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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2019 to 31 Jan 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD 414 (GLP study)
Justification for type of information:
Following ECHA decision (CCH-D-2114428300-65-01/F) on Gardenol it was requested to conduct additional toxicological studies.
The Screening study for reproductive/developmental toxicity (Annex VIII, Sections 8.6.1 and 8.7.1.; test method: OECD TG 421) in rats, oral route, and Pre-natal de-velopmental toxicity study (Annex IX, Section 8.7.2.; test method: EU B.31./OECD 414) in a first species (rat or rabbit), oral route.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
yes
Remarks:
the concentration of dosing solution was not measured during the in-life stage of the study as defined in the study plan.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-phenylethyl acetate
EC Number:
202-288-5
EC Name:
1-phenylethyl acetate
Cas Number:
93-92-5
Molecular formula:
C10H12O2
IUPAC Name:
1-phenylethyl acetate
Test material form:
liquid
Specific details on test material used for the study:
Identification: GARDENOL

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Condition:Outbred, SPF-Quality.
Source: Charles River Deutschland, Sulzfeld, Germany or Charles River Laboratories France, L'Arbresle Cedex, France.
Number of Males: 40.
Number of Females: 48 (nulliparous and non-pregnant).
Number of Pups Expected: Approximately 480 pups (40 litters x 12 pups).
Target Age at the Initiation of the Pretest Period: Females: approximately 10-12 weeks.
Target Age at the Initiation of Dosing: Males: approximately 10-12 weeks. Females: approximately 12-14 weeks.
Target Weight at the Initiation of Dosing: Males: 250 to 350 g. Females: 200 to 250 g.
ENVIRONMENTAL CONDITIONS
Temperature: 18 to 24°C.
Humidity: 40 to 70%.
Light Cycle:12-hours light and 12-hours dark (may be interrupted for designated procedures).
Ventilation: At least 10 air changes per hour.
Diet: ad libitum
Water:ad libitum
Acclimation period: at least 5 days
Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Vehicle:Corn oil

Test item dosing formulations (w/w) will be homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations will be prepared daily as a solution and dosed within 6 hours after adding the vehicle to the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No analysis of the formulations was performed during the in-life phase of this study to determine the concentration and homogeneity (see deviation in Appendix 7). The test item preparation was performed with approved procedures (see also Test Facility Study Nos. 20186563 and 20186559) and was documented in detail. Formulations were visually inspected for homogeneity prior to use and all formulations were used within 24 hours after removing the formulations from the refrigerator. Trial formulation analysis (Test Facility No. 20186563) and formulation analysis performed for the OECD421 study (Test Facility Study No. 20186559) with the same test item and vehicle showed stable, accurate and homogenous formulations when prepared according to instructions described in the Study Plan of the present study. In the OECD421 study identical dose concentrations were tested (25-50-125 mg/mL) and in the method validation trial formulations were tested at 1.09 and 264 mg/g. It can therefore be concluded that the method, used for the preparation of the formulations in the present study, results in formulations that meet the criteria for accuracy and homogeneity. The impact on the study of not having performed the formulation analysis during the in-life phase of this study was regarded/estimated to be low.
Details on mating procedure:
time-mated female Wistar Han Rats
Duration of treatment / exposure:
Day 6 to Day 20 post-coitum
Frequency of treatment:
once daily oral gavage 7 days a week
Duration of test:
15 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
vehicle (corn oil)
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
22 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
Groups (n=22) of time-mated Wistar female rats were administered gardenol at 100, 200 and 500 mg/kg/day via oral gavage. Control animals recieved corn oil. The animals were dosed 7 days per week from Day 6-Day 20 post-coitum. The dose volume for each animal was based on the most recent body weight measurement and administered using a plastic feeding tube. Dose selection was based on a previously conducted OECD 421 study in rats. In this study, up to 500 mg/kg/day were well tolerated by the females in the pre-treatment and post-coitum phase.

Examinations

Maternal examinations:
Mortality/Moribundity checks- 2x daily (morning and evening) throughout the study
Clinical observations- minimum 1x daily starting on Day2 post-coitum and up to the day prior to necropsy. The time of onset, grade and duration of observed sign was recoreded
Body weight - animals weighted individually on Days 2, 6, 9, 12, 15, 18, and 21 post-coitum
Food Consumption- measured on Days 2-6, 6-9, 9-12, 12-15, 15-18, 18-21 post-coitum
Water Consumption- monitored daily by visual inspection
Sample collection- blood ws collected on day of necropsy. Animals not fasted overnight
Thyroid hormone- blood collected and processed for serum and analyzed for T3, T4 and TSH
Terminal Procedures- necrosy performed and tissues with gross lesions collected as well as organ weights. Histology and histopathology was conducted on controls and high dose groups
Necropsy- animals subjected to external, thoracic and abdominal examination with special attention paid to reproductive organs.
Organ weights- thyroid gland and uterus were weighed at necropsy
Ovaries and uterine content:
Ovary and uterine horn dissected and examined for : number of copea lutea, weight of the gravid uterus, number of implantation sites, number and distribution of live and dead fetuses, number and distribution of emybro-fetal deaths
Fetal examinations:
External- each vialbe fetus was sexed, examined for macroscopic visible abnormalities and weight determined. Anogenital distance measured for all viable fetus
Visceral- sex was confirmed by internal examination and examined for visceral abnormalities
Skeletal examinations-
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisions were conducted using two sided tests and reported at the 1 or 5% levels.
Parametic: Dataset with at least 3 groups were compared using Dunnett-test.
Non-parametric: Dataset with at least 3 groups were compared using Steel-test. Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution were compared using the Mann Whitney test. Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.
Fisher’s exact test was used to compare all groups at the 5% significance level. Pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.

Indices:
Preimplantation loss (%)
Postimplantation loss (%)
Viable fetuses affected/litter (%)
Historical control data:
Treatment and vehicle effects were compared to historical control data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Slight salivation was observed on one or more occasions after dosing in 8/22 females treated at 200 mg/kg/day and in 18/22 females treated at 500 mg/kg/day.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
abdominal alopecia was observed at 200 mg/kg/day dose in 1 animal.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg/day, mean body weight gain was statistically significantly lower (up to 38%) compared to concurrent controls on Days 9-18 post-coitum. Mean body weight was comparable to concurrent controls throughout the dosing period.
Body weight gain corrected for the uterine weight was statistically significantly lower (37%) in females treated at 500 mg/kg/day compared to controls and was below the lower limit of the historical control data. There was no effect on mean body weight, body weight gain and body weight corrected for gravid uterus at 100 and 200 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption before and after allowance for body weight was statistically significantly lower in animals treated at 500 mg/kg/day compared to controls on Days 6-12 post-coitum (up to 26% and 24% respectively) and with values below or just above the lower limit of available historical control data. This returned to normal by Day 15 post-coitum unwards. No changes in food consumption were noted up to 200 mg/kg/day.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum levels of total triiodothyronine (Total T3), total thyroxine (Total T4), thyroid stimulating hormone (TSH) were considered to be unaffected by treatment up to 500 mg/kg/day.
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):
Thyroid weights and thyroid:body weight ratios of treated animals were comparable to those of control animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were observed at necropsy that were considered treatment related. Incidental findings among controls and treated animals were: reddish foci on the thymus as well as red-brown discoloration, reddish discoloration of the mandibular lymph node, alopecia, and diaphragmatic hernia of right median liver lobe. These findings were not dose-dependent and are ocassionally seen among rats in these types of studies.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test item-related microscopic findings observed in the thyroid glands. All microscopic findings were withing range of background in thyroid glands of rats of this age and strain.
Histopathological findings: neoplastic:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Treatment-related effects on corrected body weight gain at 500 mg/kg/day that were lower than historical control data. No effects observed at lower doses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean female and combined (male and female) body weights were lower at 500 mg/kg/day compared to concurrent controls. No effects were noted at 100 or 200 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations of bent limb bones were observed in one fetus in the control, 100, and 500 mg/kg/day and one fetus at 200 mg/kg/day had sternoschisis. Due to single occurrence amoung the groups incliuding the control, both malformations were considered chance findings. The incidence of skeletal variations aws 81.4%, 76.9%, 69.9% and 76.6% in control, 100, 200 and 500 mg/kg/day, respectively. The variation of bent ribs occured at lower incidences at 200 and 500 mg/kg/day and the mean incidences were 24.6%, 14.5%, 6.4% and 6.2% in control, 100, 200 and 500 mg/kg/day, respectively. These incidences were all within historical control data range and since control incidence was near the upper limit of the data, the effects at 200 and 500 mg/kg/day were considered to occur by chance and not due to treatment.
Visceral malformations:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Fetal anogenital distance showed no toxicological treatment-related effects at any dose tested.
Details on embryotoxic / teratogenic effects:
No toxicologically relevant treatment related effects were noted at any any dose level.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects up to the highest dose tested

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test article caused statistically significant changes in maternal body weigth gain at the highest dose tested but no effect at lower doses. All other maternal parameters remained unaffected by treatment. No toxicologically relavant changes in fetal toxicity or development were observed. Based on the results in this prenatal developmental toxicity study, the maternal No Observed Adverse Effect Level (NOAEL) for GARDENOL was established as being 200 mg/kg/day and the developmental NOAEL was established as being at 500 mg/kg/day.
Executive summary:

The potential for GARDENOL to induce developmental toxicity after maternal exposure during the critical period of organogenesis when given orally by gavage to time-mated female Wistar Han rats from Day 6 to 20 post-coitum was evaluated in this study. The dose levels in this study were selected to be 0, 100, 200, 500 mg/kg/day based on the results of an OECD 421 study in the rat (Test Facility Study No. 20186559).

The following parameters and endpoints were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (total triiodothyronine (Total T3), total thyroxine (Total T4), thyroid stimulating hormone (TSH)), gross necropsy findings, organ weights ((gravid) uterus and thyroid gland), uterine contents, histopathologic examination (thyroid gland), corpora lutea, implantation sites and pre- and postimplantation loss.

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations.

A test item-related decrease in (relative) food consumption was observed at 500 mg/kg/day on Days 6-12 post-coitum, which resulted in a slightly lower body weight gain on Days 9-18 post-coitum. However, the food consumption recovered to normal values during the remainder of the treatment period and because the effect on terminal (gravid) body weight was minimal, this was considered to be non-adverse. In addition, slightly lower fetal body weights were observed at this dose level. This was also considered to be non-adverse, as the low fetal body weight was observed in the absence of retarded growth for ossification parameters and with all values within the available historical control data.

Although no difference was observed between gravid uterine weight in the control compared to the 500 mg/kg/day groups, the corrected body weight gain in the high dose group was evidently lower (37%) compared to controls, indicating a clear effect on net maternal weight gain. Considering the magnitude of change and as the corrected body weight gain was below the available historical control data, this effect was considered to be adverse.

Salivation was seen post-treatment in 8/22 females dosed at 200 mg/kg/day and in 18/22 females dosed at 500 mg/kg/day and was considered to be of no toxicologically relevance due the nature and minor severity of the effect and time of occurrence (i.e. after dosing). No test item-related changes were noted in any of the remaining maternal parameters investigated in this study and no toxicologically relevant changes were noted in any of the developmental parameters investigated in this study.

Based on this study, the maternal NOAEL was 200 mg/kg/day due to changes in body weight gain and the developmental NOAEL was 500 mg/kg/day, highest dose tested.