Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 April 1996 - 9 January 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted under GLP conditions and according to a guideline equivalent or similar to OECD guideline 416.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997
Reference Type:
publication
Title:
Two-generation oral (diet) reproductive toxicity study of resorcinol bis-diphenylphosphate (Fyrolflex RDP) in rats
Author:
Henrich, R., Ryan, B.M., Selby, R., Garthwaite, S., Morrisey, R., Freudenthal, R.I.
Year:
2000
Bibliographic source:
International Journal of Toxicology, 19, 243-255

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 3-[(diphenoxyphosphoryl)oxy]phenyl triphenyl 1,3-phenylene bis(phosphate) and tetraphenyl 1,3-phenylene bis(phosphate)
EC Number:
701-337-2
Cas Number:
not available
Molecular formula:
C30H24O8P2
IUPAC Name:
Reaction mass of 3-[(diphenoxyphosphoryl)oxy]phenyl triphenyl 1,3-phenylene bis(phosphate) and tetraphenyl 1,3-phenylene bis(phosphate)
Details on test material:
- Name of test material (as cited in study report): Fyrolflex RDP
- Physical state: Liquid
- Lot/batch No.: Confidential information
- Storage condition of test material: In original container at room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: (P) 6 wks
- Weight at study initiation: (P) Males: 113-138 g; Females: 101-128 g
- Housing: Individually under standard conditions
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum, municipal tap water
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Batches of dosing formulation were prepared weekly by mixing amounts of Fyrolflex RDP appropriate to each dose level with powdered rodent chow.

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Purina Certified Rodent Meal
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 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 (how): Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity under the conditions of this study was confirmed.
Duration of treatment / exposure:
(P) Males: 10 weeks prior to mating, during and up to 2-3 weeks of mating and until sacrifice
(P) Females: 10 weeks prior to mating, during and up to 2-3 weeks of mating, during gestation up to weaning of F1 (postnatal day 25) and until sacrifice
(F1) Males: 11 weeks starting at weaning, during and up to 2 weeks of mating and until sacrifice
(F1) Females: 10 weeks prior to mating, during and up to 2-3 weeks of mating, during gestation up to weaning of F1 (postnatal day 25) and until sacrifice
(F2) No treatment
Frequency of treatment:
Continuous (in diet)
Details on study schedule:
- Selection of parents from F1 generation when pups were 25 days (weaning) of age.
- Age at mating of the mated animals in the study: 14 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 1000, 10000, 20000 mg/kg (ppm) food
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 49, 520, 995 mg/kg bw/day
Basis:
analytical conc.
(P) males
Remarks:
Doses / Concentrations:
0, 59, 602, 1199 mg/kg bw/day
Basis:
analytical conc.
(P) females
Remarks:
Doses / Concentrations:
0, 55, 602, 1260 mg/kg bw/day
Basis:
analytical conc.
(F1) males
Remarks:
Doses / Concentrations:
0, 63, 683, 1411 mg/kg bw/day
Basis:
analytical conc.
(F1) females
No. of animals per sex per dose:
(P) 30 males and 30 females
(F1) 60 animals
Control animals:
yes, plain diet
Details on study design:
No data
Positive control:
Not relevant

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: morbidity and mortality, also parturition in dams during gestation

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly prior to mating, but not during mating. Thereafter, males weekly until sacrifice, females on day 0, 6, 12, 18 and 20 of gestation and on postnatal day 0, 4, 7, 14 and 21.

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, weekly prior to mating, but not during mating. After mating, male food consumption was measured weekly until sacrifice, female food consumption on day 0, 6, 12, 18 and 20 of gestation, on postnatal day 0, 4, 7, 14 and 21 and weekly thereafter.
Oestrous cyclicity (parental animals):
Daily vaginal smears were collected for the (P) and (F1) females for three weeks prior to mating to ensure and evaluate cyclicity. In addition, vaginal smears were collected and evaluated from these rats for two to three days prior to, and on the day of necropsy to determine the stage of the estrus cycle at termination
Sperm parameters (parental animals):
Parameters examined in P and F1 male parental generations: testis weight, epididymis weight, sperm count in epididymides, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 4 pups/sex/litter; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals between end of mating and weaning of pups
- Maternal animals: All surviving animals after weaning of pups

GROSS NECROPSY
- Gross necropsy included examination of the external surface and pleural, peritoneal and cranial activities

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared and examined for gross lesions:
Vagina, uterus, ovaries with oviducts, cervix, stomach, pituitary, testes, epididymides, prostate, brain, liver, kidneys, adrenals, spleen, thymus, seminal vesicles.

Tissues below were also weighed:
Uterus, ovaries with oviducts, testes, epididymides, prostate, brain, liver, kidneys, adrenals, spleen, thymus, seminal vesicles.

Tissues below were also processed for histopathology:
Vagina, uterus, ovaries with oviducts, cervix, testes, epididymides, prostate, seminal vesicles.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 25 days and 21 days of age, respectively.
- These animals were not subjected to postmortem examinations

GROSS NECROPSY
- Gross necropsy of F1 included examination of the external surface and pleural, peritoneal and cranial activities. F2 generation rats were euthanized and discarded without necropsy.

HISTOPATHOLOGY / ORGAN WEIGTHS
The F1 tissues indicated below were prepared weighed and examined for gross lesions, the tissues with an asterisk (*) were also processed for histopathology.
Vagina (*)
Uterus (*)
Ovaries with oviducts (*)
Cervix (*)
Stomach
Pituitary
Testes (*)
Epididymides (*)
Prostate (*)
Brain
Liver
Statistics:
ANOVA, followed by Dunnett's test were appropriate for body weights, body weight gains, food consumption and organ weight data.
Comparison of litter parameters was complicated by the fact that pup weight is dependent upon, among other factors, litter size. Litter size was accounted for by least squares comparisons for covariance.
Reproductive indices:
Not relevant
Offspring viability indices:
Not relevant

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One animal died without clinical signs that were considered treatment related

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Male food consumption and absolute body weight were significantly decreased during first week of test substance administration in high dose males. Only on postnatal day 21 food consumption was significantly decreased in females of the mid and high dose groups. These effects were most likely associated with flavor aversion to the test substance in the food and in dams also with concomitant decreased food consumption by the weanlings.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Significant increases observed in liver weight in male and female rats in a dose-related fashion, but these were attributed to metabolic adapation after histopathology. Increased adrenal weight was presumed to be related to stress associated with the initital flavor aversion and the resulting food avoidance in the mid and high dose groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
995 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: No treatment related effects
Dose descriptor:
NOAEL
Effect level:
1 199 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: No treatment related effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)
F1: Two animals died without clinical signs that were considered treatment related

BODY WEIGHT AND FOOD CONSUMPTION (OFFSPRING)
F1: Decreased body weight observed on postnatal day 7 and 14 until weaning, but this was considered related to the taste aversion phenomenon (decreased food consumption). Mean body weights decreased (significant for mid and high dose group) during 11-week pre-mating period, induced by decreased food consumption in the first 2 weeks.
F2: Decrease in body weight observed up on to postnatal day 21, which was considered to be related to litter size

Effect levels (F1)

open allclose all
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 260 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: No treatment related effects
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 411 mg/kg bw/day (actual dose received)
Sex:
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 (plain diet) Low (1000 ppm) Medium (10000 ppm) High (20000 ppm)
ANALYSES
Actual concentration in diet Within 3% of target levels
Stability Confirmed
Homogeneity Confirmed
Test substance intake 0 mg/kg bw/day (P) M/F: 49 and 59 mg/kg bw/day (P) M/F: 520 and 602 mg/kg bw/day (P) M/F: 995 and 1199 mg/kg bw/day
0 mg/kg bw/day (F1) M/F: 55 and 63 mg/kg bw/day (F1) M/F: 602 and 683 mg/kg bw/day (F1) M/F: 1260 and 1411 mg/kg bw/day
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL
PARENTAL DATA (P)
Number of animals (M/F) 30/30 30/30 30/30 30/30
Mortality (numbers) x x x 1 female: dead during lactation
Body weight x x x Males: absolute weight significantly decreased in first week of treatment
Food consumption x x Females: significantly decreased on postnatal day 21 Males: significantly decreased in first week of treatment, Females: significantly decreased on postnatal day 21
Clinical signs x x x x
Organ weights x M/F: Significant increase in absolute and relative liver weight M/F: Significant increase in absolute and relative liver weight M/F: Significant increase in absolute and relative liver weight
Macroscopy x x x x
Microscopy Hepatic periportal hypertrophy (adaptive) Not done Not done Hepatic periportal hypertrophy (adaptive)
Sperm count x x x x
Sperm motility x x x x
Sperm morphology x x x x
Length of oestrus cycle x x x x
Ovarian primordial follicle counts 82.4 +/- 33.8 Not done Not done 73.0 +/- 29.8
Number of females successful mated 29 27 28 29
Mating index (%) 97 90 93 97
Number selected for littering 27 25 28 29
No. of litters 23 25 28 26
Gestation index (%) 85 100 100 90
Litter size per dam (mean +/- SD) 12.3 +/- 1.80 11.7 +/- 2.72 11.7 +/- 2.51 11.0 +/- 2.6
PARENTAL DATA (F1)
Number of animals (M/F) 25/25 25/25 28/28 26/26
Mortality (numbers)
Body weight x M/F: Sporadic but significant differences M/F: Significant decrease throughout study. M: mean body weight gain significantly decreased M/F: Significant decrease throughout study. M: mean body weight gain significantly decreased
Food consumption M/F: Sporadic but significant differences x x
Clinical signs x x x x
Organ weights M/F: Significant increase in absolute and relative liver weight M/F: Significant increase in absolute and relative liver weight M/F: Significant increase in absolute and relative liver weight
Macroscopy x x x x
Microscopy Hepatic periportal hypertrophy (adaptive) Not done Not done Hepatic periportal hypertrophy (adaptive)
Sperm count x x x x
Sperm motility x x x x
Sperm morphology x x x x
Length of oestrus cycle x x x x
Ovarian primordial follicle counts 73.4 +/- 18.9 Not done Not done 82.1 +/- 24.8
Number of females successful mated 25 25 26 26
Mating index (%) 100 100 93 100
No. of litters 23 25 22 24
Gestation index (%) 92 100 85 92
Litter size per dam (mean +/- SD) 12.1 +/- 3.45 12.9 +/- 3.02 12.7 +/- 1.73 11.6 +/- 2.67
 
OFFSPRING TOXICITY (F1)
Litter size (mean +/- SD) 12.3 +/- 1.80 11.7 +/- 2.72 11.7 +/- 2.51 11.0 +/- 2.6
Foetal weight (mean +/- SD) 6.78 +/- 0.327 6.84 +/- 0.428 6.89 +/- 0.528 7.05 +/- 0.466
Viability index (%) 97 99 94 96
Post natal survival until weaning (%, based on mean litter survival) 64 68 66 69
Visible abnormalities x x x x
Postnatal growth, growth rate (body weight gain) x x M/F: significant decrease PD14-21 F: significant decrease PD7-21, M: significant decrease PD14-21
Vaginal opening (F) or preputial separation (M) x x Delayed development (related to body weight) Delayed development (related to body weight)
OFFSPRING TOXICITY (F2)
Litter size (mean +/- SD) 12.1 +/- 3.45 12.9 +/- 3.02 12.7 +/- 1.73 11.6 +/- 2.67
Foetal weight (mean +/- SD) 6.84 +/- 0.501 6.69 +/- 0.448 6.73 +/- 0.410 6.87 +/- 0.385
Viability index (%) 98 98 96 97
Post natal survival until weaning (%, based on mean litter survival) 64 60 61 65
Postnatal growth, growth rate (body weight gain) x M/F: significantly decreased on PD4-21 M/F: significantly decreased on PD4-21 M/F: significantly decreased on PD4-21
x = no effects (as compared to control group)

Applicant's summary and conclusion

Conclusions:
Under the conditions of this two-generation reprotoxicity study, Fyrolflex RDP induced no adverse effects in doses up to 20,000 ppm in parental, F1 and F2 animals. Therefore, a NOAEL of 20,000 ppm was established - representing measured concentrations of 995, 1199, 1260 and 1411 mg/kg bw/day for P: males, P: females, F1: males, F1: females, respectively. The substance does not need to be classified as toxic to reproduction based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.
Executive summary:

In this two-generation reproduction toxicity study, male and female rats (P and F1) were exposed to 0, 1000, 10000 or 20000 ppm Fyrolflex RDP in diet for approximately 11 weeks prior to mating, during and up to 2 -3 weeks of mating and until sacrifice (males) or during gestation up to the weaning of the pups (females). Mortality, clinical signs, body weights, food consumption and organ weights were recorded, reproductive function was assessed and gross necropsy and histopathology were performed.

The administered concentrations were analytically determined to be 0, 49, 520 and 995 mg/kg bw/day for males and 0, 59, 602 and 1199 mg/kg bw/day for females (P). For F1, this was 0, 55, 602 and 1260 mg/kg bw/day for males and 0, 63, 683 and 1411 mg/kg bw/day for females.

No treatment related adverse effects were observed in P, F1 or F2 (no necropsy performed). Effects that were seen were related to flavor aversion to the test substance in food, resulting in a decrease in food consumption and alterations in body and organ weights. Liver effects could be attributed to a metabolic adaptation to the test substance.

Under the conditions of this two-generation reprotoxicity study, Fyrolflex RDP induced no adverse effects in doses up to 20,000 ppm in parental, F1 and F2 animals. Therefore, a NOAEL of 20,000 ppm was established - representing measured concentrations of 995, 1199, 1260 and 1411 mg/kg bw/day for P: males, P: females, F1: males, F1: females, respectively. The substance does not need to be classified as toxic to reproduction based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.