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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-11-08 to 2017-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-phenylpropan-1-ol
EC Number:
204-587-6
EC Name:
3-phenylpropan-1-ol
Cas Number:
122-97-4
Molecular formula:
C9H12O
IUPAC Name:
3-phenylpropan-1-ol

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 10 weeks (Males) and 12 weeks (Females)
- Weight at study initiation: 316.5-401.5 g at the initiation of dosing (Males)
248.4-314.9 g at the initiation of dosing (Females)
- Housing: 2 or one animal during acclimation, pre-treatment and treatment, 1 male and 1 female during mating period, 1 mated female during gestation
- Diet: ad libitum, standard rat and mouse pellet diet (Lot No.: MAR 28 162, MAY 24 162 and JUN 29 163, Lab Diet® #5053 PMI Nutrition International, USA; irradiated by gamma-ray
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was weighed and suspended in corn oil with a magnetic stirrer for about 5 min to prepare the target concentration. The high dose formulation was prepared first and then the lower dose formulations were prepared by diluting the higher dose formulation with corn oil.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was considered non-toxic with this dose volume (2 mL/kg), and it has been used in previous studies because of the solubility of the test item with this vehicle.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation analyses were conducted according to a validated method by GC in the test site. Samples for dose formulation analysis were transferred at room temperature.
Duration of treatment / exposure:
Dosing of the males was begin 14 days prior to mating and continued through the day prior to sacrifice (at least 50 days). Dosing of the females was beginning 14 days prior to mating and continued through lactation day (LD) 13. Animals in recovery group was not mated and assigned to 2 weeks of recovery period after the completion of administration.
Frequency of treatment:
once daily
Details on study schedule:

- Age at mating of the mated animals in the study: Age at mating of the mated animals in the study: 12 - 14 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
vehicle control and high dose groups: 18 males and 18 females
100 and 100 mg/kg bw/day dose groups: 12 males and 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose-range finding study
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week
Detailed clinical observations were made in all animals individually for abnormalities. Signs noted were included (but are not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, clonic and tonic movements, stereotypical and bizarre behavior, and difficult and prolonged parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption was observed once a week

ORGAN WEIGHTS:
Organs weighed at terminal and recovery sacrifice: see table 3 in the field 'any other information on material and methods incl. tables'


THYROID HORMONE ANALYSIS:
Blood samples were taken from all adult animals at termination from the caudal vena cava. Thyroid hormone (T4 and TSH) concentrations in the serum samples of the males were determined.
Oestrous cyclicity (parental animals):
A vaginal smear was taken daily for each female from the beginning of the 14 days prior to mating with continued monitoring into the mating period until there was evidence of mating. Furthermore, regularity and length of the estrus cycle during the treatment period until mating was examined.
In addition, vaginal smear of sacrificed females were taken at termination to examine the stage of the estrus cycle and allow correlation with histopathology of female reproductive organs.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, Seminal vesicles (with coagulation gland) weight, prostate weight, adrenal gland weight
The reproductive organs collected from the main group were further processed to sildes, stained with H&E, and examined microscopically.

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
All pups were monitored for mortality and morbidity once every day. All pups were monitored for general clinical signs once every day. Clinical signs including general appearance and behavior changes were recorded with date/time of finding. Body weight and sex were recorded on post-natal day (PND) 0, 4 (before culling) and 13, and it was reported as litter. Anogenital distance was measured in all pups on PND 4. Both, pup body weight and anogenital distance were collected on the same day and the anogenital distance was normalized based on the cube root of body weight. In addition, the number of nipples in all male pups was counted on PND 12.

GROSS EXAMINATION OF DEAD PUPS:
External and visceral examinations were conducted in dead pups at parturition and dead or moribund pups from birth to day 4 of lactation, if possible.

Postmortem examinations (parental animals):
SACRIFICE
All surviving main group males were euthanized on the day after final dosing using isoflurane and necropsied. All surviving main group females on LD 14 were euthanized using isoflurane.
All surviving recovery group males and females were euthanized at least 14 days after the first scheduled sacrifice of males and females using isoflurane and necropsied.
Dead animal was conducted for macroscopic findings, tissue preservation and microscopic findings.
Non-mated female was euthanized after 24 days from final mating day using CO2, macroscopic findings, tissue preservation and microscopic findings were conducted.
Non-parturition females were euthanized after GD 27 using CO2, and macroscopic findings, tissue preservation, pregnancy confirmation and microscopic findings were conducted.
Dam of which all pups are dead was euthanized as soon as possible using CO2, and macroscopic findings, tissue preservation and microscopic findings were conducted.
All scheduled sacrificed surviving main and recovery group animals were fasted more than 16 hours (overnight) prior to scheduled sacrifice.


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 1 and 2 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
All pups on PND 13 were euthanized using CO2.
Statistics:
Mean values and standard deviations were calculated in the final report. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using Pristima System or Statistical Analysis Systems. Data was considered to be significant when p<0.05 or p<0.01.
Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data was analyzed using the Analysis of Variance (ANOVA) and the significance of inter-group differences were analyzed using Dunnett’s Test. Heterogeneous data was analyzed using Kruskal-Wallis Test and the significance of inter-group differences between the control and treated groups were assessed using Dunn’s Rank Sum Test.
For comparing control group and recovery group, the data was analyzed for homogeneity for variance using F-test. Homogeneous data was analyzed using T-test and the significant difference between control and recovery group was assessed using Dunnett’s Test. Heterogeneous data was analyzed using Kruskal-Wallis Test and significant difference between control and recovery group was assessed using Dunn’s Rank Sum Test.
One-way analysis of covariance (ANCOVA) was used to analyze pup body weight. The litter size was used as the covariate. Litter data was statistically evaluated using the statistical unit as a litter.
Data presented as frequencies was analyzed by χ2-test followed by the Fisher's exact test where necessary.
Reproductive indices:
please refer to table 3
Offspring viability indices:
please refer to table 3

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In males at 300 and 1000 mg/kg bw/day, salivation was observed in 10 and 18 animals, respectively. In females at 300 and 1000 mg/kg bw/day, salivation was observed in 4 and 18 animals, respectively. It was considered test item-related but not toxicologically significant since it was considered to be attributed to the palatability of the test item.
In males and females at 1000 mg/kg bw/day, subdued behavior was observed in 14 and 7 animals, respectively and prone position was observed in 6 and 13 animals, respectively. These clinical signs occurred after dosing and then disappeared. In addition, there was no effect of test item on the body weight, food consumption, functional behavior, motor activity, macroscopic and microscopic findings. Therefore, they were not considered adverse.
Other clinical signs were observed in this study but were not considered test item-related since these findings were observed with low frequency or occurred sporadically and did not show a dose-response relationship.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male (Animal No. 60) at 1000 mg/kg bw/day was found dead on treatment day 9. In the microscopic findings, slight chronic active inflammation, marked alveolar edema and mononuclear cell infiltration and moderate type II alveolar cell hypertrophy/hyperplasia in lung, moderate acute inflammation in trachea and slight lymphoid hyperplasia in spleen were observed. Changes in lung and trachea were considered the cause of death. But, these changes were not considered related to test item as there were no correlated microscopic changes in other animals in same treated group.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant changes in body weight gains were found for a few time periods during the study, but were scattered over time, sex and showed no dose dependency and thus were not considered test item-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant changes in food consumption were found for a few time periods during the study, but did not correlated with body weight changes and thus were not considered test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decreased total red blood cell count (RBC, 93% of control) was observed in males at 1000 mg/kg bw/day. This change was not considered adverse since there were no histopathological correlates. Decreased RBC was fully reversed after recovery period.
Other statistically significant changes were not considered test item-related, because these changes were minimal, lacked a dose-relationship, were not correlated with microscopic changes and/or were observed only in recovery group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decreased blood urea nitrogen (BUN, 86% of control) in males at 1000 mg/kg bw/day, significant increased glucose (GLU, up to 1.36-folds over control) in females at 300 and 1000 mg/kg and significant increases of total bilirubin (TBIL, 1.25-fold over control), calcium (Ca, 1.09-fold over control) and inorganic phosphorus (IP, 1.23-fold over control) in females at 1000 mg/kg bw/day were observed. After the recovery period, decreased BUN was still observed in males at 1000 mg/kg bw/day (85% of control, statistically not significant) and all other changes were fully reversed. These changes were not considered adverse since there were no histopathological correlates and/or values were within the normal historical control ranges.
Other statistically significant change was not considered test item-related, because there was no dose-dependent and not correlated with microscopic changes.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic findings observed during this study were considered to be incidental or spontaneous since they were infrequent, of low severity and similarly distributed among control and test item-treated groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

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

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
An increased number of dead or cannibalized pups and a statistically significant decrease in viability index was observed at 1000 mg/kg bw/day. This was mainly affected by one whole litter loss affecting 20 pups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in body weights in F1 pups was observed on PND 4 (94% of control) and 13 (92% of control) at 1000 mg/kg bw/day.
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:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No test item-related changes in thyroid hormone (T4) were observed in pups on PND 13 during the study.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

open allclose all
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)

Applicant's summary and conclusion

Conclusions:
Adverse effects regarding reproduction/development were observed at 1000 mg/kg bw/day. The NOAEL was determined to be 300 mg/kg bw/day.
Executive summary:

This study was conducted to evaluate the potential of the test item to cause general systemic and reproductive/development toxicity. The test item was administered by oral gavage to Sprague-Dawley rats (12 animals per sex per group) at dose levels of 0, 100, 300 and 1000 mg/kg bw/day with a dose volume of 2 mL/kg in corn oil as vehicle. Males and females were dosed for two weeks prior to mating and continued through the day before sacrifice in males (at least 50 days), and continued through the lactation day (LD) 13 in females. Additional animals in recovery group at 0 and 1000 mg/kg bw/day (6 animals per sex per group) were also administered but not mated, and then assigned to 2 weeks of recovery period after the completion of administration. General systemic observations including mortality, general clinical signs, body weights, body weight gain, food consumption, functional behavior examination, motor activity examination, macroscopic findings, hematology, coagulation, clinical chemistry, organ weights and microscopic findings were measured and conducted. In addition, reproductive/developmental observations including estrus cycle, precoital time, fertility data, reproductive and littering findings, F1 pups clinical signs, body weight, anogenital distance, nipple retention and external examination were conducted. Thyroid hormone (T4) level in blood was also analyzed for adult males and pups at sacrifice. No adverse effects were found regarding general toxicity. In reproductive/developmental observations, at 1000 mg/kg bw/day, an increased number of dead or cannibalized pups and a decrease in viability index were observed. This was mainly affected by one whole litter loss affecting 20 pups. Decreased body weight in F1 pups was also observed on PND 4 (94% of control) and 13 (92% of control). Therefore, the No-Observed-Adverse-Effect Level (NOAEL) for general toxicity effects was considered to be at least 1000 mg/kg bw/day and the NOAEL for reproduction/developmental toxicity was considered to be 300 mg/kg bw/day.

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