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Description of key information

In a combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test performed according to OECD TGs 422 and 408 the following NOAELs for Piperonal or Heliotropine were established:

Parental NOAEL: 300 mg/kg bw/day (based on an adverse increase in trabecular bone in both sexes at 1000 mg/kg bw/day).

Reproduction NOAEL: 300 mg/kg bw/day (based on the absence of healthy offspring at 1000 mg/kg bw/day).

Developmental NOAEL: at least 300 mg/kg bw/day. Note: at 1000 mg/kg bw/day no litters were available for evaluation of post-natal development.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Feb 2019 - 18 Oct 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
2000
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures described in this study plan essentially conform to the following guidelines:
• OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
• EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.
• EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
• OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 2018.
• EPA OPPTS 870.3100, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(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
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-9 wks
- Weight at study initiation: Males: 194-243 g; Females: 140-172 g
- Fasting period before study: No
- Housing: On arrival and following randomization, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage enrichment, bedding material, food and water.
Animals were separated during designated procedures/activities.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Animal enrichment: For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.
- Acclimation period: 13 days prior to start of the pretest period

The feed was analyzed by the supplier for nutritional components and environmental contaminants and periodic analysis of the water was performed. It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 48-80
- Air changes (per hr): ≥10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Mar 2019 To: 19 Jul 2019
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. Formulations were prepared in amber colored glassware and/or glassware wrapped in aluminum foil. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred and protected from light until and during dosing. No adjustment was made for specific gravity of the test item. Adjustment was made for specific gravity of the vehicle (1.125). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 0, 20, 60, 200 mg/mL
- Amount of vehicle: 5 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis during week 1, 6 and 13 of treatment for concentration analysis (all dose groups) and homogeneity analysis (in low and high dose groups; The homogeneity results obtained from the top, middle and bottom for the Group 2 and 4 preparations were averaged and utilized as the concentration results). Analyses were performed using a validated analytical procedure.
Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.

Stability analysis: Stability analyses performed previously in conjunction with the method development and validation study 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:
The test item and vehicle were administered to the appropriate animals for a minimum of 13 weeks. Animals were treated minimally 8 weeks prior to mating, up to and including the day before scheduled necropsy. For females, this included 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 95-112 days.
Frequency of treatment:
once daily oral gavage 7 days a week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finder with oral administration of Piperonal or Heliotropine in rats (see other information on Materials and Methods), and in an attempt to produce graded responses to the test item.
- Fasting period before blood sampling for clinical biochemistry: F0-males only.

The dose volume for each animal was based on the most recent body weight measurement. The dosing formulations were stirred continuously during dose administration. Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. For mated females that fail to deliver viable offspring, body weights were recorded weekly from Day 20 post-coitum onwards until the day of scheduled necropsy.

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examination after application of a mydriatic agent (Tropicol 5 mg/ml solution) during Pretreatment in all animals (including spare animals), at the end of the Dosing Period in Week 13 in all males, during lactation in all females with litters, and all Group 1-4 females without viable litters.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood of F0-animals (all males, and all females with or without live offspring; except for animals which were found dead and females with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m..
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, F0-males only (overnight with a maximum of 24 hours before blood sampling, but water was available)
- How many animals: all animals
- Parameters: According to guideline.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood of F0-animals (all males, and all females with or without live offspring; except for animals which were found dead and females with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m..
- Animals fasted: Yes, F0-males only (overnight with a maximum of 24 hours before blood sampling, but water was available)
- How many animals: all animals
- Parameters: According to guideline.

THYOIRD HORMONE
- Time schedule for collection of blood: Blood of F0-animals (all males, and all females with or without live offspring; except for animals which were found dead and females with total litter loss) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m..
- Animals fasted: Yes, F0-males only
- How many animals: all animals
- Parameters: Triiodothyronine (T3), Thyroxine (T4), Thyroid Stimulating Hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on all males during Week 13 of treatment and all females during the last week of lactation (i.e. PND 6-13).
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity.
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 mating, 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. This was done for all females, except for females with total litter loss.
Sperm parameters (parental animals):
Parameters examined in P male animals: testis weight, epididymis weight. For the testes of all males of Groups 1 and 4, and all males that failed to sire or died before mating, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.
Thyroxine (T4) levels were determined in PND 14-16 pups. Assessment of T3 and T4 for PND 4 pups and T3 and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.

GROSS EXAMINATION OF DEAD PUPS:
Except for one missing pup, no pups died during the course of the study.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals; following completion of the mating period (a minimum of 13 weeks of administration).
- Maternal animals: All surviving animals PND 14-16; Females which failed to deliver with evidence of mating: Post-coitum Day 25-38; Dams with total litter loss were euthanized within 24 hours after the last pup was found dead or missing.

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Tables 1 and 2 were weighed and prepared for microscopic examination, respectively.
For the testes of all males of Groups 1 and 4, and all males that failed to sire or died before mating, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Postmortem examinations (offspring):
SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. 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. The pups selected for (complete) blood sampling were the same pups as selected for thyroid preservation.

GROSS NECROPSY
Sex was determined 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.
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. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.
Reproductive indices:
Mating index (%): (Number of females mated/Number of females paired) x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): (Number of pregnant females)/(Number of females mated) x 100
Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) x 100
Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
Viability index (%): (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Lactation index (%): (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were noted during daily detailed clinical observations or during weekly arena observations up to 1000 mg/kg bw/day.
Slight salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose groups from the second week of treatment onwards and animals of the 100 mg/kg bw/day group from the eight week of treatment onwards was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Incidental findings that were noted included scabs, chromodacryorrhoea, hunched posture, abnormal posture, piloerection and alopecia. These findings 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. At the incidence observed, these were considered not to be signs of toxicological relevance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item up to 1000 mg/kg bw/day.
One male of the 100 mg/kg bw/day group was found dead on Day 21 of the premating period. No clinical signs were observed and gross findings at necropsy included isolated, dark red focus/foci in the glandular mucosa of the stomach, enlarged liver and watery-clear fluid in the thoracic cavity. Many organs were autolytic and no cause of death could be established. Due to the single occurrence in the low dose group (100 mg/kg bw/day), this mortality was regarded as unrelated to the test item.
One female of the 1000 mg/kg bw/day group was sacrificed on Day 1 of lactation, because of a total litter loss. No abnormalities were noted for this female at macroscopic evaluation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of males and females up to 300 mg/kg bw/day were considered to have been unaffected by treatment with the test item.
Male rats treated at 1000 mg/kg bw/day:
At 1000 mg/kg bw/day, absolute body weights in males were decreased from Day 22 onwards (up to 12% compared with concurrent controls at end of treatment), reaching statistical significance from Day 43 onwards. Moreover, statistically significant reduced body weight gain was recorded for males treated at 1000 mg/kg bw/day from Day 22 onwards (up to 29% lower than concurrent controls at end of treatment).
Female rats treated at 1000 mg/kg bw/day:
No effects on body weights and body weight gain were noted during the premating and mating period. From post-coitum Day 14 onwards, a lower mean body weight and body weight gain was observed, reaching statistical significance on Day 17 and/or Day 20, respectively. This could be explained by the reduced weight gain and/or weight loss in 4/4 pregnant females, which had abnormal pregnancies (three females with implantation sites only and one female with total litter loss on PND 1). At Day 1 of lactation, mean body weight of the one female that delivered one pup (with total litter loss on PND 1) was similar to that of controls. Due to the abnormal pregnancies, no body weight data of females treated at 1000 mg/kg bw/day during lactation is available.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Male rats treated up to 1000 mg/kg bw/day:
Food consumption before or after correction for body weight was similar to the control level up to 1000 mg/kg bw/day in males during the premating and mating period.
Female rats treated up to 300 mg/kg bw/day:
During lactation from Day 4-7 onwards, relative food consumption was decreased in females treated at 300 mg/kg bw/day (8% lower than concurrent controls, statistically significant on Days 4-7 and 7-13), which was considered not toxicologically relevant, due to the minimal magnitude of the change, and/or the absence of an effect on body weight. During the post coitum period, a higher absolute and relative food consumption was noted in females treated at 300 mg/kg bw/day on Days 11-14 (up to 13% higher than concurrent controls). This change was considered to be unrelated to treatment with the test item, due to the minimal magnitude of the change, and/or no clear trend was apparent regarding dose and duration of treatment.
Female rats treated at 1000 mg/kg bw/day:
Food consumption before or after correction for body weight was increased during premating Day 8-71, although no statistical significance was achieved. In addition, absolute food consumption (of 4 pregnant females only) was increased on post coitum Days 4-7 and relative food consumption was increased from post coitum Days 4-7 onwards, reaching statistical significance on Days 4-7, 14-17 and 17-20. These changes were considered not toxicologically relevant, due to the minimal magnitude and direction of change. No food consumption data was collected for females at 1000 mg/kg bw/day during lactation, since none of the females delivered a healthy litter.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment with the test item up to 1000 mg/kg bw/day. The nature and incidence of ophthalmology findings noted during the pretreatment period and end of treatment period was similar among the groups, and occurred within the range considered normal for rats of this age and strain.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No changes in haematology parameters of treated males were noted that were considered to be related to treatment with the test item up to 300 mg/kg bw/day.
Male rats treated at 1000 mg/kg bw/day:
Mean platelet count was decreased in males (0.80x). Although no statistical significance was achieved, the mean value and individual values of 4/9 males at 1000 mg/kg bw/day were below or at the lower limit of historical control range.
In addition, the following statistically significant changes in haematology parameters distinguished treated animals from control animals.
- Mean number of reticulocytes was increased (1.30x). The mean value and individual values of 4/9 males were above the upper limit of historical control range.
- Mean corpuscular volume (MCV) was increased (1.04x). The mean value and individual values of 4/9 males were above the upper limit of historical control range.
- Mean number of red blood cells was decreased (0.93x). No statistical significance was achieved. The mean value and individual values of 4/9 males were below the lower limit of historical control range.
The decreased mean red blood cell count, accompanied by an increased mean reticulocyte count and a minimally increased mean MCV might be indicative for a slightly larger volume of the red blood cell population. However, these changes were considered not toxicologically relevant based on the absence of a correlation between these parameters on the individual animal level and/or the absence of a clear dose-related response.
The mean number of eosinophils was decreased in males at 1000 mg/kg bw/day. As values remained within the range of historical control data and no dose-related response was observed, this change was considered unrelated to treatment with the test item.

Female rats treated up to 300 mg/kg bw/day:
No changes in haematology parameters were noted that were considered to be related to treatment with the test item up to 300 mg/kg bw/day. The statistically significant increase in mean corpuscular haemoglobin concentration (MCHC) of females at 100 mg/kg bw/day was considered to be unrelated to treatment in the absence of a dose-related trend.

Female rats treated at 1000 mg/kg bw/day:
For evaluation, these values were compared with historical control data from 90-day studies (with a comparable physiologic status, i.e. non-lactating animals).
- Decreased number of red blood cells (0.86x).
- Decreased level of haemoglobin (0.89x).
- Decreased level of hematocrit (0.89x).
- Decreased mean platelet count (0.70x).
Since the historical control data from 90-day studies was obtained from fasted females and the females in this study were not fasted prior to blood collection for clinical pathology, historical control data from OECD 422 studies was additionally evaluated (both from fasted and not-fasted females). The mean values of hematocrit and platelet counts were below the lower level of historical control data from OECD 422 studies (both fasted and not fasted) as well. Despite the physiological and glycemic status of these females, levels were decreased when compared with historical control data. The changes in red blood cells and haemoglobin were within the range of historical control data of OECD 422 studies for not fasted females. These changes were probably not related to treatment with the test item, but due to the different physiological and/or glycemic status of these females.
Any other statistically significant changes in hematology parameters in females treated at 1000 mg/kg bw/day were within the range of the historical control data from 90-day studies and considered to be unrelated to treatment with the test item. These changes were probably not related to treatment with the test item, but due to the different physiological status of these females.

Coagulation
Male rats treated up to 1000 mg/kg bw/day:
No changes in coagulation parameters were noted in treated males that were considered to be test item-related up to 1000 mg/kg bw/day.
Prothrombin time (PT) was statistically significantly increased in males at 1000 mg/kg bw/day (1.07x of control). The mean value remained within the range of historical control data (mean = 17.2; P5 – P95 =15.6 – 18.8 (n= 160)) and no dose-related response was observed. Therefore, this was considered unrelated to treatment with the test item.
Female rats treated up to 300 mg/kg bw/day:
No changes in coagulation parameters were noted that were considered to be related to treatment with the test item up to 300 mg/kg bw/day.
Female rats treated at 1000 mg/kg/day:
No changes in coagulation parameters were noted that were considered to be related to treatment with the test item at 1000 mg/kg bw/day.


Historical control data for Wistar Han rats; 90-day studies (2018):
Platelets (10E9/L) males: mean = 730; P5 – P95 = 563.0 – 883.0 (n= 158).
Reticulocytes (10E9/L) males: mean = 183.9; P5 – P95 = 146.9 – 215.2 (n=40).
Eosinophils (10E9/L) males: mean = 0.1; P5 – P95 = 0.0 – 0.2 (n=49).
MCV (fL) males: mean = 51.9; P5 – P95 = 49.6 – 54.8 (n=159).
Red blood cells (10E12/L) males: mean = 9.06; P5 – P95 = 8.16 – 9.86 (n=159).
Eosinophils (10E9/L) males: mean = 0.1; P5 – P95 = 0.0 – 0.2 (n=49).
Red blood cells (10E12/L) females: mean = 8.21; P5 – P95 =7.54 – 8.79 (n= 162).
Haemoglobin (mmol/L) females: mean = 9.5; P5 – P95 =8.8 – 10.1 (n= 162).
Hematocrit (L/L) females: mean = 0.445; P5 – P95 =0.408 – 0.481 (n=162).
Platelets (10E9/L) females: mean = 731; P5 – P95 =526 – 927 (n= 161).
PT (s) males: mean = 17.2; P5 – P95 =15.6 – 18.8 (n= 160).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Male rats treated up to 1000 mg/kg bw/day:
The following, statistically significant, test item-related changes were noted in treated males.
- Mean alanine aminotransferase (ALAT) activity was increased in at 1000 mg/kg bw/day (1.47x). The mean value and individual values of 8/10 males at 1000 mg/kg bw/day were above the upper limit of historical control range.
- Mean alkaline phosphatase (ALP) was increased in males treated at 1000 mg/kg bw/day (2.00x). The mean value and individual values of all males at 1000 mg/kg bw/day were above the upper limit of historical control range.
- Mean levels of cholesterol, HDL cholesterol and LDL cholesterol were mildly decreased in males treated at 1000 mg/kg bw/day.
- Bile acids were increased at 300 and 1000 mg/kg bw/day (1.57x and 2.86x, respectively; statistically significance achieved at 1000 mg/kg bw/day). The mean value and individual values of 6/10 males at 1000 mg/kg bw/day were above the upper limit of historical control range.
- Mean inorganic phosphate was increased in males treated at 1000 mg/kg bw/day (1.34x). The mean value and individual values of 5/10 males at 1000 mg/kg bw/day were above the upper limit of historical control range.
In addition, the following (statistically significant) changes were noted in treated males.
- Decreased mean total protein in males at 1000 mg/kg bw/day (0.92x).
- Mean urea was increased at 300 and 1000 mg/kg bw/day, although not statistically significant (1.12x and 1.18x, respectively).
- Mean level of creatinine was increased in males treated at 300 mg/kg bw/day (1.09x).
- Mean potassium was increased at 1000 mg/kg bw/day (1.08x).
- Mean chloride was increased at 300 and 1000 mg/kg bw/day (1.01x and 1.02x, respectively).
While several of these changes were statistically significant, the changes in total protein, urea, creatinine, potassium and chloride were considered not toxicologically relevant, as values remained within the range of historical control data, due to the direction or magnitude of change and/or in the absence of a clear dose-response.

Female rats treated at 100 and 300 mg/kg bw/day:
In addition, the following statistically significant changes were noted in treated females.
- Mean level of albumin was increased at 100 mg/kg bw/day (1.08x).
- Mean urea was decreased at 300 mg/kg bw/day (0.84x).
These changes were considered not toxicologically relevant based on the minimal magnitude of the change and/or absence of a dose-related response.

Female rats treated at 1000 mg/kg bw/day:
For evaluation, these values were compared with historical control data from 90-day studies (with a comparable physiologic state, i.e. not lactating animals). Relative changes in mean values relative to the mean values of historical control data are indicated between parentheses.
- Increased ALP activity (2.50x).
- Decreased level of creatinine (0.88x).
- Increased level of glucose (1.53x).
Since the historical control data from 90-day studies was obtained from fasted females and the females in this study were not fasted prior to blood collection for clinical pathology, historical control data from OECD 422 studies was additionally evaluated (both from fasted and not-fasted females). The mean value of glucose was above the upper level of historical control data from OECD 422 studies (both fasted and not fasted) as well. Despite the physiological and glycemic status of these females, glucose level was increased when compared with historical control data. The changes in ALP and creatinine were within the range of historical control data of OEC 422 studies for not fasted females. These changes were probably not related to treatment with the test item, but due to the different physiological and/or glycemic status of these females.
Any other statistically significant changes in clinical biochemistry parameters in females treated at 1000 mg/kg bw/day were within the range of the historical control data from 90-day studies and considered to be unrelated to treatment with the test item. These changes were probably not related to treatment with the test item, but due to the different physiological status of these females.

Thyroid hormone analyses:
Mean serum levels of Total thyroxine (T4) were dose-dependently decreased in treated males at 300 and 1000 mg/kg bw/day (0.72x and 0.37x lower than concurrent control, respectively. Serum levels of T4 in males at 1000 mg/kg bw/day were below the lower limit of historical control range. In females, T4 serum levels were comparable among all dose groups.
For serum levels of Total triiodothyronine (T3) no results were available for the females of the control group, 2 values were available for females treated at 100 and 300 mg/kg bw/day and 5 values were available for females treated at 1000 mg/kg bw/day as values were below the reportable range. In order to have a proper evaluation, historical control data was used to complete the evaluation. For females at 1000 mg/kg bw/day, results were available for 5 out of 9 animals. As these results were within the historical control range, it was concluded that T3 was not affected by treatment.

Historical control data for Wistar Han rats; 90-day studies (2018):
ALAT (U/L) males: mean = 40.1; P5 – P95 = 27.9 – 60.8 (n=164).
ALP (U/L) males: mean = 75.0; P5 – P95 = 60.8 – 95.2 (n=164).
Cholesterol (mmol/L) males: mean = 1.97; P5 – P95 = 1.46 – 2.50 (n=164).
Bile acids (umol/L) males: mean = 20.1; P5 – P95 = 7.60 – 42.50 (n=134).
Inorganic phosphate (mmol/L) males: mean = 1.76; P5 – P95 = 1.400 – 2.170 (n=164).
Total protein (g/L) males: mean = 64.0; P5 – P95 = 58.60 – 68.10 (n=164).
Urea (mmol/L) males: mean = 5.8; P5 – P95 = 3.10 – 8.80 (n=164).
Creatinine (umol/L) males: mean = 38.9; P5 – P95 = 34.80 – 43.50 (n=164).
Potassium (mmol/L) males: mean = 3.94; P5 – P95 = 3.560 – 4.280 (n=164).
Chloride (mmol/L) males: mean = 103; P5 – P95 = 99.0 – 106.0 (n=164).
ALP (U/L) females: mean = 61; P5 – P95 =30 – 105 (n= 164)
Creatinine (umol/L) females: mean = 42.3; P5 – P95 =38.2 – 47.3 (n= 164)
Glucose (mmol/L) females: mean = 7.71; P5 – P95 =6.16 – 9.94 (n= 164)

Historical control data for Wistar Han rats; F0-animals (period 2017-2019):
Total T4 (μg/dL) males: mean = 4.51; P5-P95 = 2.85-6.37 (n= 557).

Historical control data for Wistar Han rats; 90-day studies (period 2018-2019):
Total T3 (ng/dL) females: mean = 59.7; P5-P95 = 42.29-78.91 (n= 53).
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Male rats treated up to 1000 mg/kg bw/day:
Hearing ability, pupillary reflex and static righting reflex were normal in all examined males treated up to 1000 mg/kg bw/day. Forelimb and hind limb grip strength were unaffected by treatment with the test item up to 1000 mg/kg bw/day.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period. The mean total movements appeared statistically significantly lower in males at 1000 mg/kg bw/day as compared with concurrent control (0.68x). However, as values remained within the historical control range (mean = 3609; P5 – P95 = 1990 – 5497 (n=424)) and the habituation profile remained similar to control, this change was considered unrelated to treatment with the test item.

Female rats treated up to 300 mg/kg bw/day:
Hearing ability, pupillary reflex and static righting reflex were normal in all examined females treated up to 300 mg/kg bw/day. Forelimb and hind limb grip strength were unaffected by treatment with the test item up to 300 mg/kg bw/day. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period. Mean values of total movements as well as ambulations appeared to be statistically significantly higher in females at 100 mg/kg bw/day as compared with concurrent control (1.55x and 1.92x, respectively). However, as no dose related-response was observed and the habituation profile remained similar to control, this change was considered unrelated to treatment with the test item.

Female rats treated up to 1000 mg/kg bw/day:
Functional observation parameters were considered unaffected by treatment at 1000 mg/kg bw/day.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with Piperonal or Heliotropine were noted in the liver, thymus and bones of femur and sternum of the 300 and/or 1000 mg/kg bw/day group males and females (See table additional information on results).
Thymus: A minimal degree of lymphoid atrophy was noted in 1000 mg/kg bw/day males. (Cystic) epithelial hyperplasia was noted at increased incidence and severity (up to moderate) in 1000 mg/kg bw/day females.
Liver: A minimal degree of hepatocellular hypertrophy was noted in 1000 mg/kg bw/day males. The recorded hepatocellular hypertrophy in a single 1000 mg/kg bw/day female, showing total litter loss, was considered to be within background range findings.
Bone-sternum and femur: increased trabecular bone was noted in females starting at 300 mg/kg bw/day and in males at 1000 mg/kg bw/day. In general, females were more severely affected than males, and the femur more than the sternal bone.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 1000 mg/kg bw/day.
Most females had regular cycles of 4 to 5 days during the premating period. During the premating period, one control female and one 100 mg/kg bw/day female appeared to be acyclic and for one control female estrous cycle regularity could not be determined (all with normal litters). At 1000 mg/kg bw/day, one female was acyclic and an irregular cycle was noted for one female. These females had implantation sites only. Despite the normal length and regularity of the estrous cycle for the other females treated at 1000 mg/kg bw/day, none of these females delivered a healthy litter. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Stage aware evaluation of all testes examined did not show any indication for abnormal spermatogenesis.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
There were 2/10 couples of the 100 mg/kg bw/day group, 1/10 couple of the 300 mg/kg bw/day group and 10/10 couples of the 1000 mg/kg bw/day group without healthy offspring. Microscopic examination of the reproductive organs of these rats did not reveal an explanation for their lack of healthy offspring.
Mating index was considered not to be affected by treatment with the test item. All females showed evidence of mating, resulting in mating incidences of 100% for all groups, although the mating period was extended for two females treated at 1000 mg/kg bw/day.
Precoital time was considered not to be affected by treatment with the test item. Most females showed evidence of mating within 4 days. For one female at 100 mg/kg bw/day, evidence of mating was noted after 6 days. Two females at 1000 mg/kg bw/day were re-cohabited with different males after 14 days of mating. These females were proven to be mated one or three days after re-cohabitation. At the incidence observed and in the absence of a dose-relationship, this was considered to be unrelated to treatment.
The mean number of implantation sites was decreased at 1000 mg/kg bw/day (2.3 versus 12.4 in controls), due to the low numbers of implantation sites in all 4/10 pregnant females (Two females with one implantation site, and two females with two and five implantation sites, respectively).
Treatment with the test item at 100 or 300 mg/kg bw/day did not affect the number of implantation sites.
The fertility indices were 100, 90, 90 and 40% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
At 1000 mg/kg/day, 6/10 mated females were not pregnant. There were no histopathology changes in reproductive organs or abnormalities in estrous cyclicity that could explain this.
At the lower dose levels, two females were not pregnant at 100 and 300 mg/kg bw/day, respectively. These cases of non-pregnancy were regarded as unrelated to treatment with the test item as they occurred incidentally and without related histopathology changes in reproductive organs or decrease in number of implantation sites.

Except for one female at 100 mg/kg bw/day, which had implantation sites only, all pregnant females of the control, 100 and 300 mg/kg bw/day groups had live offspring. The gestation indices were 100, 89 and 100% for the control, 100 and 300 mg/kg bw/day groups, respectively.
The failed pregnancy of female at 100 mg/kg bw/day, without related histopathology changes in reproductive organs, was judged to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.
Duration of gestation was slightly, but statistically significantly lower in females treated at 100 mg/kg bw/day (0.98x). This was considered to be unrelated to treatment with the test item, due to the lack of a dose-related trend.
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
musculoskeletal system
Organ:
bone
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment with the test item.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment with the test item up to 300 mg/kg bw/day.
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 100% for the control and 100 mg/kg bw/day groups and 99% for the 300 mg/kg bw/day group, respectively.
One pup at 300 mg/kg bw/day was missing on PND 2, which was most likely cannibalized. No toxicological relevance was attributed to this missing pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment with the test item. No pups were found dead/missing between lactation Days 5 and 13, resulting in a lactation index of 100% for the control, 100 and 300 mg/kg bw/day groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment with the test item up to 300 mg/kg bw/day.
Mean body weight of female pups and the combined weight of male and female pups was statistically significantly lower at 100 mg/kg bw/day and of female pups at 300 mg/kg bw/day on PND 1. No clear dose response was observed. As the changes in body weight remained minimal, pup body weight was comparable with the control after PND 1, and values remained within the historical control range it was considered to be of no toxicological relevance.

Historical control data for pup body weight of Wistar Han rats (period 2017-2019):
PND 1 pups (males): mean = 6.0 ; P5 – P95 = 5.3 – 7.6 (n=2590)
PND 1 pups (females): mean = 6.0; P5 – P95 = 5.0 – 7.3 (n=2623)
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 with the test item.
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 with the test item up to 300 mg/kg bw/day.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 300 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item up to 300 mg/kg bw/day.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94, 85 and 81% for the control, 100 and 300 mg/kg bw/day groups, respectively.
The survival index at 300 mg/kg bw/day was slightly lower than concurrent controls and the mean was below the range of the historical control data. This decrease was considered unrelated to treatment with the test item, as the absolute numbers of living pups at first litter check and implantation sites were both comparable to concurrent control group and within normal limits.
For two females (control), 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 14-16) days of lactation. No toxicological relevance was attached to this finding in the current study.
Litter size was considered not affected by treatment with the test item up to 300 mg/kg bw/day. Live litter sizes were 11.7, 11.6 and 9.7 living pups/litter at first litter check for the control, 100 and 300 mg/kg bw/day groups, respectively. A slightly lower mean number of living pups was recorded at 300 mg/kg bw/day (9.7 vs. 11.7 in the control group), since three pups in one litter were found dead at first litter check. As the mean remained within the range considered normal for rats of this strain and age, this was considered not to be toxicologically relevant.

Historical control data for post-implantation survival index for Wistar Han rats (period 2015 – wk 4 2019): mean = 92; P5 – P95 = 83 – 98 (n= 118).
Historical control data for living pups at first litter check for Wistar Han rats (period 2015 – wk 4 2019): mean = 11.0; P5 – P95 = 5.0-15.0 (n=468).
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects up to and including 300 mg/kg bw/day
Remarks on result:
other: at 1000 mg/kg/day no litters were available for evaluation of post-natal development.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Dose formulation analysis

In the Group 1 formulations, no formulation related signal was detected.

The concentrations analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%; actual week 1: 102%, 98% and 98%; week 6: 98%, 95% and 95% ; week 13: 97%, 98% and 97% in groups 2, 3 and 4, respectively.

The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%; actual week 1: 3.0% and 1.6%; week 6: 1.3% and 3.4%; week 13: 0.82% and 1.2% in groups 2 and 4, respectively).

Mean Percent Liver and Thymus Weight Differences from Control Groups- Males

 

Males

Dose level (mg/kg bw/day):

100

300

1000

 

 

 

 

LIVER

 

 

 

              Absolute

3

6

21**

              Relative to body weight

1

9*

41**

 

 

 

 

THYMUS

 

 

 

              Absolute

8

-12

-32**

              Relative to body weight

7

-8

-20*

*: P<0.05, **: P<0.01

Mean Percent Thymus Weight Differences from Control Groups- Females

 

Females

Dose level (mg/kg/day ):

100

300

1000a

 

 

 

 

THYMUS

 

 

 

              Absolute

18

41**

45**

              Relative to body weight

24

47**

75**

* P<0.05, **: P<0.01
aNote, none of the 1000 mg/kg/day group females did have healthy offspring, and the physiological status is therefore not comparable to the other groups.

Summary Test Item-Related Microscopic Findings

 

Males

Females

Dose level (mg/kg/day):

0

100

300

1000

0

100

300

1000b

 

 

 

 

 

 

 

 

 

THYMUSa

10

10

10

10

10

10

10

10

   Lymphoid atrophy

 

 

 

 

 

 

 

 

         Minimal

-

-

-

4

2

2

-

1

   (Cystic) Epithelial hyperplasia

 

 

 

 

 

 

 

 

         Minimal

3

1

-

-

3

2

2

4

         Slight

-

-

-

-

-

1

1

4

         Moderate

-

-

-

-

-

-

-

1

 

 

 

 

 

 

 

 

 

LIVERa

10

10

10

10

10

10

10

10

    Hepatocellular hypertrophy

 

 

 

 

 

 

 

 

         Minimal

-

-

-

7

-

-

-

1

 

 

 

 

 

 

 

 

 

BONE - STERNUMa

10

10

10

10

10

10

10

10

    Increased bonec

 

 

 

 

 

 

 

 

         Minimal

-

-

-

7

-

-

2

1

         Slight

-

-

-

-

-

-

-

9

 

 

 

 

 

 

 

 

 

BONE - FEMURa

10

10

10

10

10

10

10

10

    Increased bonec,metaphysis

 

 

 

 

 

 

 

 

         Minimal

-

-

-

1

-

-

1

-

         Slight

-

-

-

6

-

-

1

2

         Moderate

-

-

-

2

-

-

-

8

a = Number of tissues examined from each group.
b
 = Note, none of the 1000 mg/kg/day group females did have healthy offspring, and the physiological status is therefore not comparable to the other groups.
Other termsHyperostosis (proliferative or non-proliferative types); Osteopetrosis; Osteosclerosis; Trabecular hypertrophy (reference INHAND):

Conclusions:
In a combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test peformed according to OECD TGs the parental NOAEL was established at 300 mg/kg bw/day based on an adverse increase in trabecular bone in both sexes at 1000 mg/kg bw/day and reproduction NOAEL was 300 mg/kg bw/day based on the absence of healthy offspring at 1000 mg/kg bw/day.
Executive summary:

A combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed according to OECD TGs 422 and 408 and in accordance with GLP principles. Wistar Han rats were given Piperonal or Heliotropine orally by gavage for a minimum of 13 weeks. The following parameters and end points were evaluated in this study: mortality, clinical signs, functional observations, ophthalmoscopic observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormones T4, T3 and TSH (F0-animals), gross necropsy 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 and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND14-16 pups)).

Chemical analyses of formulations were conducted three times during the study and confirmed that formulations of test item in Polyethylene glycol 400 were prepared accurately and homogenously.

There was no test item-related mortality up to 1000 mg/kg bw/day.

The increased trabecular bone observed in males at 1000 mg/kg bw/day and in females at 300 and 1000 mg/kg bw/day was most obvious in the femur, just below the growth plate in the metaphysis area and to a lesser extent in the femur head. Due to the increase in bone, the spaces for the bone marrow appeared to be slightly diminished. However, this was not (yet) expressed in hematology parameters or haematopoiesis/ myelopoiesis in other organs. For the sternum, the bone adjacent to the intervertebral discs was the most affected. Given the nature and severity, this finding was considered adverse at 1000 mg/kg bw/day and non-adverse at 300 mg/kg bw/day.

Mean serum levels of Thyroxine (T4) were dose-dependently decreased at all dose levels in males. No changes were noted in the TSH values, thyroid weight or during histopathological examination of the thyroid of males in any of the dose groups. As values of males at 100 and 300 mg/kg bw/day remained within the historical control range, the changes in serum T4 in these animals were considered non-adverse. For males at 1000 mg/kg bw/day, values were outside the historical control range, however under the conditions of this screening study no adverse effect was observed that could be linked to the reduction of total T4 and therefore this reduction was not taken into account when determining the parental NOAEL. In addition, the following effects were observed at 300 and/or 1000 mg/kg bw/day, which were considered non-adverse:

- A lower mean body weight gain was observed for males treated at 1000 mg/kg bw/day from Day 22 of premating onwards, corresponding with a lower mean body weight at the end of the treatment period. As the food consumption of these animals was similar to concurrent control, this effect was considered as non-adverse.

- A few clinical pathology parameters were affected in males (platelets, alanine aminotransferase (ALAT), alkaline phosphatase (ALP), cholesterol (including HDL and LDL), bile acids and inorganic phosphate) and females (haematocrit, platelets and glucose) at 1000 mg/kg bw/day. For these parameters, mean values in this study exceeded the range of relevant historical control data. However, the changes observed in males and females were not associated with adverse anatomic pathology findings, remained within physiological ranges and/or were not observed at critically levels which would impair function (e.g. tissue oxygenation or resistance to infection). These changes were therefore regarded as nonadverse.

- Hepatocellular hypertrophy was recorded at minimal severity in 1000 mg/kg bw/day group males and was associated with higher liver weights. Since this finding was not accompanied by any other morphologic alteration indicating cell damage, it was considered non-adverse.

- In males treated at 1000 mg/kg bw/day, lymphoid atrophy in the thymus was recorded at minimal severity and related with a lower thymus weight. At this low degree, these findings were considered non-adverse. In females treated at 1000 mg/kg bw/day, an increased incidence and severity of (cystic) epithelial hyperplasia was noted in the thymus.

Reproductive and developmental results

No toxicologically relevant changes were noted in any of the reproductive or developmental parameters examined in this study after treatment with the test item up to 300 mg/kg bw/day.

In the group treated at 1000 mg/kg bw/day, a decreased number of implantation sites and a low fertility index at was observed. Despite the absence of any changes in mating index, estrous cycle, precoital time, spermatogenic profiling and histopathology of reproductive organs, none of the couples treated at 1000 mg/kg bw/day was able to produce healthy offspring.

In addition, as the non-pregnancy of one female at 300 mg/kg bw/day was observed in combination with thymus findings and bone findings similar to the findings observed in females at 1000 mg/kg bw/day (but at a lower severity), it cannot be excluded that the findings in this female are related to the test item. However, as it concerned a single female the combination of the changes observed in this female at 300 mg/kg bw/day was considered nonadverse within the current study.

In conclusion, based on the results of this combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for Piperonal or Heliotropine were established:

Parental NOAEL: 300 mg/kg bw/day (based on an adverse increase in trabecular bone in both sexes at 1000 mg/kg bw/day).

Reproduction NOAEL: 300 mg/kg bw/day (based on the absence of healthy offspring at 1000 mg/kg bw/day).

Developmental NOAEL: at least 300 mg/kg bw/day. Note: at 1000 mg/kg bw/day no litters were available for evaluation of post-natal development.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Study performed according to OECD/EC guidelines and GLP principles (Klimisch 1).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed according to OECD TGs 422 and 408 and in accordance with GLP principles. Wistar Han rats were given Piperonal or Heliotropine orally by gavage for a minimum of 13 weeks. The following parameters and end points were evaluated in this study: mortality, clinical signs, functional observations, ophthalmoscopic observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormones T4, T3 and TSH (F0-animals), gross necropsy 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 and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND14-16 pups)).

Chemical analyses of formulations were conducted three times during the study and confirmed that formulations of test item in Polyethylene glycol 400 were prepared accurately and homogenously.

 

There was no test item-related mortality up to 1000 mg/kg bw/day.

The increased trabecular bone observed in males at 1000 mg/kg bw/day and in females at 300 and 1000 mg/kg bw/day was most obvious in the femur, just below the growth plate in the metaphysis area and to a lesser extent in the femur head. Due to the increase in bone, the spaces for the bone marrow appeared to be slightly diminished. However, this was not (yet) expressed in hematology parameters or haematopoiesis/ myelopoiesis in other organs. For the sternum, the bone adjacent to the intervertebral discs was the most affected. Given the nature and severity, this finding was considered adverse at 1000 mg/kg bw/day and non-adverse at 300 mg/kg bw/day.

Mean serum levels of Thyroxine (T4) were dose-dependently decreased at all dose levels in males. No changes were noted in the TSH values, thyroid weight or during histopathological examination of the thyroid of males in any of the dose groups. As values of males at 100 and 300 mg/kg bw/day remained within the historical control range, the changes in serum T4 in these animals were considered non-adverse. For males at 1000 mg/kg bw/day, values were outside the historical control range, however under the conditions of this screening study no adverse effect was observed that could be linked to the reduction of total T4 and therefore this reduction was not taken into account when determining the parental NOAEL. In addition, the following effects were observed at 300 and/or 1000 mg/kg bw/day, which were considered non-adverse:

- A lower mean body weight gain was observed for males treated at 1000 mg/kg bw/day from Day 22 of premating onwards, corresponding with a lower mean body weight at the end of the treatment period. As the food consumption of these animals was similar to concurrent control, this effect was considered as non-adverse.

- A few clinical pathology parameters were affected in males (platelets, alanine aminotransferase (ALAT), alkaline phosphatase (ALP), cholesterol (including HDL and LDL), bile acids and inorganic phosphate) and females (haematocrit, platelets and glucose) at 1000 mg/kg bw/day. For these parameters, mean values in this study exceeded the range of relevant historical control data. However, the changes observed in males and females were not associated with adverse anatomic pathology findings, remained within physiological ranges and/or were not observed at critically levels which would impair function (e.g. tissue

oxygenation or resistance to infection). These changes were therefore regarded as nonadverse.

- Hepatocellular hypertrophy was recorded at minimal severity in 1000 mg/kg bw/day group males and was associated with higher liver weights. Since this finding was not accompanied by any other morphologic alteration indicating cell damage, it was considered non-adverse.

- In males treated at 1000 mg/kg bw/day, lymphoid atrophy in the thymus was recorded at minimal severity and related with a lower thymus weight. At this low degree, these findings were considered non-adverse. In females treated at 1000 mg/kg/day, an increased incidence and severity of (cystic) epithelial hyperplasia was noted in the thymus.

 

Reproductive and developmental results

No toxicologically relevant changes were noted in any of the reproductive or developmental parameters examined in this study after treatment with the test item up to 300 mg/kg bw/day.

In the group treated at 1000 mg/kg bw/day, a decreased number of implantation sites and a low fertility index at was observed. Despite the absence of any changes in mating index, estrous cycle, precoital time, spermatogenic profiling and histopathology of reproductive organs, none of the couples treated at 1000 mg/kg bw/day was able to produce healthy offspring.

In addition, as the non-pregnancy of one female at 300 mg/kg bw/day was observed in combination with thymus findings and bone findings similar to the findings observed in females at 1000 mg/kg bw/day (but at a lower severity), it cannot be excluded that the findings in this female are related to the test item. However, as it concerned a single female the combination of the changes observed in this female at 300 mg/kg bw/day was considered nonadverse within the current study.

In conclusion, based on the results of this combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAEL) for Piperonal or Heliotropine were established:

Parental NOAEL: 300 mg/kg bw/day (based on an adverse increase in trabecular bone in both sexes at 1000 mg/kg bw/day).

Reproduction NOAEL: 300 mg/kg bw/day (based on the absence of healthy offspring at 1000 mg/kg bw/day).

Developmental NOAEL: at least 300 mg/kg bw/day. Note: at 1000 mg/kg bw/day no litters were available for evaluation of post-natal development.

 

One (1) supplemental reproduction and developmental screening study was identified (Vollmuth, 1990) which was not performed according to OECD TG 421/422 and GLP and is considered reliable with restrictions. Groups of 10 virgin female Sprague-Dawley rats were administered Piperonal by oral gavage at dose levels of 0, 250, 500, or 1000 mg/kg bw/day. Dose administrations occurred once daily for 7 days prior to cohabitation, during the cohabitation period (up to 7 days), during gestation, and up to 4 days post-partum. The authors determined the NOAEL for both maternal and developmental toxicities to be 250 mg/kg bw/day based on mortality, clinical observations and decreased body weight (gain) in maternal animals and decreased viability and body weight gain in the offspring.

Effects on developmental toxicity

Description of key information

In a prenatal developmental toxicity study in rat performed according to OECD TG 414 the following NOAELs for Piperonal or Heliotropine were established:

Maternal NOAEL: 300 mg/kg bw/day (based on lower body weight gain and food intake, and clinical signs at 1000 mg/kg bw/day).

Developmental NOAEL: 300 mg/kg bw/day (based on two cases of 100% implantation loss at 1000 mg/kg bw/day, along with lower fetal body weights and skeletal malformations)).

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Sept 2019 -
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 - 14 weeks
- Weight at study initiation: 177 - 245 g
- Fasting period before study: No
- Housing: Individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water: Municipal tap water was freely available to each animal via water bottles.
- Animal enrichment: For psychological/environmental enrichment and nesting material, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Acclimation period: at least 5 days before the commencement of dosing

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It was considered that there were no known contaminants in the feed and water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 40 - 73
- Air changes (per hr): ten or greater
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 Sept 2019 To: 26 Sept 2019
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution, filled out in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the test item. Adjustment was made for specific gravity of the vehicle (1.125). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment is complete. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.
- Concentration in vehicle: 0, 20, 60 or 200 mg/mL
- Amount of vehicle: 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis during week 2.
For determination of accuracy, samples were taken at middle position (50% height; group 1 and 3) or at top, middle and bottom position (90%, 50% and 10% height; group 2 and 4). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations (groups 2 and 4).

Acceptance criteria:
Accuracy: mean accuracies between 90% and 110%
Homogeneity: coeeficient of variation ≤ 10%

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20172129) 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:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Day 6 to Day 20 post-coitum, inclusive
Frequency of treatment:
once daily, 7 days a week
Duration of test:
Day 2 post-coitum - Day 21 post-coitum
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
group 4
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 10-Day dose range finding study in rat (Study No. 20172126) conducted for an OECD422 study (Study No. 20172124), and on the results of a combined 90-Day study with an OECD422 study (Study No. 20172124), and were attempted to produce graded responses to the test item.
In the dose range finding study, non-pregnant rats received dose levels of 500 or 1000 mg/kg bw/day. At both dose levels, transient effects on body weight, food intake and clinical appearance were observed. No relevant macroscopic findings were observed. Liver weights were considered slightly increased at both 500 and 1000 mg/kg bw/day.
In the OECD422 study, there were no viable litters at 1000 mg/kg/day (all females showed evidence of mating); 7 females were non-pregnant, 2 females had implantation sites only and one female had total litter loss. At 300 mg/kg bw/day, there was a trend towards a decreased number of living pups at first litter check, and a temporary decrease of body weights on PND 1, but no further treatment-related effects on developmental parameters were observed. No relevant clinical signs were noted among F0 animals up to 1000 mg/kg bw/day. The lower body weights recorded for F0 females from post-coitum Day 14 onwards was attributed to the high number of females that did not deliver viable offspring. Weight gain of these females during the 10-week premating period was considered unaffected by treatment, but food intake was higher between Days 8-71 of treatment. Other parameters determined in this OECD422 study (eg. clinical pathology at end of lactation) were considered not relevant for selecting dose levels for this Developmental Toxicity study.
The absence of viable litters at 1000 mg/kg bw/day was considered to be a reproductive effect rather than a developmental effect (note that developmental parameters were essentially unaffected in the OECD 422 study). Also, changes in body weight and food intake during the 10-week premating period were considered not dose-limiting given the relatively short treatment duration of a Developmental Toxicity study. Therefore, overall, a dose level of 1000 mg/kg bw/day was considered suitable as high dose for this Developmental Toxicity study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The thyroid gland and uterus were weighed at necropsy for all scheduled euthanasia animals. Thyroid glands of all animals of Group 1 and 4 were processed and examined by a board-certified toxicological pathologist.

THYROID HORMONES:
Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected between 7:00 and 9:00 a.m., from the jugular vein in the animal facility.
Animals were not fasted overnight.
Parameters assessed: T3, T4 and TSH
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Sex of each fetus based on the anogenital distance (AGD). The AGD was normalized to the cube root of the fetal body weight. Sex of all fetuses was confirmed by internal examination.
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. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank 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.
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and postimplantation loss.
Indices:
Maternal:
Body Weight Gains: Calculated against the body weight on Day 6 post-coitum.
Corrected Body Weight Gains: Body weight on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum.

Reproduction en developmental variables
Preimplantation loss (%): (number of corpora lutea - number of implantation sites)/number of corpora lutea x 100
Postimplantation loss (%): (number of implantation sites - number of live fetuses)/number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where: Viable fetuses affected/litter (%): (number of viable fetuses affected/litter)/(number of viable fetuses/litter) x 100
Historical control data:
See attached background data
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, piloerection was recorded for 15/22 animals, between post-coitum Days 7 and 20. Two of these animals also showed hunched posture between post-coitum Days 13 and 20.
At 300 mg/kg bw/day, two animals showed hunched posture on post-coitum Day 16 or between post-coitum Days 14 and 17, and two other animals showed piloerection between post-coitum Days 14 and 20.
Salivation was recorded after dosing among all animals at 1000 mg/kg bw/day and most animals at 300 mg/kg bw/day, increasing in incidence as treatment progressed. This sign was considered to be a physiological response rather than a sign of systemic toxicity, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
No clinical signs were noted among control animals. Any other clinical signs noted among test item-treated groups 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 with the test item.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, mean body weight gain was statistically significantly lower than control means throughout the treatment period. Mean body weight gain over the treatment period at this dose level was 0.83x of the control mean. Several animals at 1000 mg/kg bw/day showed weight loss ranging from 1 to 8% over post-coitum Days 6 to 9 and one animal showed a weight loss of 7% over post-coitum Days 6 to 12. Two of these animals had implantation sites only and were therefore not included in the summary tables. Body weights corrected for gravid uterus weight of three animals were below the historical control range.
Mean weight gain corrected for gravid uterus at 1000 mg/kg bw/day however remained well within the historical control range.
At 100 and 300 mg/kg bw/day, mean body weights, body weight gain and weight gain corrected for gravid uterus were considered not affected by treatment with the test item.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, mean food consumption before and after correction for body weight was statistically significantly lower between post-coitum Days 6 to 9 (0.78x of control mean for absolute food intake). Mean absolute food intake recovered to levels similar to control means as treatment progressed. However, mean food consumption after correction for body weight at this dose level was statistically significantly higher between post-coitum Days 12 and 18 (1.10x (Days 12-15) and 1.11x (Days 15-18) of control mean, respectively). Several animals at 1000 mg/kg bw/day also showed a notable reduction in food intake over post-coitum Days 15-21 (i.e. a food intake over Days 18 to 21 that was at least 10 grams/animal/day lower than food intake over Days 15-18).
At 100 and 300 mg/kg bw/day, mean food consumption before and after correction for body weight were considered not affected by treatment with the test item.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in thyroid gland weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy showed no lesions that were considered to be related to treatment with the test item.
All necropsy findings recorded for animals are occasionally seen among rats used in this type of study, and their incidence did not indicate a relationship to treatment with the test item.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations.
The recorded microscopic findings of the thyroid glands were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Note: A number of total T3 values across all dose groups (5/21, 10/20, 9/21 and 17/22 for the control group and 100, 300 and 1000 mg/kg bw/day, respectively), and a number of total T4 values at 1000 mg/kg bw/day (6/22) were below LLOQ and reported as LLOQ/2. Therefore, mean values reported for these parameters at the respective dose levels should be interpreted with caution.
Mean total Triiodothyronine (T3) and total Thyroxine (T4) levels showed an apparent dose-related trend towards a reduction across the dose groups. At 1000 mg/kg bw/day, means reached a level of statistical significance (0.65x and 0.73x of control mean for total T3 and T4, respectively). Mean total T3 was below the historical control data range at 100, 300 and 1000 mg/kg bw/day, and mean total T4 remained within the historical control data range at these dose levels. Mean Thyroid-Stimulating hormone (TSH) levels showed an apparent dose-related trend towards an increase across the dose groups, but without reaching statistical significance. Mean TSH remained within the historical control data range across the dose groups.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, a high mean post-implantation loss (i.e. increased mean number of resorptions, primarily early resorptions) and subsequently reduced mean number of viable fetuses was recorded (not statistically significant). This was attributed to two animals, both of which had no foetuses (implantation sites only) and a 100% implantation loss. Additionally, one female at 300 mg/kg bw/day had no fetuses (implantation sites only) and therefore a 100% implantation loss. For this latter female, it could not be confirmed from available data if this was due to early and/or late resorptions.

Preimplantation loss in the control and test groups were similar and in the range of normal biological variation.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The numbers of pregnant females, corpora lutea and implantation sites in the control and test groups were similar and in the range of normal biological variation.
One female each in the control group and at 300 mg/kg bw/day, and two females at 100 mg/kg bw/day were non-gravid. The number of non-gravid females remained within the historical control range, and their incidence did not show a relationship to treatment. As such these incidences were considered not to be related to treatment with the test item.
Key result
Dose descriptor:
NOAEL
Remarks:
maternal
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
pre and post implantation loss
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, mean body weights of male and female fetuses were statistically significantly lower than control means (0.74x and 0.73x of control mean, respectively; outside historical control range).
At 100 and 300 mg/kg bw/day, fetal body weights were not statistically significantly different to the control means. However, based on mean fetal body weights per litter, there was an apparent dose-related trend towards lower fetal weights over the dose groups; the mean fetal body weight of a total of 1/21, 4/20, 10/20 and 20/20 litters was below the historical control data range. Therefore, it was considered that fetal body weights were also affected by treatment with the test item at 100 and 300 mg/kg bw/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was considered not affected by treatment with the test item.
Mean sex ratios (males:females) were 5.1:5.5, 5.6:4.9, 5.4:5.5 and 4.1:5.3 for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/day, mean litter size was slightly below the historical control data range (9.4 fetuses/litter, vs 10.6 fetuses/litter in the control group; not statistically significant). This was attributed to four females with a low litter size (i.e. one female with 1 fetus, one female witf 4 fetuses and two females had no fetuses).
At 100 and 300 mg/kg bw/day, mean litter size was considered not affected by treatment with the test item. Mean litter sizes were 10.5 and 10.9 fetuses/litter, respectively.
External malformations:
no effects observed
Description (incidence and severity):
External malformations and variations were not observed in any group.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Malformations
At 1000 mg/kg bw/day, a test item-related increase of total skeletal malformations was recorded (15.0% per litter vs. 1.6%, 1.8% and 1.8% per litter in the control, 100 and 300 mg/kg bw/day groups, respectively). Skeletal malformations that were considered to be related to treatment with the test item consisted of (in descending order of frequency) vertebral anomaly with or without associated rib anomaly, rib anomaly, vertebral centra anomaly, sternoschisis and costal cartilage anomaly. The incidence of these findings exceeded the historical control data range. Moreover, all these malformations were localised in the same thoracic body region and were therefore considered to be related to treatment with the test item.
The incidence of bent limb bones at 1000 mg/kg bw/day was similar to the incidences recorded in other groups (1.5% per litter vs. 1.6% - 1.8% per litter in other groups), and therefore this malformation was considered not to be related to treatment with the test item.

Variations
The following variations were considered to be related to treatment with the test item:
- Increased incidence of variations indicating delayed skeletal ossification (reduced ossification of the skull, unossified metacarpals and/or metatarsals, reduced ossification of vertebral centra and arches, unossified sternebrae and reduced ossification of pubis and ribs) at 300 and/or 1000 mg/kg bw/day. Statistical significance was reached at 300 and 1000 mg/kg bw/day for reduced ossification of the skull and unossified metacarpals and/or metatarsals, and at 1000 mg/kg bw/day only for reduced ossification of sternebrae and vertebral centra and arches. The higher incidences of these ossification parameters occurred in conjunction with decreased fetal body weights at 1000 mg/kg bw/day (at 100 and 300 mg/kg bw/day, an apparent trend towards reduced fetal weights was only noted based on the incidence of means of fetal body weights per litter that were below the historical control data range). Therefore, it was considered that this increased incidence of variations indicating delayed skeletal ossification, was due to lower fetal weights, and not a direct effect of the test item.
- Increased incidence of 7th cervical full ribs at 1000 mg/kg bw/day (16.2%, vs. 1.9% in the control group; exceeding the historical control range), and increased incidence of 7th cervical ossification sites at 300 and 1000 mg/kg bw/day (11.4% and 32.8%, respectively, vs. 5.4% in the control group; exceeding the historical control range). Although these two variations are strongly anatomically related, the higher incidence of 7th cervical ossification sites at 300 mg/kg/day was not accompanied by an increased incidence of 7th cervical full ribs.
- Increased incidence of bent ribs at 300 and 1000 mg/kg bw/day (61.3% and 48.4% per litter, respectively, vs. 27.6% in the control group and 28.5% at 100 mg/kg bw/day; only statistically significant at 300 mg/kg bw/day). The mean incidence at 300 and 1000 mg/kg bw/day clearly exceeded the historical control range (mean incidences of the control and 100 mg/kg bw/day marginally exceeded the maximum (27.4% per litter) of this range). Although the incidence of this variation did not show a clear dose-related trend, bent ribs were considered to be related to treatment with the test item at 300 and 1000 mg/kg bw/day based on the high incidences at these dose levels and taking into account the lower fetal body weights that were predominantly recorded at 1000 mg/kg bw/day. Moreover, bent ribs were considered to represent a transient developmental effect.
- Increased incidence of 14th full ribs at 1000 mg/kg bw/day (21.0% vs. 8.3% in the control group; not statistically significant, but exceeding the historical control range), and a caudal shift of the pelvic girdle (28.1%, vs. 7.3% in the control group; statistically significant and exceeding the historical control range). The incidence of malaligned sternebrae at 1000 mg/kg bw/day also appeared increased (39.8% vs. 22.9% in the control group; not statistically significant (insufficient historical data available)).
The ossification parameter “unossified metacarpals and/or metatarsals” was also statistically significantly increased at 100 mg/kg bw/day (5.5% versus control value 1.0% per litter). However, since reduced ossification was only recorded for metacarpals and/or metatarsals and not for other bones, and since the mean remained within the historical control data, this was considered not to be related to treatment with the test item.
Remaining skeletal variations occurred in the absence of a dose-related incidence trend, infrequently and/or in control fetuses only. Therefore, they were considered not to be related to treatment with the test item.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related variations affecting the urinary tract occurred in one fetus at 300 mg/kg bw/day and four fetuses at 1000 mg/kg bw/day. These variations were not encountered in the control group, nor at 100 mg/kg bw/day. These affected fetuses had one or two of the following findings: dilated ureter, convoluted ureter and absent or small renal papilla. The incidence of dilated ureters and absent or small renal papillae at 1000 mg/kg bw/day exceeded the historical control data range and therefore, they were considered to be related to treatment with the test item. However, it can be expected that these variations are reversible and point towards a general developmental delay associated with lower fetal body weights, as was predominantly evident at 1000 mg/kg bw/day. Therefore, the urinary tract variations may not be a direct effect of the test item.
The only other variation (small supernumerary liver lobes) occurred in the absence of a dose relationship and was considered a chance finding.
The only visceral malformations that were observed occurred in one fetus at 1000 mg/kg bw/day that had a malpositioned testis and small kidney. Although none of these malformations were previously encountered in historical control fetuses, these findings were confined to a single fetus. Therefore, these were considered not to be related to treatment with the test item.
Other effects:
no effects observed
Description (incidence and severity):
Fetal ano-genital distance (both before and after normalization for body weight) was considered not affected by treatment with the test item.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
skeletal: rib
skeletal: vertebra
Description (incidence and severity):
Adverse fetal morphological findings (skeletal malformations) were recorded at 1000 mg/kg bw/day (15.0% per litter vs. 1.6%, 1.8% and 1.8% per litter in the control, 100 and 300 mg/kg bw/day groups, respectively) and consisted of vertebral anomaly with or without associated rib anomaly, rib anomaly, vertebral centra anomaly, sternoschisis and costal cartilage anomaly. The incidence of these findings exceeded the historical control data range and all these malformations were localised in the same thoracic body region.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified

Dose formulation analyses:

Accuracy

In the Group 1 formulation, no test item was detected.

The concentrations analyzed in the formulations of Groups 2, 3 and 4 (100%, 101% and 106%, respectively) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

Homogeneity

The formulations of Groups 2 (CoV 1.3%) and 4 (CoV 0.72%) were homogeneous (i.e. coefficient of variation≤ 10%).

 Summary table results

Dose level (mg/kg bw/day)

0

100

300

1000

Females on study

22

22

21a

22

-         

0

0

0

0

-         

0

0

0

0

-         

0

0

0

0

Females examined at scheduled necropsy

22

22

21

22

-         

21

20

20

22

-         

1

2

1

0

Mean number of corpora lutea

11.9

11.5

12.1

11.9

Mean number of implantation sites

11.1

10.9

11.4

11.3

-early resorptions

-late resorptions

(total number and % per litter)

10 (4.3%)

0 (0%)

8 (3.6%)

0 (0%)

9 (3.9%)

0 (0%)

39 (13.8%)

3 (1.1%)

Dams with stillbirths, resorptions only and/or dead fetuses

0

0

0a

2

Pre-implantation loss (number and %)

16 (6.3%)

13 (7.9%)

14 (5.5%)

12 (6.7%)

Post-implantation loss (number and % per litter)

10 (4.3%)

8 (3.6%)

9 (3.9%)

42 (14.9%)

Mean body weight on day 21 pc (g)

319

318

320

307

Mean body weight gain day 6-21 pc (%)

14.2

12.0

13.8

12.1

Gravid uterine weight (g)

73.6

72.6

74.0

61.6

Mean live offspring (number and percent)

10.6 (95.7%)

10.5 (96.4%)

10.9 (96.1%)

9.4 (85.1%)

Mean fetal body weight (males; g)

5.4

5.3

5.2

4.0**

Mean fetal body weight (females; g)

5.2

4.9

4.9

3.8**

Mean fetal body weight (sexes combined; g)

5.3

5.2

5.0

3.9**

Malformations (including runts) (number and % per litter)

-external

-soft tissue

-skeletal

 

 

 

 

0 (0%)

0 (0%)

2 (1.6%)

 

 

 

 

0 (0%)

0 (0%)

2 (1.8%)

 

 

 

 

0 (0%)

0 (0%)

2 (1.8%)

 

 

 

 

0 (0%)

1 (1.0%)

16 (15.0%)*

Variations (% per litter)

-external

-soft tissue

-skeletal

 

 

0

4.0

91.9

 

 

0

0.9

87.9

 

 

0

5.6

94.1

 

 

0

5.5

94.7

aAt 300 mg/kg bw/day the number of females on study was 22, and the number of gravid females with “resorptions only” was 1; One females was excluded from these tables, since the number of implantation sites and uterine content were not documented for this animal (necropsy confirmed presence of implantation sites and absence of foetuses).

*: significantly different from the control group at 0.05

**: significantly different from the control group at 0.01

Conclusions:
In a prenatal developmental toxicity study in rat peformed according to OECD TG 414 the maternal NOAEL was established at 300 mg/kg bw/day based on lower body weight gain and food intake, and clinical signs at 1000 mg/kg bw/day and developmental NOAEL at 300 mg/kg bw/day based on two cases of 100% implantation loss at 1000 mg/kg bw/day, along with lower fetal body weights and skeletal malformations.
Executive summary:

A prenatal developmental toxicity study was performed in rat according OECD TG 414 and in accordance with GLP principles. Time-mated female Wistar Han rats were given 0, 100, 300 or 1000 mg/kg bw/day of the substance orally by gavage from Day 6 to 20 post-coitum, inclusive. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (T3, T4, 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.

Formulation analyses confirmed that formulations of test item in Polyethylene glycol 400 were prepared accurately and homogenously.

Maternal Findings

All animals survived the scheduled treatment duration. However, adverse maternal effects were recorded at 1000 mg/kg bw/day. A lower mean body weight gain was recorded throughout the treatment period (0.83x of the control mean at the end of the treatment period), with an initial slight weight loss during the first week of treatment for several animals. A few animals also had body weights corrected for gravid uterus weight that were below the historical control range. This was accompanied bya lower mean food consumption between post-coitum Days 6 to 9 (0.78x of control mean).Although mean food intake recovered to control levels as treatment progressed, several animals at 1000 mg/kg bw/day also showed a notable reduction in food intake over post-coitum Days 15-21.

Additionally, piloerection (and incidentally hunched posture) was recorded for most animals between post-coitum Days 7 and 20. Overall, these findings were considered to represent an adverse effect of the test item at 1000 mg/kg bw/day.

At both 100 and 300 mg/kg bw/day, body weights (also corrected for gravid uterus) and food intake levels were considered not affected by treatment with the test item. At 300 mg/kg bw/day, hunched posture or piloerection were recorded for a few animals during the last week of treatment. In absence of other maternal signs of toxicity these few occurrences of clinical signs were considered not to represent an adverse effect of the test item.

Mean total Triiodothyronine (T3) and total Thyroxine (T4) levels showed an apparent dose-related trend towards a reduction across the dose groups, and mean Thyroid-Stimulating hormone (TSH) levels showed an apparent dose-related trend towards an increase. At 1000 mg/kg bw/day, the decrease in mean T3 and T4 was statistically significant (0.65x and 0.73x of control mean for total T3 and T4, respectively). The mean total T3 was below the historical control data range at 100, 300 and 1000 mg/kg bw/day; possible adversity of this change could not be established within this study. Both mean total T4 and TSH remained within the historical control data range at these dose levels, and as such these changes were considered not to represent an adverse effect.There were no test item-related alterations in thyroid gland weights or thyroid histopathology.

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Developmental Findings

At 1000 mg/kg bw/day, a 100% (early) resorption rate (i.e. implantation sites only) was recorded for two animals. This 100% resorption consisted of early resorptions of implantations only and the higher mean percentage of early resorptions at 1000 mg/kg bw/day could be attributed to these two animals only. Although the number of animals with an early resorption rate that was higher than the concurrent control range appeared slightly increased at 1000 mg/kg bw/day (5 out of 22 animals), the number of animals with an early resorption rate that was higher than the historical control range was similar between 1000 mg/kg bw/day and the control group (7 out of 22 animals at 1000 mg/kg bw/day vs. 5 out of 21 animals in the control group). Nonetheless, based on historical control data, occurrence of a 100% resorption of implantations is a rare occurrence (only recorded for 4 out of 1094 litters). Therefore, although this was confined to two animals, this was considered to be related to treatment with the test item and to be adverse based on the type and severity of the effect at 1000 mg/kg bw/day. Also, at 1000 mg/kg bw/day, a lower mean litter size was recorded, which was attributed to four females with a low litter size ranging between 0 and 4 fetuses. Additionally, an adverse lower mean body weight of male and female fetuses was recorded at this dose (0.74x and 0.73x of control mean, respectively). At 100 and 300 mg/kg bw/day, mean fetal body weights and litter size were considered not affected by treatment with the test item. 

A single case of 100% resorptions was recorded at 300 mg/kg bw/day. However, there were no necropsy recordings of how many implantations were present for this animal and if the resorptions were early or late resorptions. It can therefore not be excluded that this animal had a low number of implantations, making it more likely that a 100% resorption is reached when this animal had only one or two implantations. Also, there were no concurrent adverse developmental effects noted at 300 mg/kg bw/day. As such, a relationship to treatment with the test item for this single occurrence of a 100% resorption rate was doubtful, and for this reason considered not to be adverse.

Adverse fetal morphological findings (skeletal malformations) were recorded at 1000 mg/kg bw/day (15.0% per litter vs. 1.6%, 1.8% and 1.8% per litter in the control, 100 and 300 mg/kg bw/day groups, respectively) and consisted of vertebral anomaly with or without associated rib anomaly, rib anomaly, vertebral centra anomaly, sternoschisis and costal cartilage anomaly. The incidence of these findings exceeded the historical control data range and all these malformations were localised in the same thoracic body region.

Other skeletal variations that were considered to be related to treatment with the test item (but not adverse) consisted of an increase in incidence of various ossification parameters (reduced ossification of the skull, vertebral centra, arches, pubis and ribs, and unossified metacarpals and/or metatarsals and sternebrae) at 300 and/or 1000 mg/kg bw/day. The higher incidences of these ossification parameters occurred in conjunction with decreased fetal body weights at 1000 mg/kg bw/day (at 100 and 300 mg/kg bw/day, an apparent trend towards reduced fetal weights was only noted based on the incidence of means of fetal body weights per litter that were below the historical control data range). Therefore, it was considered that this increased incidence of variations indicating delayed skeletal ossification, was due to lower fetal weights, and not a direct effect of the test item.

Other treatment-related (but non-adverse) skeletal variations consisted of increased incidence of 7th cervical full ribs, 14th full ribs, caudalshift of the pelvic girdle, and malaligned sternebrae at 1000 mg/kg bw/day, and 7th cervical ossification sites and bent ribs at 300 and 1000 mg/kg bw/day.

Treatment-related (but non-adverse) visceral variations affecting the urinary tract occurred in one fetus at 300 mg/kg bw/day and four fetuses at 1000 mg/kg bw/day, consisting of dilated ureter, convoluted ureter and/or absent or small renal papilla. These variations were considered reversible and to be indicative of a general developmental delay associated with lower fetal body weights. Therefore, the urinary tract variations may not be a direct effect of the test item.

Remaining skeletal variations occurred in the absence of a dose-related incidence trend, infrequently and/or in control fetuses only. Therefore, they were considered not to be related to treatment with the test item.

There were no external fetal lesions noted.No test item-related changes were noted in sex ratio and anogenital distance.

In conclusion, based on the results in this prenatal developmental toxicity study the following maternal and developmental No Observed Adverse Effect Level (NOAEL) for the registered substance was established:

Maternal NOAEL: 300 mg/kg bw/day (based on lower body weight gain and food intake, and clinical signs at 1000 mg/kg bw/day).

Developmental NOAEL: 300 mg/kg bw/day (based on two cases of 100% implantation loss at 1000 mg/kg bw/day, along with lower fetal body weights and skeletal malformations)).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study performed according to OECD/EC guidelines and GLP principles (Klimisch 1).
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A prenatal developmental toxicity study was performed in rat according OECD TG 414 and in accordance with GLP principles. Time-mated female Wistar Han rats were given 0, 100, 300 or 1000 mg/kg bw/day of the substance orally by gavage from Day 6 to 20 post-coitum, inclusive. The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (T3, T4, 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.

Formulation analyses confirmed that formulations of test item in Polyethylene glycol 400 were prepared accurately and homogenously.

Maternal Findings

All animals survived the scheduled treatment duration. However, adverse maternal effects were recorded at 1000 mg/kg bw/day. A lower mean body weight gain was recorded throughout the treatment period (0.83x of the control mean at the end of the treatment period), with an initial slight weight loss during the first week of treatment for several animals. A few animals also had body weights corrected for gravid uterus weight that were below the historical control range. This was accompanied by a lower mean food consumption between post-coitum Days 6 to 9 (0.78x of control mean). Although mean food intake recovered to control levels as treatment progressed, several animals at 1000 mg/kg bw/day also showed a notable reduction in food intake over post-coitum Days 15-21.

Additionally, piloerection (and incidentally hunched posture) was recorded for most animals between post-coitum Days 7 and 20. Overall, these findings were considered to represent an adverse effect of the test item at 1000 mg/kg bw/day.

At both 100 and 300 mg/kg bw/day, body weights (also corrected for gravid uterus) and food intake levels were considered not affected by treatment with the test item. At 300 mg/kg bw/day, hunched posture or piloerection were recorded for a few animals during the last week of treatment. In absence of other maternal signs of toxicity these few occurrences of clinical signs were considered not to represent an adverse effect of the test item.

Mean total Triiodothyronine (T3) and total Thyroxine (T4) levels showed an apparent dose-related trend towards a reduction across the dose groups, and mean Thyroid-Stimulating hormone (TSH) levels showed an apparent dose-related trend towards an increase. At 1000 mg/kg bw/day, the decrease in mean T3 and T4 was statistically significant (0.65x and 0.73x of control mean for total T3 and T4, respectively). The mean total T3 was below the historical control data range at 100, 300 and 1000 mg/kg bw/day; possible adversity of this change could not be established within this study. Both mean total T4 and TSH remained within the historical control data range at these dose levels, and as such these changes were considered not to represent an adverse effect. There were no test item-related alterations in thyroid gland weights or thyroid histopathology.

The numbers of pregnant females, corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.

Developmental Findings

At 1000 mg/kg bw/day, a 100% (early) resorption rate (i.e. implantation sites only) was recorded for two animals. This 100% resorption consisted of early resorptions of implantations only and the higher mean percentage of early resorptions at 1000 mg/kg bw/day could be attributed to these two animals only. Although the number of animals with an early resorption rate that was higher than the concurrent control range appeared slightly increased at 1000 mg/kg bw/day (5 out of 22 animals), the number of animals with an early resorption rate that was higher than the historical control range was similar between 1000 mg/kg bw/day and the control group (7 out of 22 animals at 1000 mg/kg bw/dayvs. 5 out of 21 animals in the control group). Nonetheless, based on historical control data, occurrence of a 100% resorption of implantations is a rare occurrence (only recorded for 4 out of 1094 litters). Therefore, although this was confined to two animals, this was considered to be related to treatment with the test item and to be adverse based on the type and severity of the effect at 1000 mg/kg bw/day.Also, at 1000 mg/kg bw/day, a lower mean litter size was recorded, which was attributed to four females with a low litter size ranging between 0 and 4 fetuses. Additionally, an adverse lower mean body weight of male and female fetuses was recorded at this dose (0.74x and 0.73x of control mean, respectively). At 100 and 300 mg/kg bw/day, mean fetal body weights and litter size were considered not affected by treatment with the test item. 

A single case of 100% resorptions was recorded at 300 mg/kg bw/day. However, there were no necropsy recordings of how many implantations were present for this animal and if the resorptions were early or late resorptions. It can therefore not be excluded that this animal had a low number of implantations, making it more likely that a 100% resorption is reached when this animal had only one or two implantations. Also, there were no concurrent adverse developmental effects noted at 300 mg/kg bw/day. As such, a relationship to treatment with the test item for this single occurrence of a 100% resorption rate was doubtful, and for this reason considered not to be adverse.

Adverse fetal morphological findings (skeletal malformations) were recorded at 1000 mg/kg bw/day (15.0% per litter vs. 1.6%, 1.8% and 1.8% per litter in the control, 100 and 300 mg/kg bw/day groups, respectively) and consisted of vertebral anomaly with or without associated rib anomaly, rib anomaly, vertebral centra anomaly, sternoschisis and costal cartilage anomaly. The incidence of these findings exceeded the historical control data range and all these malformations were localised in the same thoracic body region.

Other skeletal variations that were considered to be related to treatment with the test item (but not adverse) consisted of an increase in incidence of various ossification parameters (reduced ossification of the skull, vertebral centra, arches, pubis and ribs, and unossified metacarpals and/or metatarsals and sternebrae) at 300 and/or 1000 mg/kg bw/day. The higher incidences of these ossification parameters occurred in conjunction with decreased fetal body weights at 1000 mg/kg bw/day (at 100 and 300 mg/kg bw/day, an apparent trend towards reduced fetal weights was only noted based on the incidence of means of fetal body weights per litter that were below the historical control data range). Therefore, it was considered that this increased incidence of variations indicating delayed skeletal ossification, was due to lower fetal weights, and not a direct effect of the test item.

Other treatment-related (but non-adverse) skeletal variations consisted of increased incidence of 7th cervical full ribs, 14th full ribs, caudalshift of the pelvic girdle, and malaligned sternebrae at 1000 mg/kg bw/day, and 7th cervical ossification sites and bent ribs at 300 and 1000 mg/kg bw/day.

Treatment-related (but non-adverse) visceral variations affecting the urinary tract occurred in one fetus at 300 mg/kg bw/day and four fetuses at 1000 mg/kg bw/day, consisting of dilated ureter, convoluted ureter and/or absent or small renal papilla. These variations were considered reversible and to be indicative of a general developmental delay associated with lower fetal body weights. Therefore, the urinary tract variations may not be a direct effect of the test item.

Remaining skeletal variations occurred in the absence of a dose-related incidence trend, infrequently and/or in control fetuses only. Therefore, they were considered not to be related to treatment with the test item.

There were no external fetal lesions noted. No test item-related changes were noted in sex ratio and anogenital distance.

 

In conclusion, based on the results in this prenatal developmental toxicity study the following maternal and developmental No Observed Adverse Effect Level (NOAEL) for Piperonal or Heliotropine was established:

Maternal NOAEL: 300 mg/kg bw/day (based on lower body weight gain and food intake, and clinical signs at 1000 mg/kg bw/day).

Developmental NOAEL: 300 mg/kg bw/day (based on two cases of 100% implantation loss at 1000 mg/kg bw/day, along with lower fetal body weights and skeletal malformations)).

Justification for classification or non-classification

Based on the available information, the registered substance is not classified for toxicity to reproduction according to CLP Regulation EC (No.) 1272/2008.

Additional information