Registration Dossier

Administrative data

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 NOAEL parental 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
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 oral 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
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.
Observations and examinations performed and frequency:
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
- How many animals: all animals
- Parameters: See additional information on materials and methods

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: See additional information on materials and methods

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 (overnight with a maximum of 24 hours before blood sampling, but water was available)
- 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.

ESTROUS CYCLE: Yes
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.
Sacrifice and pathology:
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 additional material on materials and methods 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.
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.
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 (mean = 730; P5 – P95 = 563.0 – 883.0 (n= 158)).
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 = 40.1; P5 – P95 = 27.9 – 60.8 (n=164)).
- 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 = 75.0; P5 – P95 = 60.8 – 95.2 (n=164)).
- Mean levels of cholesterol, HDL cholesterol and LDL cholesterol were mildly decreased in males treated at 1000 mg/kg bw/day (0.66x, 0.76x and 0.73x, respectively).
- 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 = 20.1; P5 – P95 = 7.60 – 42.50 (n=134)).
- 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 (mean = 1.76; P5 – P95 = 1.400 – 2.170 (n=164)).
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
Description (incidence and severity):
Males
Test item-related higher liver weights and lower thymus weights (absolute and relative to body weights) were noted in the 300 and/or 1000 mg/kg bw/day group males (see table additional information on results). Some organ weight differences in males were statistically significant when compared to the control group but were considered to be the result of a test item-related effect on final body weight (-14%). This was the case for the pituitary gland, kidney, adrenal glands, the prostate gland and the epididymides. The brain and heart weights showed no dose-response and therefore their statistically significance at 300 and/or 1000 mg/kg bw/day was considered unrelated to treatment.

Female rats treated up to 1000 mg/kg bw/day:
Test-item related higher thymus weights (absolute and relative to body weights) were noted at 300 and 1000 mg/kg bw/day (See table additional information on results).
At 1000 mg/kg bw/day, organ weights of the brain, heart, liver, thyroid gland, kidneys, adrenal gland, ovaries and uterus were statistically significant different from the control group mean values. Since there was no evidence of test item related microscopic lesions in these organs and since all females of the 1000 mg/kg bw/day group were not pregnant or did not produce healthy offspring, these organ weight differences were regarded to be caused by their difference in physiologic status and/or lower body weight, rather than by a direct effect of the test item on the organ weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations.
All recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
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.
Other effects:
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.
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
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

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.
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.

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.

In conclusion, based on the results of this combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the No Observed Adverse Effect Level (NOAEL) parental for Piperonal or Heliotropine was 300 mg/kg bw/day based on an adverse increase in trabecular bone in both sexes at 1000 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles (Klimisch 1).
System:
musculoskeletal system
Organ:
bone

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test performed according to OECD TGs 422 and 408 and in accordance with GLP principles is available. Wistar Han rats were given Piperonal or Heliotropine at 0, 100, 300 or 1000 mg/kg bw/day orally by gavage for a minimum of 13 weeks.

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.

In conclusion, based on the results of this combined 90-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the No Observed Adverse Effect Level (NOAEL) parental for Piperonal or Heliotropine was 300 mg/kg bw/day based on an adverse increase in trabecular bone in both sexes at 1000 mg/kg bw/day.

 

Three (3) supplemental repeated dose toxicity studies were identified, one of which was reported in 3 separate publications (U.S. FDA, 1954; Haganet al., 1965; Haganet al., 1967; Bar and Griepentrog, 1967; Trubek, 1958). All of the studies pre-date OECD test guidelines and GLP and are considered reliable with restrictions. No adverse effects were observed up to and including the highest dose levels tested.

Justification for classification or non-classification

Based on the available information, the registered substance does not have to be classified for repeated dose toxicity according to CLP Regulation EC (No.) 1272/2008.