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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 April 2003 - 2 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to GLP and an equivalent of OECD guideline 421.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Phosflex 61B
- Physical state: Liquid
- Analytical purity: No data
- Storage condition of test material: At room temperature protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: (P) 8 wks
- Weight at study initiation: (P) Males: 226-262 g; Females: 156-193 g
- Housing: Individually, in accordance with the Guide for the Care and Use of Laboratory Animals (1996)
- Diet (e.g. ad libitum): Ad libitum, certified rodent diet
- Water (e.g. ad libitum): Ad libitum, municipal water
- Acclimation period: approx. 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-26.1
- Humidity (%): 30-70
- Air changes (per hr): min. of 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared approximately every 4 weeks at graded concentrations of 25, 125 and 500 mg/ml and adminstered at a dosing volume of 2 ml/kg.

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

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Cage side observations: moribundity and mortality, general appearance, overt signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

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

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

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

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

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

Evaluation of histopathology and organ weights was performed for the ovaries, testes and epididymides from animals in the vehicle control and high-dose groups (10/sex/group).
Postmortem examinations (offspring):
Not performed
Statistics:
- Mean and SD
- One-way ANOVA (homogeneity in variance)
- Post-hoc comparisons using Dunnett's test (when main effect differences were found)
- Chi-square test (mating performance and absolute pup survival)
Reproductive indices:
Not calculated
Offspring viability indices:
Not calculated

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
not considered treatment related
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
not considered treatment related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality and no treatment related clinical signs observed.

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

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

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

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

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

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No treatment related effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
not considered treatment related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The post-implantation loss was higher in the mid and high dose groups (not statistically significant). However, a statistically significant increase in the absolute number of stillbirths in the mid and high dose groups was noted. For the mid dose group this was caused by 10 deaths in one litter. The number of litter deaths between PD0-4 was significantly decreased for all treatment groups. The overall number of litter deaths was only significantly decreased for the low dose group. Due to the lack of a dose related response and similar overall deaths across the groups, the effects were considered unrelated to treatment.

BODY WEIGHT (OFFSPRING)
No effects were noted on the mean body weights of litters during PD0-4.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No treatment related effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

RESULTS OF TEST DOSING GROUPS
Control Low (50 mg/kg bw/day) Medium (250 mg/kg bw/day) High (1000 mg/kg bw/day)
ANALYSES
Stability Greater than 7 weeks
Concentration Within 5% of target concentrations
Homogeneity Within 5% of target concentrations
 
PATERNAL TOXIC EFFECTS BY DOSE LEVEL 
Number of animals (M) 12 12 12 12
Mortality X X X X
Clinical signs X X X X
Body weight X X X X
Body weight gain X X X X
Food consumption X X X Significantly increased in week 5
Gross necropsy X X X X
Organ weights changes (relative and absolute) X X X X
Histopathology X X X X
MATERNAL TOXIC EFFECTS BY DOSE LEVEL 
Number of animals (F) 12 12 12 12
Mortality X X X X
Clinical signs X X X X
Body weight X Significantly increased on GD 7 and 14 Significantly increased on GD 0, 7 and 14 and at termination Significantly increased on GD 0, 7 and 14 and at termination
Body weight gain X Significantly increased during week 1 of precohabitation Significantly increased during week 1 of precohabitation (also resulting in increased total gain) Significantly increased during week 1 of precohabitation (also resulting in increased total gain)
Food consumption X Significantly increased in week 1 X Significantly increased on GD 14 and 20
Number pregnant per dose level 11 12 11 12
Number of corpora lutea 148 159 146 176
Pre-implantation loss 6 8 8 10
Number of implantations 142 151 138 166
Post-implantation loss 12 8 30 20
Duration (days) of pregnancy (mean +/- SD) 22.1 +/- 0.7 22.3 +/- 0.62 22.5 +/- 0.82 22.1 +/- 0.29
Number of pups born 130 145 121 (109 after correction for 1 litter, which was excluded due to water deprivation) 154
Number of stillborn fetuses 0 2 13* (increase, but caused by 10 deaths in one litter) 9* (increase)
Number alive on day 0 130 143 108 (96 after correction for 1 litter, which was excluded due to water deprivation) 145
Gross necropsy X X X X
Organ weights changes (relative and absolute) X X Significantly increased fasted weight Significantly increased fasted weight
Histopathology X X X X
FETAL DATA 
Number of litters 11 12 11 12
Mean litter size 11.8 12.1 11.0 12.8
Sex ratio (M/F) 0.9:1 1.4:1 1.1:1 0.8:1
Body weights per litter X X X X
Mean postnatal survival per litter (day 0-4) 10.7 11.6 8.7 11.8
Mean postnatal deaths per litter (day 0-4) 1.1 0.2 1.2 0.3
Absolute numbers of postnatal survival (day 0-4) 118 141 96 (one litter excluded, due to water deprivation) 145
Absolute numbers of postnatal deaths (day 0-4) 12 2* (decrease) 0* (decrease) 4* (decrease)
Total number of deaths 12 4* (decrease) 13 (caused by 10 deaths in 1 litter) 13
X = no effects (as compared to control group)
* = significant difference (as compared to control group)

Applicant's summary and conclusion

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

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

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

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

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