Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

None

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 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.
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
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.

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.
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.
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
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
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: 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.
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.
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
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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.
Dose descriptor:
other: Not determined
Generation:
F1
Remarks on result:
not measured/tested
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Reproductive effects observed:
not specified

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

 

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.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
See discussion below
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There are two 28-day screening studies available for the substance that assess reproductive toxicity. Adverse effects on fertility were noted in that effects on fertility and reproductive performance were documented during the study. These effects were reversible, however, and were not apparent in the recovery phase of the study. Test article-related reductions in male fertility and copulation indices were noted. Decreased epididymal weights were considered test article-related.   As these are screening studies, a further assessment of prolonged effects is proposed. The registrant originally proposed to conduct the OECD 416 study on this particular substance to allow comparison with other existing data on analogous substances, plus given that the OECD 443 study is a relatively “new” guideline.

However, a request was received from the EC Commission (reference ENV.B2/fvr/ARES(2016)-6427555 to alter this proposal in accordance with the Draft Commission Implementing Decision on the examination of testing proposals for reproductive toxicity. As such, the registrant has altered the study proposal in accordance with this request.

It should be noted that the basic test design is proposed. The registrant considers that there is sufficient information already available for neurotoxicity on the substance, and immunotoxicity is not considered to be a concern on the basis of the data available. It is considered therefore that the proposed basic test design will be adequate to determine the potential reproduction effects noted in the 28-day screening studies.

 

Without further clarification, the reproduction toxicity triggered classification under the CLP Regulation (EC No 1272/2008) as follows:

 

Repr. Cat 2, H361: Suspected of damaging fertility or the unborn child < Fertility - effects on epididymides (oral)

Short description of key information:

Screening for oral reproduction toxicity only is discussed.

Justification for selection of Effect on fertility via oral route:

See discussion below

Effects on developmental toxicity

Description of key information

Developmental toxicity only is discussed.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 25, 2014 to April 28, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & US EPA test guidelines in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
A total of 112 time-mated female CD® [Crl:CD®(SD)] rats (approximately 8 to 10 weeks of age) were received from Charles River Laboratories, Portage, Michigan on February 25 and 26, 2014. The animals were randomly assigned to study and dosing began on GD 0. Prior to the initiation of dose administration, the Study Director reviewed the GD 0 body weight and detailed observation data for all animals and gave final approval for assignment to study.

Randomization, Assignment to Study, and Maintenance
Using a standard, by weight, measured value randomization procedure, 100 female animals (weighing 179 to 254 g, at randomization) were assigned to the control and treatment groups identified in the following table.
Group Assignments
Group Number Dose Level (mg/kg/day) Number of Time-mated Females
1 0 25
2 100 25
3 200 25
4 400 25

Animals assigned to study had body weights within ±20% of the mean body weight. Extra animals obtained, but not placed on study, were euthanized via carbon dioxide inhalation. Euthanasia was confirmed via exsanguination of the abdominal vena cava, and the carcasses were discarded.
Each animal was assigned an animal number to be used in the Provantis™ data collection system and was implanted with a microchip bearing a unique identification number. The individual animal number, implant number, and study number comprised a unique identification for each animal. Each cage was identified by the animal number, study number, group number, and sex.
The animals were individually housed in solid bottom cages with nonaromatic bedding in an environmentally controlled room. Animal enrichment was provided according to SOP. Fluorescent lighting was provided for approximately 12 hours per day. Temperature and humidity were continuously monitored, recorded, and maintained to the maximum extent possible within the ranges of 68 to 79°F and 30 to 70%, respectively. The actual temperature and humidity findings are not reported but are maintained in the study file.
Block Lab Diet® (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum. The lot number from each diet lot used for this study was recorded. Certification analysis of each diet lot was performed by the manufacturer. Tap water was available ad libitum via an automatic watering system. The water supply is monitored for specified contaminants at periodic intervals according to SOP. The results of food and water analyses are retained in the Archives. The Study Director is not aware of any potential contaminants likely to be present in the diet or water that would have interfered with the results of the study. Therefore, no analyses other than those stated above were conducted.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The vehicle and test article were administered once a day from GD 0 to 19 at approximately the same time each day (±2 hours from the GD 0 dose) via oral gavage. The dose levels for the treated groups were 100, 200, and 400 mg/kg/day at a dose volume of 5 mL/kg/day. The control group received the vehicle in the same manner as the treated groups. The dosing formulations were stirred throughout administration. Individual doses were based on the most recent body weights.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples (see following table) were collected using a positive displacement pipette, while stirring, and placed into amber glass bottles. Following acceptance of the analytical results (signing of the final report) by the Study Director, or at the discretion of the Study Director, backup samples will be discarded. Tabulated data is listed below under "any other information"

Samples were stored refrigerated at 2 to 8°C until shipped on gel packs to the MPI Research, Inc., site at State College, Pennsylvania, for analysis. All analytical work was conducted by MPI Research, Inc., using an analytical method developed by MPI Research, Inc.

Objective: To determine the homogeneity and concentration of Reofos 35 in dosing formulations.
Analytical Method Number and Title: 399-246-A-02 (V0008648-3): Determination of Reofos 35 in Corn Oil. Dose Formulations by GC-FID
Reference Standard: Reofos 35; Lot Number: 2012123125; MPI Research Inventory ID: SP0014320; Storage: Room temperature; Correction Factor: None
Data Collection and Analysis Software: Empower™ 2: Build 2154 (Waters Technologies Corporation®). ExyLIMS Version 3.0 (MPI Research, Mattawan, Michigan)
GC Conditions: Gas chromatography system equipped with a Hewlett Packard HP-1 column, 30 m x 0.25 μm, 0.25 μm thickness with a flame ionization detector.
Analysis Description: Prior to analysis, samples were diluted with 2-propanol (diluent) to within the range of the calibration curve. Vehicle samples were diluted with diluent using a dilution factor of 8. An aliquot of each sample was injected into the GC-FID system for analysis.
Regression Type: Linear unweighted
Study Sample Receipt(s): Number of Samples: 48; Date(s) of receipt by Analytical Department:
February 26, 2014 to March 12, 2014
Study Sample Storage Conditions: Refrigerated (2 to 8°C)
Storage Stability: All samples were analyzed within the established storage stability determined under MPI Research Study Number 399-243.
Analysis Period: March 6, 2014 to March 27, 2014

Run Acceptance Criteria
System Suitability Test Standards:
1. Injection repeatability (peak area and retention time) ≤5% RSD (relative standard deviation)
2. Resolution between the analyte peak and any adjacent peaks must be ≥1.5
3. Tailing Factor (T) ≤2
4. Theoretical Plates (N) ≥2000
Calibration Standards:
1. Accuracy within ±10% of the nominal concentration
2. Coefficient of determination (R2) ≥0.995
Performance Check Standards (Same preparation as the System Suitability Standard):
1. Periodically injected so that no more than 10 samples are bracketed by performance check standards. Samples are considered valid if bracketed by passing performance check standard injections.
2. Accuracy within ±10% of the nominal concentration
Blank Injections: ≤20% of the limit of quantitation (LOQ)

Assessments
Homogeneity:
1. Average concentration within ±10% of the nominal concentration
2. Precision ≤5% RSD
Concentration:
1. Average concentration within ±10% of the nominal concentration
2. Precision ≤5% RSD
3. Vehicle (control) samples < LOQ

Results and Discussion
Conclusion: A total of 36 samples were analyzed for Reofos 35 in dosing formulations using a validated method. The analytical data demonstrated acceptable performance of the method for all reported results. In dataset 030614, there was a programming error in the analytical sequence, which resulted in some samples (Group 1 Week 1 and Group 1 Week 2) not being injected and other samples (Group 2 Week 1) being injected multiple times. All data generated in this dataset is reported; therefore, there are additional replicates for the homogeneity and concentration analyses for the Group 2 Week 1 samples. All samples met the acceptance criteria, so there is no suspected impact.
Deviations: This study phase was conducted in accordance with the protocol with the exception of the following deviations:
Standard Operating Procedure (SOP) Deviations:
SOP deviations were acknowledged by the Study Director and documented in the raw data.
In the opinion of the Study Director, none of the SOP deviations were considered to have affected the quality or integrity of the study.
Details on mating procedure:
Time-mated females used in the study - no details specified in the study report.
Duration of treatment / exposure:
19 days
Frequency of treatment:
Daily
Duration of test:
20 days
Remarks:
Doses / Concentrations:
0, 100, 200, and 400 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Fresh vehicle, corn oil, was dispensed for use on study weekly and was stored refrigerated at 2 to 8°C when not in use.
The test article, Reofos 35, was used as received from the Sponsor, and no adjustment was made for purity. Formulations of the test article were prepared weekly at nominal concentrations of 20, 40, and 80 mg/mL, and were stored refrigerated at 2 to 8°C when not in use.

Analysis of Dosing Formulations
Dosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples (see table in Any other information) were collected using a positive displacement pipette, while stirring, and placed into amber glass bottles. Following acceptance of the analytical results (signing of the final report) by the Study Director, or at the discretion of the Study Director, backup samples will be discarded.
Stability has been established for at least 10 days under refrigerated (2 to 8°C) conditions under MPI Research, Inc., Study Number 399-246.

Justification for Route of Administration
The oral route is one of the potential routes of human exposure to this test article.

Justification of Dose Levels
The dose levels were selected by the Sponsor on the basis of available data from a recently conducted pilot prenatal developmental toxicity study (MPI Research, Inc., Study Number 399-245) and available data from a previously conducted reproduction/developmental screening study that included Reofos 35 (MPI Research, Inc., Study Number 1038-004). In the pilot study (MPI Research, Inc., Study Number 399-245), mortality (60%), lower body weights and body weight gain, lower food consumption, and reduced fetal body weights were observed in time-mated females treated daily from GD 0 through GD 20 with Reofos 35 at 500 mg/kg/day.
In the screening study (MPI Research, Inc., Study Number 1038-004), animals were treated daily for 8 weeks with Reofos 35 at 400 mg/kg/day. Clinical findings (salivation), lower body weights and body weight gain, reproductive effects, organ weight changes, and microscopic observations were observed.
Based on the available data, dose levels up to 400 mg/kg/day were evaluated in time-mated females from GD 0 to 20. The low- and mid-dose are at approximate log intervals and were chosen to provide a graded response.
Maternal examinations:
In-life Examinations
Cage-side Observations: All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily. On occasion, veterinary consultations were conducted during the course of the study. All treatments and observations were recorded. The medical treatments and observations are not reported but are maintained in the study file.
Detailed Clinical Observations: Daily from GD 0 through 20 (60 to 90 minutes post dose on dosing days), each animal was removed from the cage and given a detailed clinical examination. On occasion, clinical observations were recorded at unscheduled intervals. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, reactivity to handling, and unusual behavior.
Body Weights and Body Weight Changes: Body weights for all animals were measured and recorded on GD 0, 3, 6, 9, 12, 15, 18, and 20. Individual body weight change was calculated for the following GD intervals: 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-20, and 0-20. Adjusted body weight (GD 20 body weight minus gravid uterine weight) and adjusted body weight change (GD 0 to 20) were also calculated.
Food Consumption: Food consumption was measured and recorded on the corresponding body weight days and calculated for the same.

Postmortem Study Evaluations
Maternal Necropsy: A complete necropsy was performed on all dams under procedures approved by a veterinary pathologist. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced the pregnancy. The presence of lesions or other abnormal conditions in the dam were noted and described in the study records.
Ovaries and uterine content:
Ovarian and Uterine Examination
On GD 20, each female was euthanized by carbon dioxide inhalation, followed by exsanguination of the abdominal vena cava and immediately subjected to a cesarean section. Dams had uterine examinations conducted without knowledge of the treatment group. The skin was reflected from a ventral midline incision to examine mammary tissue and locate any subcutaneous masses. The abdominal cavity was then opened, and the uterus was exposed. The uterus was excised, and the gravid uterine weight was recorded. Beginning at the distal end of the left uterine horn, the location of viable and nonviable fetuses, early and late resorptions for each uterine horn, and the total number of implantations were recorded. The number of corpora lutea on each ovary was also recorded.
Each implant was categorized according to the following criteria.
Viable fetuses responded to touch. Nonviable fetuses did not respond to touch and had no signs of autolysis. Late resorptions were characterized by recognizable fetal form, but undergoing autolysis. Early resorptions were characterized as implantation sites that had no recognizable fetal characteristics.
The fetuses were removed by making a dorsal incision longitudinally along both uterine horns. The embryonic membrane of each fetus was gently removed, and each fetus was pulled away from the placenta, fully extending the umbilical cord. The placentae were examined grossly.
Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantation sites. If no foci were detected, the female was considered to be non-pregnant.
Fetal examinations:
Fetal Examinations
Each fetus was individually weighed, sexed, tagged, and examined for external malformations and variations. Fetuses were then euthanized by intraperitoneal injection of euthanasia solution. Fetal external, visceral, and skeletal examinations were conducted without knowledge of the treatment group.
Approximately one-half of the fetuses in each litter were placed in Bouin’s solution and the remaining fetuses were fixed in alcohol. All fetuses fixed in Bouin’s solution were examined for soft tissue defects using the Wilson razor-blade sectioning technique. The fetuses fixed in alcohol were macerated in potassium hydroxide, stained with Alizarin Red S, and cleared with glycerin by a method similar to that described by Dawson for subsequent skeletal examination. Fetal findings were classified as malformations or developmental variations under procedures approved by a developmental toxicologist. On occasion, additional information to clarify or identify a visceral or skeletal observation was documented. These comments are not reported, but are maintained in the study data. Mechanical artifacts (i.e., tail removed and discarded) occurred during examination and processing of the fetuses.
These artifacts are not reported, but are maintained in the study data.
Statistics:
See Any other information for Statistics data.
Indices:
Fetal and litter incidences are reported, but only the litter incidences were statistically analyzed.
Historical control data:
Historical Control Developmental Toxicity Data
Sprague Dawley Rat
07/2006 to 07/2011
Number of Studies: 50
Number of Litters Evaluated: 1230
Number of Fetuses Evaluated – External: 14180
Number of Fetuses Evaluated – Visceral: 7072
Number of Fetuses Evaluated – Skeletal: 7106
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was observed in each of the Reofos 35-treated groups and considered a test article-related effect. It was observed at least once in 4, 8 and 15 animals in the 100, 200, and 400 mg/kg/day dose groups, respectively. Its occurrence on study was likely a pharmacologic response to the test article and was not considered adverse. Other clinical findings observed at dose levels ≤200 mg/kg/day occurred at low incidence or with similar frequency as controls and considered unrelated to the test article. In the 400 mg/kg/day dose group, additional clinical findings observed within the first several days of dosing (GD 1 to 3) and considered test article related included decreased activity (one animal), red material around the nose/mouth (nine animals), hunched posture (one animal), thin appearance (two animals), hair on the face discolored brown (two animals), and hair on the forelimbs discolored brown (two animals). These clinical findings were transient and were not observed among the 400 mg/kg/day animals for the remainder of the study.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 400 mg/kg/day dose group, a mean body weight gain of 0.1 g over GD 0 to 3 differed statistically from the mean weight gain of 14.0 g in the control group. This low weight gain in the 400 mg/kg/day dose group was considered test article related and adverse correlating with a period of low food consumption.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 200 mg/kg/day dose group, mean food consumption throughout gestation was higher relative to mean control values and these differences were statistically significant over GD 3 to 6 (+23%), GD 6 to 9 (+18%), GD 9 to 12 (+17%), GD 12 to 15 (+10%), GD 18 to 20 (+10%), and GD 0 to 20 (+13%). The increase in food consumption in the 200 mg/kg/day dose group may have been test article related but the effect was not considered adverse and only over GD 0 to 20 did it correlate with statistically higher body weight gain relative to controls. In the 400 mg/kg/day dose group, mean food consumption over GD 0 to 3 at 9.1 g/day was statistically lower than the mean control value of 13.6 g/day. This was considered test article related and adverse correlating with a period of statistically lower body weight gain.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: red or white foci and swollen mucosa of the nonglandular portion of the stomach

Details on maternal toxic effects:
In-life Examinations
Mortality
All animals survived to scheduled termination on GD 20.

Detailed Clinical Observations
Salivation was observed in each of the Reofos 35-treated groups and considered a test article-related effect. It was observed at least once in 4, 8 and 15 animals in the 100, 200, and 400 mg/kg/day dose groups, respectively. Its occurrence on study was likely a pharmacologic response to the test article and was not considered adverse. Other clinical findings observed at dose levels ≤200 mg/kg/day occurred at low incidence or with similar frequency as controls and considered unrelated to the test article. In the 400 mg/kg/day dose group, additional clinical findings observed within the first several days of dosing (GD 1 to 3) and considered test article related included decreased activity (one animal), red material around the nose/mouth (nine animals), hunched posture (one animal), thin appearance (two animals), hair on the face discolored brown (two animals), and hair on the forelimbs discolored brown (two animals). These clinical findings were transient and were not observed among the 400 mg/kg/day animals for the remainder of the study.

Body Weights and Body Weight Changes
No test article effect at the 100 and 200 mg/kg/day dose levels was observed on gestation body weights or body weight gain. A statistically significant increase in mean weight gain over GD 0 to 20 in the 200 mg/kg/day dose group relative to the control mean value (170.7 g vs.156.7 g in controls) was considered unrelated to the test article but did correlate with statistically higher food consumption relative to the controls over this period. In the 400 mg/kg/day dose group, a mean body weight gain of 0.1 g over GD 0 to 3 differed statistically from the mean weight gain of 14.0 g in the control group. This low weight gain in the 400 mg/kg/day dose group was considered test article related and adverse correlating with a period of low food consumption. However, the effect was transient and over the ensuing intervals of GD 3 to 6 and GD 6 to 9, mean body weight gains in the 400 mg/kg/day dose group of 22.7 g and 22.8 g were statistically higher than the mean body weight gains of 15.2 g (49% higher) and 19.5 g (17% higher) respectively, in the control group. For the remainder of gestation and over the entire GD 0 to 20 period, mean weight gains in the 400 mg/kg/day dose group were comparable to mean control values.

Food Consumption
No adverse effect of test article at the 100 and 200 mg/kg/day dose levels was observed on gestation food consumption. Mean food consumption throughout gestation in the 100 mg/kg/day dose group was comparable to mean control values. In the 200 mg/kg/day dose group, mean food consumption throughout gestation was higher relative to mean control values and these differences were statistically significant over GD 3 to 6 (+23%), GD 6 to 9 (+18%), GD 9 to 12 (+17%), GD 12 to 15 (+10%), GD 18 to 20 (+10%), and GD 0 to 20 (+13%). The increase in food consumption in the 200 mg/kg/day dose group may have been test article related but the effect was not considered adverse and only over GD 0 to 20 did it correlate with statistically higher body weight gain relative to controls. In the 400 mg/kg/day dose group, mean food consumption over GD 0 to 3 at 9.1 g/day was statistically lower than the mean control value of 13.6 g/day. This was considered test article related and adverse correlating with a period of statistically lower body weight gain. The effect on food consumption at 400 mg/kg/day was transient and for the remainder of gestation, mean food consumption in this group was 4% to 17% higher than the mean control values with statistical significance identified over GD 3 to 6 (+15%), GD 9 to 12 (+17%), and GD 12 to 15 (+13%).

Postmortem Study Evaluations
Uterine and Ovarian Examinations
The pregnancy indices were 96%, 96%, 96%, and 92% in the control, 100, 200, and 400 mg/kg/day dose groups providing 24, 24, 24, and 23 GD 20 litters with fetuses for evaluation, respectively. No test article effect was observed from GD 20 uterine implantation data. In the 100 mg/kg/day dose group, the mean pre-implantation loss index was 14.63% and statistically higher than the 5.01% in controls but in the absence of a similar increase in this index at the higher dose levels, this was considered incidental and unrelated to the test article. All other mean uterine implantation parameters (corpora lutea, implantation sites, viable fetuses, post-implantation loss, litter size, and resorption sites [early, late, and total]) in the treated groups were comparable to mean control values. Mean gravid uterine weights in the 100 and 400 mg/kg/day dose groups were comparable to mean control values. In the 200 mg/kg/day dose group, the mean gravid uterine weight at 80.26 g was statistically higher than the mean control value of 71.24 g. This was considered related to the slightly greater number of fetuses in utero, and unrelated to the test article. The mean adjusted GD 20 body weights (GD 20 body weight minus the gravid uterine weight) and adjusted body weight change GD 0 to 20 in the treated groups were comparable to mean control values.

Maternal Macroscopic Observations
No test article effect at 100 and 200 mg/kg/day was observed from the maternal macroscopic examinations. Macroscopic findings observed among these animals occurred at low incidence (single animals affected), and were considered incidental and unrelated to the test article. In the 400 mg/kg/day dose group, red or white foci and swollen mucosa of the nonglandular portion of the stomach were observed in 3/25 animals. Their occurrence in the 400 mg/kg/day group was considered test article related as similar findings were not observed among control animals or animals at the lower dose levels. No other macroscopic findings were observed among the 400 mg/kg/day animals.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal Evaluations
Fetal Sex Ratio
No test article effect was observed from fetal sex ratios (% male fetuses/animal). Mean fetal sex ratios in the treated groups ranged from 45.4% to 53.4% and were comparable to the mean of 54.0% in the control group.
Fetal Body Weights
No test article effect was observed on fetal body weights. In the 100 mg/kg/day dose group, fetal body weights, distinguished by sex and for the combined sexes, were comparable to mean control values. In the 200 and 400 mg/kg/day dose groups, mean fetal body weights were higher than mean control values and although the differences were slight (5% to 7%) in most instances they were statistically significant and outside the ranges of recent historical control data for the laboratory. The increase in mean fetal body weights observed in the 200 and 400 mg/kg/day dose groups although statistically significant relative to control values, were still relatively slight 5 to 7% and not considered adverse or indicative of a test article-related response.
External Examinations
No test article effect was observed from the fetal external examinations. No malformations or developmental variations were observed among the control or treated fetuses from the external examinations.
Visceral Examinations
No test article effect was observed from the fetal visceral examinations. No visceral malformations were observed among the control or treated fetuses. The only visceral developmental variation observed was dilated ureter. This variation has a high historical presence in this laboratory (Appendix O, maximum litter incidence of 20.8%) and its occurrence in a single fetus in the 400 mg/kg/day dose group (litter incidence 4.3%) was considered incidental and unrelated to the test article.
Skeletal Examination
No test article effect was observed from the fetal skeletal examinations. The few skeletal malformations observed among fetuses in the treated groups (one fetus in the 200 mg/kg/day dose group and two fetuses from a single litter in the 400 mg/kg/day dose group) were dissimilar in appearance and considered unrelated to the test article. No skeletal malformations were observed in fetuses from the 100 mg/kg/day dose group; one control fetus had a skeletal malformation. Skeletal developmental variations observed in the treated groups occurred at low incidence or with similar frequency as in controls and no effect of the test article on skeletal development was indicated from these data.
Dose descriptor:
other: Not determined
Remarks on result:
not measured/tested
Abnormalities:
not specified
Developmental effects observed:
not specified

 Homogeneity

Analysis of the low- and high-dose formulations (20.0 and 80.0 mg/mL, respectively) used for dosing the first week of study confirmed they were homogeneous as prepared and therefore met the laboratory’s acceptance criteria of 100±10% of nominal, and percent relative standard deviation (RSD) ≤5. These analytical results are summarized in the table below.

Homogeneity

Dose Level (mg/kg/day)

Nominal Concentration (mg/mL)

Average Calculated Concentration (mg/mL)

Average %Recovery

%Relative Standard Deviationc

100

400

20.0

80.0

18.7372a

81.6908b

93.7a

102.1b

1.9

1.5

aRepresents an average of seven samples from the mixing container (two top, two middle and three bottom. Samples were collected while the formulation was stirring.

bRepresents an average of six samples from the mixing containers (two top, two middle and two bottom). Samples were collected while the formulation was stirring.

cAverage %recovery was calculated from the nominal concentration.

Concentration

Mean concentrations of formulations used for dosing each of the three weeks of study ranged between 93.2 and 102.1% of nominal confirming that animals were receiving the appropriate dose levels when the dose was administered at 5 mL/kg. No test article was found in the control samples. These results are summarized in the table below.

Concentration

Dose Level (mg/kg/day)

Nominal Concentration (mg/mL)

Average Calculated Concentrationa(mg/mL)

Average %Recoverya,b

0

100

200

400

0.0

20.0

40.0

80.0

BLQ

18.6350 – 19.1496

37.4113 – 38.8650

81.0502 – 81.6550

NA

93.2 – 95.7

93.5 – 97.2

101.3 – 102.1

aResults are the range of values determined during Weeks 1, 2 and 3.

bAverage %recovery was calculated from the nominal concentration.

BLQ – Below the Limit of Quantification (<0.4 mg/mL)

NA – Not Applicable

 

Summary of Gestation Detailed Clinical Observations+

Days 0 to 20

Observation

0 mg/kg/day

100 mg/kg/day

200 mg/kg/day

400 mg/kg/day

Number of Animals Observed

25

25

25

25

Behavior/Activity

               Activity decreased

               Salivation

 

0/0

0/0

 

0/0

6/4

 

0/0

36/8

 

2/1

85/15

External Appearance

               Discharge, Red, Anogenital region

               Ear/portion of ear missing, Ear/left

               Material around mouth, Red

               Material around nose, Red

               Posture hunched

               Thin

 

0/0

6/1

0/0

0/0

0/0

0/0

 

0/0

0/0

0/0

0/0

0/0

0/0

 

1/1

5/1

0/0

0/0

0/0

0/0

 

0/0

0/0

1/1

11/9

2/1

3/2

Pelage/Skin

               Hair, discolored, Brown, Face

               Hair, discolored, Brown, Forelimb/left

               Hair, discolored, Brown, Forelimb/right

               Hair, discolored, Black, Thoracic region

               Hair, sparse, Abdominal region

               Hair, sparse, Cervical region

               Hair, sparse, Forefoot/left

               Hair, sparse, Forefoot/right

               Hair, sparse, Forelimb/left

               Hair, sparse, Forelimb/right

               Hair, sparse, Hind foot/left

               Hair, sparse, Hind foot/right

               Hair, sparse, Hind limb/left

               Hair, sparse, Hind limb/right

               Hair, sparse, Inguinal region/left

               Hair, sparse, Lumbar region

               Hair wet, Anogenital region

               Scabbed area, Cervical region

               Scabbed area, Forelimb/right

 

0/0

0/0

0/0

0/0

14/1

0/0

47/7

42/6

1/1

5/1

9/2

9/2

7/1

7/1

0/0

1/1

0/0

0/0

0/0

 

0/0

0/0

0/0

0/0

0/0

0/0

10/3

19/4

12/1

12/1

4/1

4/1

0/0

0/0

6/1

9/1

0/0

2/1

0/0

 

0/0

0/0

0/0

0/0

0/0

30/3

20/2

23/4

54/7

42/5

0/0

0/0

6/1

10/1

0/0

5/1

0/0

0/0

8/1

 

4/2

3/2

3/2

1/1

21/3

0/0

1/1

5/1

35/3

14/1

0/0

0/0

27/3

14/1

0/0

9/1

2/1

0/0

6/2

+ Number of times observed/Total number of animals affected.

 

Summary of Gestation Body Weight Values

Endpoint

Study Interval (Day)

0 mg/kg/day

100 mg/kg/day

200 mg/kg/day

400 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Body Weight Values g

 

 

 

 

 

 

 

 

 

 

 

 

 

0

214.1

18.94

24

214.8

18.47

24

215.3

20.20

24

215.0

20.55

23

3

228.2

17.73

24

230.3

17.49

24

231.4

20.78

24

215.2

20.71

23

6

243.3

18.60

24

245.3

18.44

24

248.9

22.21

24

237.9

21.35

23

9

262.9

19.04

24

265.9

18.52

24

270.5

23.94

24

260.7

21.19

23

12

283.3

20.42

24

285.6

18.85

24

292.8

27.78

24

284.6

22.81

23

15

301.1

20.34

24

304.3

18.85

24

313.6

29.01

24

303.9

25.00

23

18

336.2

26.04

24

338.9

21.33

24

350.7

33.29

24

336.3

27.33

23

20

370.8

30.31

24

372.3

23.66

24

386.0

36.84

24

368.8

28.85

23

N – Number of measures used to calculate mean

SD – Standard Deviation

 

Summary of Gestation Body Weight Change Values

Endpoint

Study Interval (Day)

0 mg/kg/day

100 mg/kg/day

200 mg/kg/day

400 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Body Weight Values g

 

 

 

 

 

 

 

 

 

 

 

 

 

0 – 3

14.0

4.47

24

15.5

5.49

24

16.1

8.51

24

0.1b

9.69

23

3 – 6

15.2

4.09

24

15.0

4.91

24

17.5

4.27

24

22.7b

5.86

23

6 – 9

19.5

4.67

24

20.6

3.86

24

21.6

5.24

24

22.8a

4.01

23

9 – 12

20.4

4.78

24

19.7

4.70

24

22.4

5.45

24

23.8

4.77

23

12 – 15

17.8

5.30

24

18.7

3.29

24

20.8

4.29

24

19.3

4.47

23

15 – 18

35.1

7.86

24

34.7

6.49

24

37.1

5.99

24

32.4

4.75

23

18 - 20

34.6

6.70

24

33.3

5.62

24

35.3

5.29

24

32.5

4.50

23

0 - 20

156.7

19.12

24

157.5

17.40

24

170.7a

21.25

24

153.7

18.35

23

N – Number of measures used to calculate mean        aSignificantly different from control; (p<0.05)

SD – Standard Deviation                                                   bSignificantly different from control; (p<0.01)

 

Summary of Gestation Food Consumption Values

Endpoint

Study Interval (Day)

0 mg/kg/day

100 mg/kg/day

200 mg/kg/day

400 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Food Consumption g/animal/day

 

 

 

 

 

 

 

 

 

 

 

 

 

0 – 3

13.6

1.89

24

13.8

2.13

24

14.2

3.48

24

9.1b

3.23

23

3 – 6

17.1

2.31

24

19.2

1.73

24

21.1b

2.52

24

19.6a

4.92

23

6 – 9

18.5

2.27

24

19.4

1.43

24

21.9b

3.30

24

20.5

4.27

23

9 – 12

19.1

2.15

24

20.1

1.64

24

22.4b

2.87

24

22.3b

2.66

23

12 – 15

22.3

2.04

24

23.3

1.56

24

24.6b

3.03

24

25.3b

3.22

23

15 – 18

24.5

3.17

24

25.1

2.21

24

26.6

3.32

24

25.6

3.17

23

18 - 20

23.1

3.35

24

24.8

2.12

24

25.4a

3.56

24

24.2

2.85

23

0 - 20

19.6

1.97

24

20.6

1.34

24

22.2b

2.66

24

20.8

2.70

23

N – Number of measures used to calculate mean        aSignificantly different from control; (p<0.05)

SD – Standard Deviation                                                   bSignificantly different from control; (p<0.01)

 

Summary of Maternal Developmental Observations at Uterine Examination

Endpoint

 

0 mg/kg/day

100 mg/kg/day

200 mg/kg/day

400 mg/kg/day

No. Females on Study

25

25

25

25

No. Not Pregnant

1

1

1

2

No. Pregnant

24

24

24

23

Pregnancy Index Percent

96.0

96.0

96.0

92.0

No. Females with Viable Fetuses Day 20 Gestation

24

24

24

23

Corpora Lutea

               No. per Animal

Mean

SD

N

12.9

2.08

24

13.5

2.08

24

13.5

1.53

24

13.8

2.84

23

Implantation Sites

               No. per Animal

Mean

SD

N

12.3

2.09

24

11.3

1.57

24

12.7

1.95

24

12.3

1.42

23

Preimplantation Loss

               % per Animal

Mean

SD

N

5.01

7.506

24

14.36b

13.246

24

5.67

7.263

24

9.51

11.254

23

Viable Fetuses

               No. per Animal

Mean

SD

N

11.5

2.04

24

10.8

1.84

24

12.0

1.43

24

11.4

1.50

23

Fetal Sex Ratio

               % Males per Animal

Mean

SD

N

54.0

16.36

24

49.8

15.02

24

53.4

16.60

24

45.4

18.37

23

Postimplantation Loss

               % per Animal

Mean

SD

N

6.27

7.070

24

4.73

7.509

24

4.94

7.831

24

6.79

5.383

23

Nonviable Fetuses

               No. per Animal

Mean

SD

N

0.0

0.00

24

0.0

0.00

24

0.0

0.00

24

0.0

0.00

23

Litter Size

               No. per Animal

Mean

SD

N

11.5

2.04

24

10.8

1.84

24

12.0

1.43

24

11.4

1.50

23

Resorptions: Early + Later

               No. per Animal

Mean

SD

N

0.8

0.88

24

0.5

0.78

24

0.6

1.01

24

0.8

0.65

23

Resorptions: Early

               No. per Animal

Mean

SD

N

0.8

0.85

24

0.5

0.78

24

0.6

0.97

24

0.8

0.65

23

Resorptions: Late

               No. per Animal

Mean

SD

N

0.0

0.00

24

0.0

0.00

24

0.0

0.00

24

0.0

0.00

23

No. – Number                                                                                      bSignificantly different from control; (p<0.01)

N – Number of measured used to calculate mean

SD – Standard Deviation

Conclusions:
This study was conducted to evaluate the potential adverse effects of the test article, Reofos 35, on systemic toxicity, female reproductive performance, and development of the fetus following repeated exposure to pregnant rats. Dose levels evaluated were 0 (corn oil vehicle), 100, 200, or 400 mg/kg/day and animals were treated orally with a single dose daily from GD 0 to 19. All animals survived to scheduled termination. The No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity was 200 mg/kg/day and the No-Observed-Effect Level (NOEL) for developmental toxicity was 400 mg/kg/day, the highest dose level evaluated. Reofos 35 was not teratogenic in the rat at the dose levels tested.
Executive summary:

The study was conducted for Chemtura Corporation to evaluate the potential adverse effects of the test article, Reofos 35, following repeated exposure to pregnant animals from Gestation Day (GD) 0 to 19, including systemic toxicity, female reproductive performance, and evaluation of the fetuses.

The study was conducted in accordance with Standard Operating Procedures (SOPs) and the protocol as approved by the Sponsor. All SOP deviations were acknowledged by the Study Director and documented in the raw data. In the opinion of the Study Director, none of the SOP deviations affected the quality or integrity of the study. This study was based on Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., 2011, the United States EPA, Office of Prevention, Pesticides, and Toxic Substances, Guideline 870.3700, Prenatal Developmental Toxicity, August 1998, and the OECD 414 Guideline for the Testing of Chemicals, January 2001.

 

Observations of the animals included clinical signs, body weights, food consumption, and anatomical pathology including a uterine examination on GD 20. All fetuses were given the appropriate external, visceral, and/or skeletal examination.

Homogeneity of the dosing formulations was confirmed at the low- and high-dose levels in Week 1 and concentrations of test formulations used for dosing study ranged from 93.2 to 102.1% of nominal. No test article was found in control samples.

All control and treated animals survived to scheduled termination. Salivation was observed in each of the Reofos 35-treated groups and considered a test article-related effect. Its occurrence was attributed to a pharmacologic response to the test article and was not considered adverse. At dose levels ≤200 mg/kg/day, no adverse effects of the test article were observed from maternal clinical findings, gestation body weights, body weight change, food consumption, or macroscopic examinations. At 400 mg/kg/day, maternal toxicity was restricted to early in the treatment period (GD 0 to 3) and included clinical findings (i.e., decreased activity, red material around the nose/mouth, hunched posture, thin appearance, hair on the face discolored brown, and hair on the forelimbs discolored brown), low gestation weight gain, and low food consumption. No adverse effect of treatment with Reofos 35 at the dose levels evaluated was observed on pregnancy indices, uterine implantation data, fetal sex ratios, fetal body weights, or fetal external, visceral, or skeletal evaluations. In the 400 mg/kg/day dose group, a low incidence of red or white foci and swollen mucosa of the nonglandular portion of the stomach was observed in the maternal macroscopic evaluations and considered test article-related.

Thus, in this rat oral prenatal developmental toxicity study with Reofos 35, the No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity was 200 mg/kg/day and the No-Observed–Effect Level (NOEL) for developmental toxicity was 400 mg/kg/day, the highest dose level evaluated. Reofos 35 was not teratogenic in the rat at the dose levels tested.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
See discussion below
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A NOAEL of 200 mg/kg/day is documented for this endpoint on the basis of the maternal toxicity observed.

 

Observations of the animals included clinical signs, body weights, food consumption, and anatomical pathology including a uterine examination on GD 20. All fetuses were given the appropriate external, visceral, and/or skeletal examination.

 

All control and treated animals survived to scheduled termination. Salivation was observed in each of the test substance-treated groups and considered a test article-related effect. Its occurrence was attributed to a pharmacologic response to the test article and was not considered adverse. At dose levels ≤200 mg/kg/day, no adverse effects of the test article were observed from maternal clinical findings, gestation body weights, body weight change, food consumption, or macroscopic examinations. At 400 mg/kg/day, maternal toxicity was restricted to early in the treatment period (GD 0 to 3) and included clinical findings (i.e., decreased activity, red material around the nose/mouth, hunched posture, thin appearance, hair on the face discolored brown, and hair on the forelimbs discolored brown), low gestation weight gain, and low food consumption. No adverse effect of treatment with the test substance at the dose levels evaluated was observed on pregnancy indices, uterine implantation data, fetal sex ratios, fetal body weights, or fetal external, visceral, or skeletal evaluations. In the 400 mg/kg/day dose group, a low incidence of red or white foci and swollen mucosa of the nonglandular portion of the stomach was observed in the maternal macroscopic evaluations and considered test article-related.

 

Thus, in this rat oral prenatal developmental toxicity study with the test substance, the No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity was 200 mg/kg/day and the No-Observed–Effect Level (NOEL) for developmental toxicity was 400 mg/kg/day, the highest dose level evaluated. The test substance was not teratogenic in the rat at the dose levels tested.

Justification for selection of Effect on developmental toxicity: via oral route:

See discussion below; based on maternal toxicity observed.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP and in compliance with agreed protocols. Some of the reports do not detail a specific method; however it documents dose levels and responses in detail, so is deemed appropriate for use in the support of a formal registration.  Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds in the absence of definitive data for this endpoint.

Justification for classification or non classification

Without further clarification, the reproduction toxicity triggered classification under the CLP Regulation (EC No 1272/2008) as follows:

 

Repr. Cat 2, H361: Suspected of damaging fertility or the unborn child < Fertility - effects on epididymides (oral)