Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Version / remarks:
1983
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
2001
Deviations:
yes
Remarks:
no selection of F1 weanlings for further treatment
Principles of method if other than guideline:
This one-generation reproduction toxicity study was not only performed according to OECD 415 (1983), but also parameters of OECD 416 of 2001 were included taking into account the updated guideline requirements.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetraphenylphosphonium phenolate
EC Number:
239-480-3
EC Name:
Tetraphenylphosphonium phenolate
Cas Number:
15464-47-8
Molecular formula:
C24H20P.C6H5O
IUPAC Name:
tetraphenylphosphanium benzenolate
Constituent 2
Chemical structure
Reference substance name:
Phenol
EC Number:
203-632-7
EC Name:
Phenol
Cas Number:
108-95-2
Molecular formula:
C6H6O
IUPAC Name:
phenol
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:(Wi)WU BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 5-6 wks
- Weight at study initiation: (P) Males: 87 - 144 g; Females: 88 - 124 g
- Fasting period before study: none
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +- 2
- Humidity (%): 55 +- 5
- Air changes (per hr): 10 at minimum
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: March 2002 To: August 2002

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The test item was blended (using a mixing granulator) with diet used (Provimi Kliba 3883.9.25). To minimize dust formation 1 % peanut oil (DAB 10) was added. The amounts of the test item were calculated on the basis of an content of 91 % instead of 92 %, which is based on an effor. The diet mixtures were prepared once a week.
Details on mating procedure:
F0 animals were pretreated with the test item for about 10 weeks up to the cohabitation period. Within the weeks 8-10 of this premating period investigations on estrus cycle were performed. The females were cohoused with males at a maximum of 12 times during the 3-week mating period. Insemiated females were not further co-housed. Insemination was established by investigating vaginal smears prepared in the morning.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test item in the diet was analytically proven prior to the study for 50 ppm, 100, and 10000 ppm. Accuracy check was done for all doses.
Duration of treatment / exposure:
10 weeks before mating, and during mating (up to 3 weeks) for males, plus during pregnancy and up to weaning of pups to an age of 4 weeks for females - all together about 20 weeks for females and 13 weeks for males.
Frequency of treatment:
continuously via diet
Details on study schedule:
After a gestation period of about 22 days litters were born and the dams were allowed to rear them. If necessary, gour days after birth the F1 litters were reduced (culled) to eight pups according to random lists. If possible, four male and four female pups remained per litter. At an age of 4 weeks the pups were necropsied. F0 females were also killed at that time. F0 males were killed after the mating period partly in the course of spermatological investigations.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg diet
Remarks:
according to an actual dose of 7 mg/kg bw/day for males and 11 mg/kg bw/day for females
Dose / conc.:
900 mg/kg diet
Remarks:
according to an actual dose of 66 mg/kg bw/day for males and 89 mg/kg bw/day for females
Dose / conc.:
8 100 mg/kg diet
Remarks:
according to an actual dose of 880 mg/kg bw/day for males and 1069 mg/kg bw/day for females
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Details on study design:
dose selection was based on results of a subacute toxicity study in Wistar rats with 100 -1000 ppm in the diet.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes, weekly; the daily food intake per animal is calculated
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Oestrous cyclicity (parental animals):
Within the weeks 8-10 of the 10 week premating period investigations on estrus cycle were performed by taking vaginal smears.
Sperm parameters (parental animals):
Parameters examined in [P] male parental generations:
weight: testes, seminal vesicles with coagulation glands, prostate, epididymides
spermatologcial investigations: in all living F0 males of the control and high dose group:
spermatozoa motility and viability, spermatozoa morphology, quantitative determination of spermatozoa in epididymis, quantitative determination of homogenization resistant spermatid heads in the testis,
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities]

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities;
Postmortem examinations (parental animals):
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

ORGAN WEIGHT: Yes
brain, pituitary gland, liver, kidneys, adrenals, spleen, thyroid, uterus, seminal vesicles with coagulation glands, prostate, epididymides, testes and ovaries
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
ORGAN WEIGHTS:
brain, spleen thymus, uterus weights were determined in the first male and female living F1 weanling of each litter
Statistics:
yes, several tests
Reproductive indices:
insemination index, fertility index, gestation index, rearing index,
Offspring viability indices:
live birth index, viability index, lactation index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Description (incidence and severity):
at 900 and 8100 ppm an increasing number of rats exhibited food spillage; at 8100 ppm feces excretion was increased and reduced reactivity and/or piloerection was recorded for some rats
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
severe body weight depression at 8100 ppm in both sexes
Description (incidence and severity):
at 8100 ppm females consumed slightly more diet per animal than controls probably due to food spillage

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
incidences for insemination, fertility, gestation, viability and rearing as well as the means for the duration of pregnancy and percentage of males born were not affected;
The mating performance was unchanged up to 900 ppm. At 8100 ppm rats need a slightly prolonged time up to their matings.
Number of implantation sites (per litter 13.20 in control versus 9.91 in high dose or total 264 versus 218 in high dose) and pups born (231 versus 181) were significantly reduced at 8100 ppm.

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
100 mg/kg diet
Based on:
test mat.
Remarks:
according to 7 mg/kg bw/day in males and 11 mg/kg bw/day in females
Sex:
female
Basis for effect level:
other: no effects at this dose
Dose descriptor:
NOAEL
Effect level:
900 mg/kg diet
Based on:
test mat.
Remarks:
according to 66 mg/kg bw/day in males and 89 mg/kg bw/day in females
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
gross pathology
Dose descriptor:
NOAEL
Effect level:
900 mg/kg diet
Based on:
test mat.
Remarks:
according to 89 mg/kg bw/day
Sex:
female
Basis for effect level:
other: slightly prolonged time up to mating and number of implantation sites and pups born significantly reduced at 8100 ppm
Dose descriptor:
NOAEL
Effect level:
8 100 mg/kg diet
Based on:
test mat.
Remarks:
according to 880 mg/kg bw/day in males and 1069 mg/kg bw/day in females
Sex:
male/female
Basis for effect level:
other: no effects on reproductive performance

Target system / organ toxicity (P0)

Lowest effective dose / conc.:
8 100 mg/kg diet
System:
gastrointestinal tract

Results: F1 generation

General toxicity (F1)

Description (incidence and severity):
from 900 ppm onwards an increasing number of pups without a milk spot (pups affected: 0/0/4/15) and more smaller (pups affected: 0/2/6/1) and or thin (pups affected: 0/5/3/24) were found
Mortality / viability:
no mortality observed
Description (incidence and severity):
Birth weight and pup weight development, number of implantation sites and pups born, litter size at birth, clinical appearance and lactation index were not influenced negatively by the treatment up to 900 ppm and were significantly reduced at 8100 ppm.
Description (incidence and severity):
organ weight measurements in pups revealed no remarkable changes up to 900 ppm; at 8100 ppm absolute and/or relative organ weight changes due to the body weight depressen were evident for each organ.
Description (incidence and severity):
no test substance-related gross pathological findings were observed in F1 offsprings up to 900 ppm; as a correlate to depressed body weight development an increase in number of thin pups were observed at necropsy in the 8100 ppm group
no malformation became obvious
Description (incidence and severity):
at no point effects on the skeletal development of the pups or weanlings were observed up to 8100 ppm
Description (incidence and severity):
Birth weight, pup weight development, number of implantation sites and pups born, litter size at birth, clinical appearance and lactation index were not influenced negatively by the treatment up to 900 ppm and were significantly reduced at 8100 ppm.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
900 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: pup birth weight, pup weight development, number of implantation sites and pups born, litter size at birth, clinical appearance and lactation index were significantly affected at 8100 ppm

Target system / organ toxicity (F1)

Critical effects observed:
not specified
Lowest effective dose / conc.:
8 100 mg/kg diet
System:
other: general toxicity

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Executive summary:

A one-generation study was performed with the test item to evaluate possible effects to the entire reproduction process in Wistar rats. The study followed OECD 416 with the exception that there was no selection of F1 weanlings for further treatment.

The test item was administered to groups of 25 male and 25 female rats each at concentrations of 0 (control), 100, 900, and 8100 ppm in their diet (actual doses: 7, 66, 880 mg/kg/day (males) or 11, 89, 1069 mg/kg/day (females)).

Parental FO animals were pretreated over about 10 weeks and allowed to mate over a period of up to three weeks. F1 pups were nursed up to an age of four weeks.

Clinical signs, body weights, food intake, mating performance, fertility, duration of pregnancy, estrus cycling and sperm parameters were examined in FO rats. Litter size, percentage of males bom and pup weight at birth as weil as viability, rearing, lactation, clinical signs and body weight gain were studied in F1 offspring. Necropsies were done in all rats and implantation sites were recorded in FO females. Selected organs were weighed in FO and F1 rats.

There were no changes in mortality of FO rats up to 8100 ppm. At 900 and 8100 ppm an increasing number of rats exhibited food spillage. At 8100 ppm more FO rats exhibited a reduced or increased feces excretion as weil as some females showed macroscopically a thickened wall of the duodenum and/or a dilated cecum indicating changes in intestinal digestion and/or peristaltic by the treatment. Furthermore, some 8100 ppm rats exhibited a reduced reactivity and/or piloerection. These effects are clearly attributed to the treatment.

There was a severe body weight depression at 8100 ppm in both sexes. Accordingly, there were some 8100 ppm females showing emaciation at their necropsy.

There were no other gross findings in FO rats in relation to the treatment. At 8100 ppm females a sligthtly higher food intake per animal was measured than in controls and the body weight related food intake was distinctly increased in both sexes.

At 8100 ppm there were increased relative testis weights as weil as reduced weights of ovaries and uteri (each absolute and relative and ps0.05) in FO rats. The liver weights of FO rats were not toxicologically changed up to 900 ppm. At 8100 ppm significantly reduced absolute liver weights occurred in both sexes. The sperm parameter data of 8100 ppm FO males were comparable with those of the control group.

No changes in estrus cycling due to the treatment could be detected up to 8100 ppm. The indices for insemination, fertility, gestation, viability and rearing as well as the means for the duration of pregnancy and percentage of males born were not affected at levels of up to 8100 ppm. At 8100 ppm rats needed a slightly prolonged time until their successful matings.

The pup birth weight, pup weight development, number of implantation sites and pups born, litter size at birth, clinical appearance and lactation index were not influenced negatively by the treatment up to 900 ppm and significantly affected at 8100 ppm. At this dose more pups lacking a milk spot, smaller and/or thin pups were observed during lactation and at necropsy and changes in absolute and/or relative organ weight due to the body weight depression were evident for each organ.

The skeletal development of the pups or weanlings was unaffected at dose levels of up to 8100 ppm at any time point.

Thus, the dietary concentration of 100 ppm is established as the no observed (adverse) effect level (NOEL = NOAEL) for the parent animals. The NOEL for the

reproduction parameters was 900 ppm. The fertility was unchanged up to 8100 ppm.