1-phenylethyl acetate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 July 2019 - 8 Nov 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD 421 (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 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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

Details on test material:

- Name of test material: 1-phenylethyl acetate- Name of test material (as cited in study report): USA2850 Methyl Phenyl Carbinyl Acetate

Specific details on test material used for the study:

Identification: Gardenol

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

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

Stability for at least 24h at room temperature protected from light and at least 8 days in the refrigerator is confirmed over the concentration range 1 to 250 mg/mL (solutions).
Supplier: Sigma-Aldrich
Specific gravity: 0.92

Details on mating procedure:

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:

(i) Homogeneity analysis, (ii) Stability analysis and (iii) Concentration analysis are conducted;
Accuracy
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 (25 mg/ml, 50 mg/ml and 125 mg/ml) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
No test item was detected in the Group 1 formulation.
Homogeneity
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation
≤ 10%)
Stability analyses performed previously in conjunction with the method development and
validation study (Test Facility Study No. 20186563) demonstrated that the test item is stable
in the vehicle when prepared and stored under the same conditions at concentrations
bracketing those used in the present study

Duration of treatment / exposure:

Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period.
Females that delivered were treated for 50-65 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.
Females which failed to deliver or had a total litter loss were treated for 39-54 days

Frequency of treatment:

once daily oral gavage 7 days a week

Details on study schedule:

The Study Director signed the study plan on 11 Mar 2019, and dosing of the Main study was initiated on 05 Jun 2019. The in-life phase of the Main study was completed on 09 Aug 2019.
The experimental start date was 21 Mar 2019, and the experimental completion date was 03 Oct 2019.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

0 mg/kg diet

Remarks:

vehicle (corn oil)

No. of animals per sex per dose:

10 males, 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Groups of 10 male and female Wistar Han rats were administered Gardenol at 100, 200, and 500 mg/kg/day while control animals received corn oil via gavage once per day 7 days per week. Males were treated for 29 days up to and including the day before necropsy (minimum of 14 days prior to mating and during the mating period). Females that deliivered were treated for 50-65 days, this included 14 days prior to mating, the duration of pregancy and at least 13 days after delivery and the day before necropsy. Females that failed to deliver or had total litter loss were treated for 39-54 days.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), macroscopic findings, organ weights and histopathologic examinations. In addition the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio.

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.

Sperm parameters (parental animals):

For the testes of all males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Litter observations:

Pups were observed daily for general health/mortality and the number of live and dead pups was determined on PND 1 and daily thereafter. The following parameters were evaluated: clinical observation, body weight, sex determination, anogenital distance and areola/nipple retention.

Postmortem examinations (parental animals):

All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females.
The following tissues were collected: cervix, epididymisa, coagulation gland, mammary gland, parathyroid gland, pituitary gland, prostate gland, seminal vessicle, thyroid, gross lesions/masses, ovaries, testes, uterus and vagina

Postmortem examinations (offspring):

Sex determination both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter and the
thyroid from two pups per litter (if possible one male and one female pup) was preserved in
10% buffered formalin.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

Reproductive indices:

Mating index, precoital time, fertility index, gestation index, duration of gestation, post-implantation survival, percentage of live females at first litter check, percentage of live males at first litter check.

Offspring viability indices:

Viability index, , live birth index, lactation index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Slight salivation occured after dosing in all males and females at 200 and 500 mg/kg/day throughout the treatment period but this effect was not considered of toxicological relevance due to the low severity and time of occurrence (ie, after dosing). This effect was considered to be a physiological response related to the taste of the test article rather than a sign of toxicity. Other incidental findings of piloerection, scabs, alopecia and pale appearance were within the range of background findings expected in rats of this age and strain and were not considered signs of toxicological relevance.

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A lower body weight (up to 0.94x of controls) and body weight gain was observed for females treated at 500 mg/kg/day during the lactation period. No changes relative to controls were noted for the males.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower food consumption (0.83x of controls) was noted for females treated at 500 mg/kg/day during the lactation period. During the first week of the pre-mating phase slightly lower food consumption (absolute and relative) were noted for males and females treated at 500 mg/kg/day. Also, high food consumption was noted for males at 200 and 500 mg/kg/day during the mating period. These changes were minimal and lacked consistency in time and direction of change therefore were not considered to be toxicologically relevant.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum levels of T4 in F0-males were considered unaffected by treatment with the test item up to 500 mg/kg/day.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In male rats there were signs of bilateral hypertrophy of the follicular cells of the thyroid gland; tubular vacuolation and degeneration of germ cell in the testes, inflammatory infiltrate, luminal cell debris and sperm granuloma of the epididymides; decreased content of the seminal vesicles; atrophy of the coagulation gland; ulceratation, pustule and inflammatory infiltrate was seen in the skin; plasmacytosis in the iliac lymph node and hyaline droplets in the kidney as well as renal pelvis dilation. These effects were seen in controls and treated groups and not considered treatment-related.

Females showed signs of thyroid gland follicular cell hypertropy; hyperplasia of the ovaries; luminal debris and hemorrhages of the uterus; mucification of the cervix and vagina; lobulaalveolar development of the mammary gland; inflammatory infiltrate of the skin; thymus congestion, mucusal atropy of the jejunum; dilation and inflammatory infiltrate of the clitoral gland. These effects as in the male rats were seen in controls and across treatment groups and were not considered treatment-related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

-Mating index - were 100% for all groups.
-Precoital time was not affected by treatment
-Number of preimplantation sites were not affected by treatement.
Fertility index not affected by treatment the fertility index was 100, 90, 90 and 100% for control, 100, 200 and 500 mg/kg/day, respectively.
-Gestation index and duration- The gesational indices were 90, 100, 100, 100 and 100 for the control, 100, 200 and 500 mg/kg/day groups, respectively.
-Post-implantation survival index was 84, 89, 95 and 87% for control, 100, 200 and 500 mg/kg/day groups, respectively.
-Litter size- Live litter sizes were 9.6, 10.2, 11.3 and 10.8 living fetuses/litter for the control, 100, 200 and 500 mg/kg/day groups, respectively.
-Live birth index was considered not affected by treatment. The live birth indices were 92, 100, 98 and 94% for the control, 100, 200 and 500 mg/kg/day groups, respectively.
-Viability indices were 98, 100, 100 and 95% for the control, 100, 200 and 500 mg/kg/day groups, respectively.
-Lactation index- The lactation indices were 94, 100, 100 and 100% for the control, 100, 200 and 500 mg/kg/day groups, respectively.

Details on results (P0)

-Mating index was not affected by treatment as all females showed evidence of mating.
-Precoital time- alll females showed signs of mating within 4 days with the exception of 2 females at 100 mg/kg/day that mated after 14 days and 1 female at 500 mg/kg/day that mated after 12 days.
-Number of preimplantation sites- One control which had a total loss on lactation day 2 had a single implantaiton site.
-Fertility index- One female in the 100 mg/kg/day and one at 200 mg/kg/day were not pregnant and in the absence of dose response this effect was considered not to be related to treatment.
-Gestation index and duration were considered not affected by treatment. Except for one control animal, all pregnant females had live offsprings.
-Post-implantation survival index- For 1 fmale treated at 200 mg/kg/day), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 16 days of lactation. This was not toxicologically relevant.
-Litter size- not affected by treatment.
-Live birth index- 1 Female of the control group had total litter loss on PND 1 (in total seven pups) and one pup of a litter of the control group was found dead on PND 1. In the 200 mg/kg/day group, two pups of 1 litter were found dead on PND 1. In the 500 mg/kg/day group, a total of seven pups of 3 litters were found dead on PND 1. The mortality incidence was the same for control as for treatment therefore not considered of toxicological relevance.
-Viability index - Two pups of the control group and five pups of the 500 mg/kg/day group were missing on PND 2 or 3. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.
-Lactation index- Four pups of a litter of the control group, were missing between PND 12-14. Pups missing were most likely cannibalized. As this occurred in a litter of the control group, this was not test item-related

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

Lower food consumption for the females at 500 mg/kg/day resulted in lower body weight gain compared to controls (0.94x of controls). This slight reduction resulted in no other toxicologically relevant change.

2

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Organ:

other: lower food consumption and lower body weight gain

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 500 mg/kg/day group, scales were seen for most pups within the period of PND 1-8. The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Two pups of the control group and five pups of the 500 mg/kg/day group were missing on PND 2 or 3. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range
considered normal for pups of this age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly lower body weights of pups of the 500 mg/kg/day group (up to 0.81x of controls) were noted.

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 specified

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 500 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment.

Histopathological findings:

not specified

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

At 500 mg/kg/day there was a signficant decrease in body weight compared to controls (0.81x of controls) in the pups from PND1 to PND8 and severity increased up to PND13. However, the effects on pup weight were with the range of historical controls for the lab and were seen in presence of a reduction in maternal body weight. Data from the acompanying prenatal development study (OECD 414) showed also a slight decrease in body weight gain among pups treated at 500 mg/kg/day but was not considered adverse because they were in the range of historical controls and there were no effects on organs or ossification

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw (total dose)

System:

other: body weight gain

Organ:

other:

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this reproduction/developmental toxicity screening test, the following no-observed-adverse-effect level (NOAEL) of GARDENOL were established:
Parental NOAEL: at least 200 mg/kg/day
Reproduction NOAEL: at least 500 mg/kg/day
Developmental NOAEL: 200 mg/kg/day
Although there were decreases in body weight gain in the pups at 500 mg/kg/day, there were no other changes in fetal mortality or development. These effects on pup weight may be attributed to the lower maternal food consumption and decrease body weight gain at 500 mg/kg/day.

Executive summary:

Wistar Han rats were treated with GARDENOL by daily oral gavage at dose levels of 100, 200 and 500 mg/kg/day. The rats of the control group received the vehicle, corn oil, alone. The objectives of this study were to determine the potential of GARDENOL when given orally by gavage to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development. In addition, parental, reproducton (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The following parameters and end points were evaluated in this study: mortality/moribundity, clinical signs, body weight and food consumption, estrous cycle determination,clinical pathology, measurement of thyroid hormone T4 (F0-males), macroscopic findings, organ weights and histopathologic examinations as well as reproduction/developmental parameters (mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio) and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, macroscopy, and measurement of thyroid hormone T4 (PND 14-16 pups).

Parental results: Parental toxicity was observed up to 500 mg/kg/day. A lower food consumption (0.83x of controls) was noted for females treated at 500 mg/kg/day during the lactation period, which resulted in a lower body weight (up to 0.94x of controls) and no other toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, clinical appearance, T4 thyroid hormone levels, macroscopic examination, organ weights, and microscopic examination).

Reproductive results: No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, and histopathological examination of reproductive organs).

Developmental results: At 500 mg/kg/day scales were noted for most pups between PND 1 and 8, and a significantly lower body weight (up to 0.81x of controls) was noted. No toxicologically significant changes were noted in any of the other developmental parameters investigated in this study.

Based on the results of this reproduction/developmental toxicity screening test, the NOAELs for parental, reproduction and development are 200 mg/kg/day, 500 mg/kg/day and 200 mg/kg/day respectively.