1-phenylethyl acetate

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Data from the Reproductive/Developmental toxicity study in rats (OECD 421) showed lowered maternal food consumption and body weight gain resulting in a maternal NOAEL of 200 mg/kg/day; however, this had no effect on reproduction as the NOAEL for reproductive toxicity was 500 mg/kg/day, the highest dose tested. In the prenatal developmental toxicity test (OECD 414), there was also a reduction in maternal body weight gain, the NOAEL for this endpoint was 200 mg/kg/day but no effects on reproduction parameters including fertility indices. In both studies, although there were effects on maternal body weight, this did not impact fertility as evidenced by the lack of effect on the fertility indices.

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identification: Gardenol

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

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.

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

200 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

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

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.

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: 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.

-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

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.

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

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

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

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

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

Key result

Reproductive effects observed:

no

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline study (OECD 421)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

In the Prenatal Developmental toxicity test (OECD 414), adverse effects in the developing pups was decrease in body weight albeit very slight and the effects were not considered adverse as they were within the range of historical controls and had no effect on the developing organs or on ossification.  In this study, the developmental NOAEL was 500 mg/kg/day. In the Reproductive/Developmental toxicity test, the developmental NOAEL was 200 mg/kg/day based on decreased body weight in the pups at 500 mg/kg/day (0.81x control). This reduction was observed along with a decreased in maternal body weight and food consumption and no other toxicologically relevant effect was seen. Therefore the effects seen in the pups can be attributed to the effects on the dams and not a result of direct impact on development.     
    
    
    
    
    
    
    
    
    
    

The effects on in pups was decreased body weight, the NOAEL was 200 mg/kg/day, the effects increased up to PND13 (0.81x of controls); however, the effects were within historical control range and were seen along with decreased maternal body weight.


















  
    
  
  
  
  
  
  
  
  
  
  

The effects on development seen in pups was decreased body weight, the NOAEL was 200 mg/kg/day, the effects increased up to PND13 (0.81x of controls); however, the effects were within historical control range and were seen along with decreased maternal body weight.






















  

Link to relevant study records

Reference

Reference 1

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.

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

Specific details on test material used for the study:

Identification: GARDENOL

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

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

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.

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

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

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.

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

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.

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

Key result

Developmental effects observed:

no

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.

Reference 2

Endpoint:

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)

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.

Qualifier:

according to guideline

Guideline:

other: OECD 421 (Reproduction/Developmental screening test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identification: Gardenol

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han

Details on test animals or test system and environmental conditions:

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.

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

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

Details on mating procedure:

- M/F ratio per cage: 1:1 within the same treatment groups.
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: Ejected copulation plug and sperm in vaginal smear. Day 0 of gestation was when positive evidence of mating was detected (daily checks).
- Further matings after unsuccessful attempts: No.
- After successful mating each pregnant female was caged (how): Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding.
- Any other deviations from standard protocol: No

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

Duration of test:

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.

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

200 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

0 mg/kg bw/day

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.

Maternal 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.

Ovaries and uterine content:

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.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
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:

Offspring viability indices including; post-implantation survival index, live birth index, viability index, lactation index and sex ratio.

Historical control data:

available

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

Gross pathological findings:

no effects observed

Description (incidence and severity):

All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not specified

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; lumina l 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

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 84, 89, 95 and 87% for the control, 100, 200 and 500 mg/kg/day groups, respectively. For 1 female in the 200 mg/kg/day treatment group, 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. No toxicological
relevance was attached to this finding.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be 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. One female in the control had total litter loss on PND1 (7 pups total) and one pup from another litter in the control was found dead on PND1. In the 200 mg/kg/day group 2 pups from a litter were found dead on PND1. In the 500 mg/kg/day group a total of 7 pups across 3 different litters were found dead on PND1. No toxicological relevance was attributed to these dead pups since the mortality incidence was in the same range as control animals.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Fertility index was considered not to be affected by treatment. The fertility indices were 100, 90, 90 and 100% for the control, 100, 200 and 500 mg/kg/day groups, respectively.One female at 100 mg/kg/day and one female at 200 mg/kg/day were not pregnant. In the bsence of a dose-related incidence of non-pregnancy, this was considered not to be related to treatment.

Other effects:

not specified

Details on maternal toxic effects:

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).

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

food consumption and compound intake

Fetal body 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. However, the effects were within range of the historical controls.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was considered not to be 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. One female in the control had total litter loss on PND1 (7 pups total) and one pup from another litter in the control was found dead on PND1. In the 200 mg/kg/day group 2 pups from a litter were found dead on PND1. In the 500 mg/kg/day group a total of 7 pups across 3 different litters were found dead on PND1. No toxicological relevance was attributed to these dead pups since the mortality incidence was in the same range as control animals.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment. Viability indices were 98, 100, 100 and 95% for the control, 100, 200 and 500 mg/kg/day groups, respectively. 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.

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

At 500 mg/kg/day scales were noted for most pups between PND 1 and 8, and a significantly ower body weight (up to 0.81x of controls) was noted. As severity increased between PND 1and 13 and based on the incidence and magnitude of the change observed this was considered adverse.No toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

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 pr esence 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 m g/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

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The database includes two recent OECD guideline studies (OECD 421 and 414)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the criteria outlined in Regulation 1272/2008 and Directive 67/548/EEC, the substance does not meet the criteria for classification as toxic to reproduction (both for developmental effects and fertility).