on which all pups were delivered was designated as LD O. The litters were examined as soon as possible after delivery and parameters including litter size, number of dead and live pups, and gross abnormalities of the pups were recorded. Any intact pups found dead at birth or during lactation were preserved in Bouin's solution for possible future visceral evaluation. Pups were weighed, sexed, and examined for abnormalities on LD 0 and 4. Any abnormal behavior observed in the pups was recorded daily. On LD 4, the pups were euthanized via intraperitoneal injection of sodium pentobarbital solution and preserved intact in Bouin's solution for possible future visceral evaluation.
Doses / concentrations
Remarks:
Doses / Concentrations:
400 mg/kg/day at a dose volume of 5 ml/kg
Basis:
actual ingested
No. of animals per sex per dose:
12 male and female.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose level for each test article was selected by the Sponsor on the basis of available data from previous studies.
- Rationale for animal assignment (if not random): Prior to selection for study, each animal was given a detailed clinical examination, body weights were measured and recorded, and sex was verified. Animals considered suitable for study were randomized, by sex, using body weights, into treatment groups using a simple randomisation procedure. All animals placed on study had body weights that fell within ± 20% of the mean body weight for each sex. Extra animals obtained for this study, but not placed on study, were euthanized via carbon dioxide inhalation and discarded. Eighty two male (weighing 350 to 409 g at randomization) and 82 female rats (weighing 221 to 261 g at randomization) were assigned to the control or treated groups as described in the table in the method section.
Positive control:
No data.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily
- Cage side observations: Morbidity, mortality, signs of injury, and availability of food and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The onservations were conducted once, at approximately 60-90 minutes postdose.
- Clinical Observations included: changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity, (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and reactivity to handling, as well as the presence of clonic or tonic movements, stereotypics (e.g., excessive grooming, repetitive circling), diffcult or prolonged partrition or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights for the males and females were recorded the day after arrval, prior to randomization, at initiation of test article administration, weekly during the premating period, weekly for males, unmated females, and recovery animals (both sexes) through to termination of the main study animals. Recovery animals continued to be weighed weekly during the two-week premating period, mating and postmating periods (males and unmated females). All mated main study and recovery females were weighed on GD 0, 7, 14, and 20, and on LD 0 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data
- Time schedule for examinations: Individual food consumption was measured and recorded weekly for all main and recovery
animals during the study except during the respective mating period. Food consumption was not measured during the mating period, because the animals were cohabited. Following the cohabitation period, food consumption was recorded weekly for males and unmated females until euthanasia. Food consumption was recorded for all mated females on GD 0, 7, 14, and 20 and LD 0 and 4.
Oestrous cyclicity (parental animals):
Not examined. Historical Control Fertility and Reproduction Data for theCRL:CD(SD) IGS BR Rats is present in the report appendices under 'Attached Background Material.'
Sperm parameters (parental animals):
Not examined.
Litter observations:
STANDARDISATION OF LITTERS: No data

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: The litters were examined as soon as possible after delivery and parameters including litter size, number of dead and live born pups, and gross abnormalities of the pups were recorded.
- Time schedule for observations: Pups were weighed, sexed, and examined for abnormalities on LD 0 and 4. Any abnormal behavior observed in the pups was recorded daily. On LD 4, the pups were euthanized via intraperitoneal injection of sodium pentobarbital solution and preserved intact in Bouin's solution for possible future visceral evaluation.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE:
- Male animals: All surviving animals at test termination.
- Maternal animals: All surviving animals at test termination.

GROSS NECROPSY:
All main study and recovery animals were euthanized via carbon dioxide inhalation and examined carefully for external abnormalities, including palpable masses. The skin was reflected from a ventral midline incision and any subcutaneous abnormalities were identified
and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities, and the organs were removed and examined. Special emphasis was placed on organs ofthe reproductive system. Implantation sites (scars) along the uterus were counted and recorded. All tissues were fixed in neutral buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGHTS:
The tissues Adrenal, Epididymis, Gonads, ovary, testis, Gross lesions, Liver (3 sections collected; 2 examined) and Uterus (both homs)/Cervix were prepared for microscopic examination and weighed, respectively. Body weights and protocol-specified organ weights were recorded at scheduled necropsy and appropriate organ weight ratios were calculated (relative to body weights). Organs weighed were: Adrenal, Epididymis, Gonads: ovary, testis and Liver. Paired organs were weighed together. Organs were not weighed for animals dying spontaneously.
Postmortem examinations (offspring):
A full body visceral evaluation was conducted on a total of 22 selected pups found dead or euthanized on LD 4. Five pups were selected from the control group, one pup found dead at birth (litter number 284), one pup found dead during the 4-day lactation period (litter number 289), and three randomly selected pups from those that survived and were euthanized on LD 4. Nine pups were selected from the Reofos 65 washed group, one pup dead at birth (litter number 333), two pups randomly selected from the eight pups found dead at birth (litter number 335), three randomly selected pups (litter numbers 335, 337, and 339) from the 22 pups that died over the 4-day lactation period, and three randomly selected pups from those that survived and were euthanized on LD 4. Eight pups were selected from the mpIPTPP group, one dead pup at birth (one each from litter numbers 353,355, and 360), two pups that died during LD 0-4 (one each from litter numbers 362 and 364), and three randomly selected pups from those that survived to LD 4 and were euthanized. The pups were evaluated for visceral irregularities using a free-hand razor blade sectioning technique similar to that described by Wilson for the evaluation of term rat fetuses.
Statistics:
Tests used are listed below. The methods are detailed in the method section.
- T-Test Comparisons
- Arcsin-Square-Root Transformation
- Fisher's Exact Test
- Covariate Analysis

MPI Research Computer Systems:
Data Collection System: Provantis ™
HPLC and GC Analysis: Millenium32
Statistical Analysis: SAS
Reporting: SAS and Microsoft Office Professional
Reproductive indices:
- Mating Index - Males:
((No. males with evidence of mating) / ( No. males paired)) x 100

-Mating Index - Females:
((No. females with evidence of mating) / (No. females paired)) x 100

- Gestation Index:
((No. females delivering at least 1 live pup) / (No. females pregnant)) x 100
Offspring viability indices:
- Live Birth Index:
((No. live pups at birth) / (No. pups born)) x 100

- Dead Day 0 Index:
((No. dead pups on Day 0) / (No. pups born)) x 100

- Viabilty Index - Day 4:
((No. pups surviving 4 days (preculling)) / (No. live pups at birth)) x 100

- Lactation Index - Day 21:
((No. pups surviving 21 days (weaning)) / (No. live pups at 4 days (postculling))) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
MALES: During the daily clinical examinations of male rats, the only effect seen from treatment with the various isopropylated triphenyl phosphate mixtures tested was an increased incidence of salivation.
FEMALES: Similar to the males, salivation was also seen with increased frequency in female rats treated with the various isopropylated triphenyl phosphate mixtures tested. This was a prominent finding during the premating/mating and gestation phases of study and became less obvious during lactation (LD 0-4).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Reofos 35: Male body weights in the Reofos 35 group were significantly lower than controls at Weeks 3 through 8. In the Reofos 35 females, mean body weights and body weight gain during the premating, gestation, and lactation periods were comparable to controls and unaffected by treatment.
- Reofos 65: Mean body weights for the Reofos 65 males were slightly lower than controls throughout the study and only over the last two weeks (Weeks 7-8), when body weights were about 7% lower than controls, were differences statistically significant. In the Reofos 65 females, mean body weights and body weight gain during the premating, gestation, and lactation periods were considered comparable to controls and unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
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, treatment-related
Description (incidence and severity):
In males, adrenal and liver weights absolute and relative to body weight were increased in the Reofos 35 (exclusive of absolute liver weight), Reofos 65, Reofos 65 washed, and Reofos 120 groups. In females, the liver weights, absolute and relative to body weight, were increased for these same groups and in the mpIPTPP group. Additionally, in females, the adrenal weights, absolute and relative to body weight, were increased in the Reofos 35, Reofos 65, Reofos 65 washed, and Reofos 120 groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test article-associated fatty change was observed in the adrenal glands of males and females in Groups 2, 3,4, and 5; vacuolation of interstitial cells of the ovaries in Groups 2,3,4,5; and centrilobular hepatocellular hypertrophy of livers of females in Groups 2,3,4, and 5.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Even though the term fatty change was used for adrenal gland and vacuolation was used for the interstitial cells of the ovary, the material that accumulated to cause the change was probably the same in each organ and may be cholesteroI. Whereas both males and females had increased liver weight, only females had a distinct centrilobular hypertrophy in which cells had a small increase in acidophilic cytoplasm.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
An adverse effect on fertility and reproductive performance was seen in the Reofos 65 group.
Fertility and fecundity indices were 50% in this group and statistically lower than controls (50% vs. 100%). These effects were reversible, however, and were not apparent in the recovery phase of the study. Although not statistically lower than controls, a slight decrease of 25% for both fertility and fecundity indices were seen in the Reofos 35 group. This difference was considered suggestive of an effect on fertility and reproductive performance. No effects on fertility or reproductive performance were seen in the remaining treated groups.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): MALES: During the daily clinical examinations of male rats, the only effect seen from treatment with the various isopropylated triphenyl phosphate mixtures tested was an increased incidence of salivation. This was seen at least once during the study in most of the males from each treatment group, but not among the control males. Swollen lips were seen in some males in several of the treatment groups. A single exception (Reofos 120 male number 263 Days 9 and 10) of this was seen only during one day of study (i.e., Day 9). It was not seen in the control males or males from the Reofos 35 group and its occurrence in the remaining treatment groups ranged from 2/12 males in the Reofos 65 group to 9/12 animals in the mpIPTPP group. Because the finding was transient and not seen either prior to this day or for the remainder of the study, it was not considered toxicologically meaningful or related to treatment. Clinical examinations of males in the Reofos 65 group retained into the recovery phase of study were unremarkable. Salivation prevalent during the treatment period in these males (5/5 males) was seen in just one animal on a single occasion during the recovery phase when animals were not being treated.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): FEMALES: Similar to the males, salivation was also seen with increased frequency in female rats treated with the various isopropylated triphenyl phosphate mixtures tested. This was a prominent finding during the premating/mating and gestation phases of study and became less obvious during lactation (LD 0-4). Other findings seen among the treated females throughout the study occurred at low incidence or with similar frequency as controls or were transient (i.e. swollen lips) and not considered related to treatment. These findings were considered consistent with those seen commonly in this laboratory with this strain and age of animal. Clinical examinations of females in the Reofos 65 group retained into the recovery phase of study were unremarkable. Salivation prevalent during the treatment period in these animals (5/5 females) was not seen during the recovery phase, when animals were not being treated.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
- Reofos 35: Male body weights in the Reofos 35 group were significantly lower than controls at Weeks 3 through 8. Body weights over this period ranged from about 6% lower than control at Week 4 to about 11 % lower at Week 8. These animals also gained less weight than control during the study and for many of these weekly intervals these differences were statistically significant. These changes in body weight and weight gain for these males did not, however, correlate with any change in food consumption. In the Reofos 35 females, mean body weights and body weight gain during the premating, gestation, and lactation periods were comparable to controls and unaffected by treatment.
- Reofos 65: Mean body weights for the Reofos 65 males were slightly lower than controls throughout the study and only over the last two weeks (Weeks 7-8), when body weights were about 7% lower than controls, were differences statistically significant. Weekly body weight gain for these males was generally comparable to controls throughout the study. The only statistically significant change from controls was a lower weight gain for Week 6-7. In the Reofos 65 females, mean body weights and body weight gain during the premating, gestation, and lactation periods were considered comparable to controls and unaffected by treatment. Mean weight gain in this group over the second week of the premating period was statistically higher than controls, but considered spurious and unrelated to treatment.
- Mean body weights and body weight gain for the Reofos 65 washed, Reofos 120 and mpIPTPP males and females were considered comparable to controls and unaffected by treatment. No effect of treatment with Reofos 65, Reofos 65 washed, Reofos 120 or mpIPTPP was evident from food consumption data.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): An adverse effect on fertility and reproductive performance was seen in the Reofos 65 group.
Fertility and fecundity indices were 50% in this group and statistically lower than controls (50% vs. 100%). These effects were reversible, however, and were not apparent in the recovery phase of the study. Although not statistically lower than controls, a slight decrease of 25% for both fertility and fecundity indices were seen in the Reofos 35 group. This difference was considered suggestive of an effect on fertility and reproductive performance.No effects on fertility or reproductive performance were seen in the remaining treated groups.

ORGAN WEIGHTS (PARENTAL ANIMALS): In males, adrenal and liver weights absolute and relative to body weight were increased in the Reofos 35 (exclusive of absolute liver weight), Reofos 65, Reofos 65 washed, and Reofos 120 groups. In females, the liver weights, absolute and relative to body weight, were increased for these same groups and in the mpIPTPP group. Additionally, in females, the adrenal weights, absolute and relative to body weight, were increased in the Reofos 35, Reofos 65, Reofos 65 washed, and Reofos 120 groups. The increased adrenal weights in males and females and liver weights in females were associated with microscopic changes in these organs. In the recovery phase, adrenal to body weight ratio for the Reofos 65 females was decreased.

GROSS PATHOLOGY AND HISTOPATHOLOGY (PARENTAL ANIMALS): Test article-associated fatty change was observed in the adrenal glands of males and females in Groups 2, 3,4, and 5; vacuolation of interstitial cells of the ovaries in Groups 2,3,4,5; and centrilobular hepatocellular hypertrophy oflivers of females in Groups 2,3,4, and 5. Fatty change was similar in the adrenal glands of males and females and was characterized by cells in the zona fasciculata and zona reticularis that were enlarged by the presence of increased small or large clear vacuoles. In a similar fashion, the cytoplasm of interstitial cells in the medulla of the ovaries was expanded by numerous small clear vacuoles. Even though the term fatty change was used for adrenal gland and vacuolation was used for the interstitial cells of the ovary, the material that accumulated to cause the change was probably the same in each organ and may be cholesteroI. Whereas both males and females had increased liver weight, only females had a distinct centrilobular hypertrophy in which cells had a small increase in acidophilic cytoplasm. Males may have had diffuse liver cell enlargement rather than a zonally recognizable type of enlargement. Other microscopic observations were of low incidence, were not present in a dose-response pattern, and are incidental or usual in rats.

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
< 400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
haematology
organ weights and organ / body weight ratios
gross pathology
reproductive performance
Remarks on result:
not determinable
Remarks:
no NOAEC identified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
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:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No effect of treatment with the various isopropylated triphenyl phosphate mixtures tested was evident from pup clinical findings at birth or LD 4. All 12 pups in the litter of Reofos 65 washed female number 333 were described as cold (i.e., skin cold to touch) at birth, but this was not noted in the 10 pups that survived to LD 4 in this litter.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- Reofos 35: Five of the nine litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls.
- Reofos 65: Four of the six litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls..
- Reofos 65 washed: Six of the 10 litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls.
- Reofos 120:. Six of 11 litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls. Two litters experienced particularly high pup mortality over LD 0-4.
-mpIPTPP: No effect of treatment was evident from this treatment on pup survival to LD 4.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Reofos 65: In this group, mean pup body weights, distinguished by sex and as a combined sex, were comparable to controls at LD O. On LD 4 pup weights were about 7-8% lower than controls and these differences were statistically significant for females and the combined sexes.
- Reofos 65 washed: Mean pup body weights, distinguished by sex and as a combined sex, in the Reofos 65 washed group were comparable to controls at LD 0 but notably lower than controls (14-18%) at LD 4. Only for female pups, however, was this difference (18%) at LD 4 statistically significant.
- Reofos 35, mpIPTPP, Reofos 120: Mean pup body weights, distinguished by sex and as a combined sex, in these groups at LD 0 and 4 did not differ statistically from controls and were considered comparable between the two groups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY (OFFSPRING):
- Reofos 35: The mean pup survival index over LD 0-4 in the Reofos 35 group (92.43%) was statistically lower than controls (99.36%). Five of the nine litters in this group experienced somc pup mortality over this period in comparison to two of 12 litters in controls.
- Reofos 65: The mean pup survival index over LD 0-4 in the Reofos 65 group (86.63%) was statistically lower than controls (99.36%). Four of the six litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls. In the Reofos 65 recovery phase, the pup survival index for LD 0-4 was 100% and 100% in controls. If the lower pup survival to LD 4 in the main study was treatment-related it was not
apparent in the recovery animals.
- Reofos 65 washed: The mean pup survival index over LD 0-4 in the Reofos 65 washed group (77.95%) was statistically lower than controls (99.36%). Six of the 10 litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls. All 17 pups in the litter of female number 336 from the Reofos 65 washed group died over this 4-day period.
- Reofos 120: The mean pup survival index over LD 0-4 in the Reofos 120 group (78.63%) was lower than controls (99.36%) but this difference was not statistically significant. Six of 11 litters in this group experienced some pup mortality over this period in comparison to two of 12 litters in controls. Two litters experienced particularly high pup mortality over LD 0-4. Female number 342 had 11 live pups at Day 0 and only one survived to LD 4, and female number 343 had 13 pups at birth and none survived to LD 4.
-mpIPTPP: The mean pup survival index over LD 0-4 in the mpIPTPP group was 96.81 % and comparable to controls (99.36%). No effect of treatment was evident from this treatment on pup survival to LD 4.

CLINICAL SIGNS (OFFSPRING): No effect of treatment with the various isopropylated triphenyl phosphate mixtures tested was evident from pup clinical findings at birth or LD 4. All 12 pups in the litter of Reofos 65 washed female number 333 were described as cold (i.e., skin cold to touch) at birth, but this was not noted in the 10 pups that survived to LD 4 in this litter. Other findings seen among the live pups in the treated groups at birth or LD 4 occurred at low incidence and were considered unrelated to treatment.

BODY WEIGHT (OFFSPRING):
- Reofos 65: In this group, mean pup body weights, distinguished by sex and as a combined sex, were comparable to controls at LD O. On LD 4 pup weights were about 7-8% lower than controls and these differences were statistically significant for females and thc combined sexes. In the Reofos 65 recovery phase, mean pup weights in the treated group were higher than the recovery controls at LD 0 and 4 and some of these differences were statistically significant. If the lower pup weights on LD 4 in the main study were treatment-related it was not
apparent in the recovery animals.
- Reofos 65 washed: Mean pup body weights, distinguishcd by sex and as a combined sex, in the Reofos 65 washed group were comparable to controls at LD 0 but notably lower than controls (14-18%) at LD 4. Only for female pups, however, was this difference (18%) at LD 4 statistically significant.
- Reofos 35, mpIPTPP, Reofos 120: Mean pup body weights, distinguished by sex and as a combined sex, in these groups at LD 0 and 4 did not differ statistically from controls and were considercd comparable between the two groups.

GROSS PATHOLOGY AND HISTOPATHOLOGY (OFFSPRING):
No effect of treatment with the various isopropylated triphenyl phosphate mixtures tested was evident from external macroscopic examinations of pups found dead during lactation or at scheduled LD 4 euthanasia. The few findings seen among pups from the treated groups occurred at low incidence and were considered spurious and unrelated to treatment. No effect of treatment with Reofos 65 washed or mpIPTPP was evident from the pup visceral evaluations. Folded retina was the only finding seen during these special examinations and was observed with similar frequency among the control and treated pups. It was seen with greatest frequency among pups found dead on LD 0 or over the LD 0-4 period, but was also seen in a few pups euthanized on LD 4.

Effect levels (F1)

Dose descriptor:
other: Not determined
Generation:
F1
Remarks on result:
not measured/tested

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Results:

The majority of the results tables can be found in the 'Attached Background Material Section' in the document 'Reofos 35 Oral Repro-Developmental Tox Rat Results Tables.pdf.'

Homogeneity of formualtions used in study:

Homogeneity

Test Article

Nominal Concentration (mg/mL)

Concentration Found (mg/mL)

Percent Recovery (a)

RSD (%)

Mean

Range

Mean

Range

Reofos 35

80.0

79.6

78.6-80.4

99.5

98.3-100.5

0.7

Reofos 65

80.0

81.7

80.7-82.8

102.0

100.9-103.6

0.9

Reofos 65 washed

80.0

80.7

78.6-82.0

100.8

98.2-102.5

1.4

Reofos 120

80.0

80.9

79.2-81.8

101.1

99.0-102.2

1.2

mpIPTPP

80.0

79.7

78.8-80.2

99.6

98.5-100.2

0.7

a: Mean Percent Recovery was calculated from the nominal concentration.

RSD: Relative Standard Deviation

 

Stability of formulations used in the study:

Stability

Test Article

Nominal Concentration

(mg/mL)

Mean Found Concentration (mg/mL) (Day)

Mean % Recovery from Day 0

 

 

0a

7

14

7 days(b)

14 Days(b)

Reofos 35

80.0

79.6

79.8

80.6

100.2

101.3

Reofos 65

80.0

81.7

80.6

81.1

98.8

99.3

Reofos 65 washed

80.0

80.7

80.3

81.2

99.6

100.7

Reofos 120

80.0

80.9

81.6

81.9

100.8

101.2

mpIPTPP

80.0

79.7

81.3

82.0

102.0

102.9

a: Values expressed are the mean homogeneity concentrations.

b: Day 7 and 14 mean % recovery values were calculated from the respective Day 0 value

 

Analysis of doses used in the study:

Periodic Analysis of Dosing Formulations Used on Study

Test Article

Nominal Concentration (mg/mL)

Found Concentration (a) [mg/mL] (% of nominal)

Week 1 (b)

Week 2

Week 3

Week 4

Week 8

Vehicle

0

0 (NA)

0 (NA)

0 (NA)

0 (NA)

0 (NA)

Reofos 35

80.0

79.6

(99.5)

79.6 (99.5)

79.9 (99.8)

79.5 (99.4)

78.1 (97.6)

Reofos 65

80.0

81.7     (102.1)

81.0 (101.3)

79.5 (99.3)

81.5 (101.8)

79.2 (99.0)

Reofos 65 washed

80.0

80.7    (100.8)

81.2 (101.5)

81.8 (102.3)

80.8 (101.1)

78.6 (98.3)

Reofos 120

80.0

80.9     (101.1)

79.3 (99.1)

80.1 (100.1)

79.2 (99.0)

80.5 (100.6)

mpIPTPP

80.0

79.7       (99.6)

80.3 (100.4)

80.8 (101.0)

79.5 (99.4)

74.6 (93.2)

a: Mean of duplicate analyses

b: Mean of the six homogeneity samples

NA: Not Applicable

 

Applicant's summary and conclusion

Conclusions:
Each test article (Reofos 35, Reofos 65, Reofos 65 washed, Reofos 120, and mpIPTPP) evaluated was administered orally by gastric intubation at a dose level of 400 mg/kg/day and dose volume of 5 mL/kg in a study following OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test). Salivation was seen commonly among all the treated groups. Body weight and weight gain effects (lower) were seen in the Reofos 35 and Reofos 65 males. Poor reproductive performance was seen only in the Reofos 65 group but the response was reversible, and not apparent in a mating of recovery animals. Reproductive performance, parturition data, and litter size data for the remaining test groups were considered unaffected by treatment. Test article-associated organ weight effects were seen for the adrenals and liver and microscopically, test article-associated fatty changes were observed in thc adrenals of males and females in the Reofos 35, Reofos 65, Reofos 65 washed, and Reofos 120 groups.
Executive summary:

Each test article (Reofos 35, Reofos 65, Reofos 65 washed, Reofos 120, and mpIPTPP) evaluated was administered orally by gastric intubation at a dose level of 400 mg/kg/day and dose volume of 5 mL/kg in a study following OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test). Salivation was seen commonly among all the treated groups. Body weight and weight gain effects (lower) were seen in the Reofos 35 and Reofos 65 males. Poor reproductive performance was seen only in the Reofos 65 group but the response was reversible, and not apparent in a mating of recovery animals. Reproductive performance, parturition data, and litter size data for the remaining test groups were considered unaffected by treatment. Test article-associated organ weight effects were seen for the adrenals and liver and microscopically, test article-associated fatty changes were observed in thc adrenals of males and females in the Reofos 35, Reofos 65, Reofos 65 washed, and Reofos 120 groups.