Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 421
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
A mixture of: triphenylthiophosphate and tertiary butylated phenyl derivatives
EC Number:
421-820-9
EC Name:
A mixture of: triphenylthiophosphate and tertiary butylated phenyl derivatives
Cas Number:
192268-65-8
Molecular formula:
Unspecified
IUPAC Name:
reaction mass of: triphenylthiophosphate and tertiary butylated phenyl derivatives
Details on test material:
- Physical state, appearance: Liquid, colorless, clear
- Storage conditions: Room temperature
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 303 to 335 g; Females: 196 to 222 g
- Fasting period before study: none
- Housing: Single cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70%
- Air changes (per hr): 10 – 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14-Apr-2011 To: 02-Jun-2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance was weighed into a brown glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (2, 4 hrs and 48 hrs). During the second last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice and stored there at -20 ± 5 °C until analysis.
The samples were analyzed by analytical procedure provided by the Sponsor and adapted at at the CRO. The test item was used as the analytical standard.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 11 days
- Proof of pregnancy: daily vaginal smear was sperm positive or a copulation plug was observed
- Further matings after two unsuccessful attempts: not needed
- After successful mating each pregnant female was caged in single cages
Duration of treatment / exposure:
Males: Minimum 4 weeks
Females: Approximately 7 weeks
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a 28-day oral toxicity study in which the NOAEL was established at 200 mg/kg body weight/day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Viability/mortality was checked twice daily.
Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

OTHER:
For males: Food consumption: Weekly during pre-pairing and after pairing periods.
For females: Pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.

SACRIFICE
- Male animals: All surviving animals (day 28)
- Maternal animals: All surviving animals (post partum day 4)

GROSS NECROPSY
- For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea were recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites

HISTOPATHOLOGY / ORGAN WEIGHTS
The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.
The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Organ weights: testes and epididymides
Histological examination of ovaries was carried out in female no. 55 that did not give birth. In addition, microscopic examination of the reproductive organs of the infertile male no. 15 was made.
Fetal examinations:
PARAMETERS EXAMINED
The following parameters were examined in 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 abnormalities

SACRIFICE
- The offspring was sacrificed on day 4 post partum.
Statistics:
Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Indices:
fertility, conception and gestation indeces, viability indices (day 0-4)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs of discomfort such as salivation and pushing the head through the bedding material were observed at 50 and/or 250 mg/kg in males and females over the study. At 10 mg/kg body weight/day, no test item-related clinical signs were observed during the study. The only observation was hair loss on the dorsal side of the neck in female no. 60 starting on day 20 of the gestation until the termination. In control group, one male was noted to have a wound on the dorsal side of the neck at termination.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No test item-related adverse effects were observed in mean body weight and mean body weight gain of males at all dose levels. At 50 and 250 mg/kg body weight/day, the statistically significantly higher mean body weight gain during the pre-pairing and pairing periods, were considered to be incidental and not indicative of any test item effect. In females, at 250 mg/kg body weight/day, mean body weight gain was statistically significantly lower on days 2, 6 and 14 of the pre-pairing period and between days 1 and 4 and on day 12 of the gestation period. These were effecs of the treatment with the test item, which did not affect the mean body weight, therefore not considered to be adverse. At 50 mg/kg body weight/day, mean body weight gain was statistically significantly lower on days 3 and 11 of the pre-pairing period and on days 1 and 2 of the gestation period. These were
transient fluctuations, which did not affect mean body weight and therefore considered not to be adverse.
At 10 mg/kg body weight/day, no test item-related effects were observed during the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No test item-related effects were observed in mean food consumption of males at all dose levels during the whole study. At 250 mg/kg body weight/day, during the pre-pairing period mean food consumption was statistically significantly lower during the first week of the pre-pairing period (-11.8% compared to the control) and slightly lower during the second week. This was a transient reduction and considered not to be adverse as, mean food consumption recovered and was similar to the control group during the gestation and lactation periods. At 10 and 50 mg/kg body weight/day, no test item-related effects were observed in mean food consumption.
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:
no effects observed
Description (incidence and severity):
Weights (absolute and relative to body weight) of testes and epididymides were not affected by the treatment with the test item in any groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No test item-related abnormal findings were observed in males during the necropsy. The only findings noted were sores and kidney pelvic dilation in one male in the control group. In females, dark red or reddish discoloration of the ovaries was observed in one female at 10 mg/kg body weight/day, six females at 50 mg/kg body weight/day and three females at 250 mg/kg body weight/day. There were no hisopathologic correlates.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All findings recorded were within the range of normal background lesions, which may be recorded in animals of this strain and age. And also, no test item-related histological finding was recorded in ovary of one female (no. 55) that did not give birth and in reproductive organs of one infertile male (no. 15). No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Basis for effect level:
other: no effects observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean pup weights on day 1 post partum and mean pup weight development during the lactation to day 4 post partum were unaffected by treatment with the test item. Mean pup weights on day 4 post partum were 9.0, 8.3, 9.2 and 9.0 g for combined data of male and female pups in order of ascending dose level.
Details on embryotoxic / teratogenic effects:
The number of live pups at first litter check was unaffected by treatment with the test item. The mean number of live pups per litter was: 13.0, 14.3, 11.5 and 11.2 in order of ascending dose level.
At 10 mg/kg body weight/day, one female pup was found dead on day 2 post partum and at 50 mg/kg body weight/day one female pup was missing on day 4 post partum. The resulting viability indices were: 100.0%, 99.2%, 99.1% and 100.0% in order of ascending dose levels. No abnormal findings were noted at macroscopic examination of the pups. Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item. The proportion of males on day 4 post partum was 49%, 52%, 52% and 58% in order of ascending dose level.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the NOEL (No Observed Effect Level) for reproduction/ developmental toxicity was considered to be 250 mg/kg body weight/day.
Executive summary:

In a GLP-compliant reproduction screening study following OECD guideline 421, four groups of 10 males and 10 females were treated by gavage with the test item once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum. The following dose levels were used: Group 1: 0 mg/kg body weight/day (control group); Group 2: 10 mg/kg body weight/day; Group 3: 50 mg/kg body weight/day; Group 4: 250 mg/kg body weight/day; A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water). All animals survived until the scheduled necropsy. Signs of discomfort such as salivation at dose level of 50 and 250 mg/kg body weight/day and pushing the head through the bedding material at dose level of 250 mg/kg body weight/day were observed during the course of the study. No test item-related effects were observed in mean food consumption of males during the entire study at all dose levels. At 250 mg/kg body weight/day, mean food consumption was statistically significantly lower in females during the first week of the pre-pairing period, thereafter it progressively recovered, and therefore it was not considered to be adverse. No test item-related effects were observed in mean body weight and mean body weight gain of males at all dose levels during the entire study. In females, at 250 mg/kg body weight/day, mean body weight gain was statistically significantly lower on three occasions during the pre-pairing period and at the beginning of the gestation. This was considered an effect of the test item but not to be adverse as mean body weight was not affected. At 50 mg/kg body weight/day, the occasional lower body weight gain observed during the study was not indicative of any test item-related effect. Mating performance, fertility, conception and gestation indeces were not affected by treatment with the test item. The mean duration of gestation was also unaffected by treatment with the test item. The mean number of corpora lutea, the mean number of implantations per dam, and the incidence of post-implantation losses were not affected by treatment with the test item. No test item-related effects were observed in mean values of absolute and relative weight of testes and epididymides. There was no histological evidence of toxicological properties in the organs and tissues examined. No differences on the completeness of stages or cell populations of the testes were recorded between controls and high dose animals. The number of live pups at first litter check and on day 4 post partum was unaffected by the treatment with the test item. No test item-related clinical signs were observed at first litter check and during the lactation. Mean pup weights and weight development were also not affected by the treatment with the test item. Taking into account the lower food consumption and the occasional lower mean body weight gain which were not considered to be adverse in females at 250 and 50 mg/kg body weight/day, the general NOAEL (No Observed Adverse Effect level) was established at 250 mg/kg body weight/day for both males and females. Under the conditions of this study, the NOEL (No Observed Effect Level) for reproduction/ developmental toxicity was considered to be 250 mg/kg body weight/day.