Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Rats
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 August 2017 - 28 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
yes
Remarks:
On the day of necropsy, no vaginal lavage was taken to determine the stage of estrus from the only female with total litter loss. Yet, the result of the vaginal lavage would not have influenced the evaluation of the observations made for this animal.
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650
Version / remarks:
2000
Deviations:
yes
Remarks:
On the day of necropsy, no vaginal lavage was taken to determine the stage of estrus from the only female with total litter loss. Yet, the result of the vaginal lavage would not have influenced the evaluation of the observations made for this animal.
Guideline:
other: OECD 421, Repr./Dev. Tox Screening Test EPA OPPTS 870.3550, Repr./Dev. Tox Screening Test EC No 440/2008, B.7 Rep. Dose 28 d Tox (oral) OECD 407, Rep. Dose 28-d Oral Tox Study in Rodents EPA OPPTS 870.3050, Rep. Dose 28-d Oral Tox Study in Rodents
GLP compliance:
yes
Remarks:
Any portion of this study conducted according to OECD and GLP princples, with one exception: analyses conducted to support the information cited in the Certificate of Analysis were conducted in compliance with Good Manufacturing Practice regulations.
Limit test:
no

Test material

Constituent 1
Test material form:
liquid: viscous
Details on test material:
- Name of test material: Reaction product of 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane and aqueous phosphoric acid, 2-butoxyethanol, 2-(dimethylamino)ethanol
- Trade name: Uradil DD80
- State of aggregation: Colourless to light yellow high viscous liquid
- Batch: 616F-2072
- Density: 1.3 Pa.s
- Purity: 97%
- Water content: 0.3 w/w%
- Residual solvent: 2.7 w/w%
- Test item storage: At room temperature
- Stable under storage conditions until: 31 December 2017 (expiry date). After QC check executed in December 2017 the renewed expiry dateis 1 July 2018. This renewed expiry dated is cited in study reports of studies performed after 31 December 2017.

Test animals

Species:
rat
Strain:
other: Wistar Han
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 10 weeks (males) and 13 weeks (female)
- Weight at study initiation: between 271 and 305 g (males) and between 199 and 236 g (females)
- Fasting period before study: No
- Housing: Group housing of 5 animals per sex in Macrolon cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages
During the lactation phase, females were housed in Macrolon plastic cages. 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 be 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
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, tap water
- Acclimation period: 8 days

ENVIROMENTAL CONDITIONS
- Temperature: 18 -24°C . Actual daily mean temperature : 20 to 22°C
- Relative humidity: 40 - 70%. Actual daily mean relative humidity : 41 to 64%.
- Photoperiod (hrs dark / hrs light): 12/12except when interrupted for designated procedures.
- Air changes (per hr): 10 or more, with 100% fresh air (no air recirculation)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
Specific gravity: 1.036
Details on oral exposure:
PREPARATION OF DOSING SOLUTION
Formulations were prepared once a week as clear colorless solutions .
After homogenizing, each bulk formulation was then divided into 7 aliquots for daily dosing and stored in the refrigerator until use (maximum storage for 8 days in the refrigerator).
Before dosing, the dosing formulations were removed from the refrigerator and stirred for at least 30 minutes to homogenize and acclimatize. If practically possible, they were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item

VEHICLE
Propylene glycol
Amount of vehicle (gavage): 5 mL/kg body weight
The vehicle was prepared and stored in a similar manner as the test item dose formulations, and divided into aliquots before daily dosing.

EXPOSURE DURATION
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days.
- Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period.
- Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 - 15 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 43-44 days.

ADMINISTRATION TO PUPS
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

DEVIATIONS
Female no.71 (Group 4), was not dosed on one occasion as this female was littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.

ADMINISTRATION SCHEDULE
The first day of dosing was designated as Day 1 (exception: alternate animals used for replacement after Day 1 was assumed the day of the animal being replaced).
Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

DOSE VOLUME
5 mL/ kg

DOSE CONCENTRATION
The dose concentration for each animal was based on the most recent body weight measurement.

DOSE LEVELS
The dose levels were selected based on the results of a 14-day oral dose range finder with oral exposure of the test item in rats

DOSE APPLICATIONS
The doses were given using a plastic feeding tube.
The dosing formulations were stirred continuously during dose administration.
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

DEVIATION DURING DOSE RANGE FINDINGS
The use of the dose control system DCS was not documented properly during the conduct of the dose range finder.
Evaluation: DCS is used as an additional tool to check the dosing procedure and other (dosing) checking procedures remained intact.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dosing formulations were stirred continuously during dose administration.
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

STABILITY ANALYSIS
Analyses were conducted on a single occasion during treatment phase, according to a validated method (project 516851).
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Stability in vehicle was also determined (concentration range 1 to 200 mg/mL) for:
- at least 5 hours and room temperature under normal laboratory light conditions
- at least 8 days in the refrigerator
- at least 3 weeks in the freezer ≤ -15°C

ANALYTICAL METHOD
Analyses were performed by using a validated analytical procedure

CONCENTRATION ANALYSIS
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

HOMOGENEITY ANALYSIS
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was +/- 10%.
Duration of treatment / exposure:
Minimum 28 days:
- Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period.
- Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 - 15 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 43-44 days.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle - Group 1
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 males and 10 females for every dose concentration
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELECTION RATIONALE
Dose selection was based on Dose Range Finding Study (N 516970), that was conducted before start of Main study.

DOSE RANGE FINDING STUDY
Three females per group were exposed to 500 or 1000 mg/ kg bw/ day (5 mL volume) for 14 days, once daily.
No mortality occurred in any of the groups.
At 500 mg/kg bw/ day, single incidence of piloerection in all three animals observed at 3 hours after dosing on Day 13.
At 1000 mg/kg bw/ day, Hunched posture and/or piloerection in the three animals from Day 13 onwards
No unexpected changes in body weight gain occurred, food consumption was normal and no abnormalities were noted at macroscopic examination for both groups. Liver and kidney weights were considered to be normal for all animals.

Based on the results of this range finding study, dose levels for the Main study were chosen to be 100, 300 and 1000 mg/kg body weight.
Since no clear peak effect of occurrence of clinical signs was observed in the dose range finder, it was concluded that in the main study the clinical observations and functional observations should be performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
Positive control:
Not required

Examinations

Observations and examinations performed and frequency:
F0 GENERATION

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations were included.
DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Once daily from the first day of administration of teh test item up tp the day prior the necropsy
ARENA OBSERVATION
- Once weekly
BODY WEIGHT
- Time schedule for examinations: on the first day of treatment prior dosing, then weekly
- 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.
FOOD CONSUMPTION:
- once 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 AND COMPOUND INTAKE:
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as
no effect was suspected.
FUNCTIONAL TESTS:
- On 5 males selected during week 4 of treatment and 5 females selected during the last week of lactation (i.e. PND 11, 12 or 13).
- The tests were: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity
ESTROUS CYCLES DETERMINATION
- by examining the vaginal cytology of samples obtained by vaginal lavage.
MATING PROCEDURE
- After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Once mating had occurred, the males and females were separated.
GENERAL REPRODUCTION DATA
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
Females were allowed to litter normally, undisturbed.
Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care

F1 GENERATION

MORTALITY / MORIBUNDITY CHECKS – F1-Generation
- how many pups: all
- how often: daily from PND 1
CLINICAL OBSERVATIONS: F1-Generation
- how many pups: all
- how often: once a day, at least
BODY WEIGHT – F1-Generation
- how many pups: all live pups
- when: on PND 1, 4, 7 and 13.
SEX– F1-Generation
- how many pups: all
- when: on PND 1 and 4.
ANOGENITAL DISTANCE - F1-Generation
- how many pups: all live pups
- when: on PND 1
AREOLA/NIPPLE RETENTION – F1-Generation
- how many pups: all male pups
- when: on PND 13
CULLING – F1-Generation
- how many pups: 8 pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
- when: on PND 4

LABORATORY EVALUATIONS

BLOOD COLLECTION F0-animals:
- time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (maximum 24 hours, water was available)
- How many animals: all for thyroid hormone
Note: 5 animals/sex/group were selected for hematology, coagulation, clinical chemistry, functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology
MEASURED PARAMETERS: as described in the test guideline

BLOOD COLLECTION F1-animals:
- time schedule for collection of blood: on PND 4 and PND 13-15
- Anaesthetic used for blood collection: on PND 4 Not (by decapitation), on PND 13-15 Yes (isoflurane)
- How many animals: 2 per litter on PND 4 (from two surplus pups per litter - when available, preferably one male and one female ); 2 per litter on PND 13-15 (from two pups per litter - one male and one female ).
MEASURED PARAMETERS: as described in the test guideline


HEMATOLOGY F0-animals:
- time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (maximum 24 hours, water was available)
- How many animals: 5 animals/sex/group were selected for hematology, coagulation, clinical chemistry, functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology
- Measured parameters: as described in the test guideline
CLINICAL CHEMISTRY F0-animals:
- time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (maximum 24 hours, water was available)
- How many animals: 5 animals/sex/group were selected for hematology, coagulation, clinical chemistry, functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology
- Measured parameters: as described in the test guideline
COAGULATION F0-animals:
- time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (maximum 24 hours, water was available)
- How many animals: 5 animals/sex/group were selected for hematology, coagulation, clinical chemistry, functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology
- Measured parameters: as described in the test guideline
CLINICAL PATHOLOGY F0-animals:
- time schedule for collection of blood: just before necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (maximum 24 hours, water was available)
- How many animals: 5 animals/sex/group were selected for hematology, coagulation, clinical chemistry, functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology
- Measured parameters: as described in the test guideline
THYROID HORMONE (F0 and F1 animals)
- time schedule for collection of blood: just before necropsy
- Hiow many animals: 2 pups/litter on PND 4, 2 pups/litter on PND 13, all males and all females (except for females with total litter loss, or females which were found dead)
- Samples for T4 of F0-males and PND 13-15 pups were analysed.
- Samples for T4 of F0-females and PND 4 pups and samples for TSH of F0-males, F0-females and PND 13-15 pups were not analysed.
- For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, no microscopic findings were found in the thyroid gland in F0-females, and no treatment related changes in thyroid weight were recorded (both sexes). For the F1 generation, assessment of T4 for PND 4 pups and TSH for PND 13-15 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 13-15
Sacrifice and pathology:
POSTMORTEM EXAMINATION (F0 GENERATION):
- 5 animals/sex/group were selected for: necropsy, tissue collection, organ weights, hematology, coagulation, clinical chemistry, functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list), histology and histopathology. Note: histology and histopathology: full list for 5 animals/sex selected from group 1 and group 4; gross lesion for 5 animals/sex selected from group 2 and group 3.
- ≤ 5 animals/sex/group (non selected) for: necropsy, tissue collection, organ weights, histology and histopathology. Note: histology and histopathology:Gross lesions, Selected tissues (Selected tissues were applicable for males that failed to sire and females that failed to deliver pups (i.e. non-pregnant females) and females with total litter loss.
UNSCHEDULED DEATHS (or found dead)
- necropsy, tissue collection, histology and histopathology (full list)
SCHEDULED NECROPSIE:
- Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
- Females which delivered: PND 14-16.
- Females which failed to deliver, With evidence of mating: Post-coitum Day 27
- Females with total litter loss: euthanized within 24 hours after the last pup was found dead or missing.
IMPLANTATION LOST
- The numbers of former implantation sites were recorded for all paired females.
- In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.
NECROPSIE
- Full mortem examination: yes, all animals, special attention being paid to the reproductive organs.
ORGAN WEIGHTS
- According to Guidelines
TISSUE COLLECTION AND PRESERVATION
- According to Guidelines
HISTOLOGY
- The animals were divided in four groups: i) Selected animals and unscheduled deaths (found dead); ii) Males that failed to sire (except for males which were selected), females that failed to deliver pups and females with total litter loss; iii) Females with total litter loss; Remaining animals
- Analysis performed according to Guidelines
HISTOPATHOLOGY
- All tissues as defined under Histology were examined for histopathology
- For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

POST-MORTEM EXAMINATION (F1 GENERATION)
- Necropsie for unscheduled Deaths: examined externally and sexed (both externally and internally). The stomach of pups was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
- Euthanasia: by decapitation for pups younger than 7 days; by an intraperitoneal injection of sodium pentobarbital for all remaining pups (PND13 and 15); exception: two pups per litter (PND 13 and 15) selected for blood collection were anesthetized using isoflurane followed by exsanguination.
- necropsie for scheduled euthanasia: sex was determined (both externally and internally); descriptions of all external abnormalities were recorded (particular attention to the external reproductive genitals); blood collected from two pups / litter, thyroid from two pups / litter (one male and one female pup) preserved. Note: The pups selected for blood sampling were the same pups as selected for thyroid preservation.



Statistics:
PARENTAL VARIABLES
- Body Weight Gains
- Relative Food Consumption
- Organ Weight Relative to Body Weight

REPRODUCTION AND DEVELOPMENTAL VARIABLES
Mating (%)
Precoital time
Fertility index (%)
Gestation index (%)
Duration of gestation:
Post-implantation survival index (%)
Live birth index (%)
Percentage live males at First Litter Check (%)
Percentage live females at First Litter Check (%)
Viability index (%)
Lactation index (%)

APPLIED STATISTICAL METHODS
- Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
- Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
- The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group.
- 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 is significant.

STATISTICAL ANALYSIS
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% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- from 100 mg/kg: mostly slight salivation, ith a slight dose-related increase in onset and incidence. Taking into account the nature and minor severity of the salivation 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
- in a single male and female at 1000 mg/kg: breathing difficulties, comprising rales, on a few occasions. This latter finding was possibly related to treatment, but since it occurred temporarily in two high dose animals only it was considered of no toxicological significance.

Mortality:
mortality observed, non-treatment-related
Description (incidence):
- One female from group 4 was found dead in the day before scheduled necropsy.
Observations: advanced autolysis, menlarged liver, black discoloration of the uterus and the stomach and caecum distended with gas; the thoracic cavity was filled with reddish fluid; severe congestion of the liver. Furthermore, no relevant clinical signs or changes in body weight and food consumption were observed preceding the death of this female.
Based on these results and the fact that it was an isolated incident, this premature death was considered as unrelated to treatment with the test-item.

- No further mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- in males at 1000 mg/kg: statistically significantly lower, from Day 8 of the pre-mating period onwards - resulting in approximately 7% lower body weights at end of treatment in comparison with controls. Body weights and body weight gain cannot be correlated with changes in food consumption
- in females at 1000 mg/kg: less clear trend:
During the first week of treatment a minimal body weight gain, the further growth during the treatment period was comparable to that in the other groups.
On one occasion at the end of the post-coitum period, the mean body weights of females at 1000 mg/kg was statistically significantly lower in comparison with controls.
Slightly lower body weights, not statistically significantly different, were also observed during lactation in females at 1000 mg/kg compared to controls.
The 3% lower body weights observed in females at 1000 mg/kg at termination of treatment, was considered minimal, indicating that the effects on body weights and body weight gain at 1000 mg/kg were of no toxicological significance.
- in males and females up to 300 mg/kg: same range as controls
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- for females at 1000 mg/kg: post-coitum: slightly lower over the 0-4 days post-coitum period, and in comparison with controls over days 17-20 post-coitum; pre-mating and lactation period: normal
- for males up to 1000 mg/kg and for females up to 300 mg/kg: similar to control levels
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
-Pupillary reflex was normal in all examined animals.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The following changes distinguished females at 1000 mg/kg from control animals:
- Slightly decreased red blood cell and reticulocyte counts (0.95x and 0.88x control),
- Slightly increased red blood cell distribution width (0.91x control),
- Decreased haemoglobin and haematocrit concentrations, achieving levels of statistical significance when compared to controls (0.91x and 0.96x control),
- A tendency towards a dose related decrease in the red blood cell parameter derived indices and mean corpuscular haemoglobin concentration (MCHC), reaching a level of statistical significance in females at 1000 mg/kg for MCHC.
All mean values remained well within the range considered normal for animals of this strain and age.

- males up to 1000 mg/kg and females up to 300 mg/kg: Haematological parameters not affected by treatment

- Coagulation parameters: not affected by treatment.

- No alterations were observed in the bone marrow after histopathological examination.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, the following changes distinguished treated animals from control animals:
- Alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) activity increased in males (1.35 and 1.48x control, respectively), and ALAT (not statistically significant) and alkaline phosphatase (ALP) activity increased in females (1.28x and 2.28x control, respectively),
- Total bilirubin and creatinine concentration increased in males (2.18x and 1.09x control, respectively),
- Glucose levels increase in females (1.32x control),
- Cholesterol levels increased in males and females (both 2.14x control),
- Bile acids increased in males (4.6x control),
- Sodium levels decreased in males (0.98x control),
- Inorganic phosphate levels decreased in males and females (0.88x and 0.89x control, respectively; not statistically significant),

At 300 mg/kg, the following changes distinguished treated animals from control animals:
- Bile acids increased in males (2.0x control, not statistically significant).
- Inorganic phosphate levels decreased in males (0.88x control; not statistically significant)

For males at 100 mg/kg and females up to 300 mg/kg: clinical biochemistry parameters not affected by treatment.

Thyroid hormone analyses:
- in F0 males: Serum levels of T4 not affected by treatment.
A tendency towards a dose related increase in mean T4 value in males was observed, with a 33% increase at 1000 mg/kg (without reaching a level of statistical significance) in comparison with the control value.
Since the increase T4 values in high dose males still remained within the historical control range and no microscopic alterations were observed in the thyroid gland of these males the changes in T4 were considered of no toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- in Females 1000 mg/kg: higher liver weights (absolute and relative to body weights)

- in male: lower weights for the prostate gland and seminal vesicles - statistically significant difference for the absolute weights of the prostate at all dose levels and of the seminal vesicles at 300 and 1000 mg/kg. In the absence of a dose relationship for these organ/body weight ratios, no microscopic alterations were found and all organ weights were within the historical control range , these changes in prostate and seminal vesicle weights were considered to have occurred as a result of slightly high control weights rather than indicative of a treatment related effect.

- in male at 1000 mg/kg:statistically significantly higher heart/body weight ratio, considered the result of the low terminal body weights in these males.

Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the liver, duodenum, jejunum and mesenteric lymph node:
- Vacuolation of the Ito cells of the liver in males and females treated at 300 and 1000 mg//kg/day (up to slight).
- Lymphangiectasis in the jejunum of 300 and 1000 mg/k/day treated males (up to moderate).
- Lymphangiectasis in females in the jejunum from 100 mg/kg/day (up to moderate), in the duodenum from 300 mg/kg/day (minimal) and in the mesenteric lymph node of 1000 mg/kg/day treated females (up to slight).
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
FUNCTIONAL TEST:
No treatment realted effects observed.
All those parameters were similar between control and treated animals: hearing ability, pupillary reflex and static righting reflex, grip strength, motor activity

REPRODUCTIVE PERFORMANCE
At 1000 mg/kg, 2 couples without offsprings.
At 1000 mg/kg: 1 total litter loss
Reproductive organs and spermatogenic staging profiles were all normal

REPRODUCTION CYCLE
- Estrous Cycle: not affected by treatment. All females had regular cycles of 4 days.
- Mating index: not affected by treatment. All females showed evidence of mating.
- Precoital time: not affected by treatment. All females showed evidence of mating within 4 days.
- Number of implantation sites: not affected by treatment.
At 300 and 1000 mg/kg, a statistically significantly lower number of implantation sites was noted when compared to controls. As this occurred in absence of a dose response relationship, and as values remained well within the normal range that could be expected , this was considered to be unrelated to treatment. - Fertility index: Two females at 1000 mg/kg were not pregnant, resulting in a slightly low fertility index (80%, compared to 100% for controls).
The fertility index was not considered to be affected by treatment up to 300 mg/kg. All mated females at 100 and 300 mg/kg were pregnant

DEVELOPMENTAL DATA
- Gestation index and duration: not affected by treatment
- Parturition/maternal care: not affected by treatment
- Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites): 94% in control group, 84% in 100 mg/kg group,92% in 300 mg/kg group, and 74% in 1000 mg/kg treated group.
- Individual difference between number of implantation sites and number of pups born (unaccounted for (implantation) sites): in the 10 females in the control group between 0 and 2; in the 100 mg/kg group, seven females with 0-2 and one female with 3 and two females with 5 unaccounted for sites; in the 300 mg/kg group, nine females with 0-2 and one female with 3 unaccounted for sites; in the 1000 mg/kg group, four females with 0-1 and four females with 4 or more unaccounted for sites, i.e. one with either 4, 5, 6 or 9. Note: For one female (300 mg/kg group ) the number of pups born was higher than the number of implantation sites, i.e. 12 and 11 respectively. This phenomenon is occasionally observed and is caused by normal resorption of these areas during (the 14 days of) lactation. No toxicological relevance was attached to this finding in the current study.
- Litter size (mean number of living pups): 13.3 in the control group, 10.6, 10.9 and 9.1 at 100, 300 and 1000 mg/kg groups, respectively,
- Live birth index: not affected by treatment. Note: One pup of the control group, 6 pups at 100 mg/kg and one pup at 1000 mg/kg were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
- Viability index (number of live offspring on Day 4 before culling compared to the number of offspring on Day 1): in the 1000 mg/kg group was 89% (compared to 98% in control). Note: Two pups of the control group, 6 pups at 100 mg/kg, one pup at 300 mg/kg and 8 pups at 1000 mg/kg were found missing on PND 2, 3 or 4.
No toxicological relevance was attributed to the missing pups at 100 and 300 mg/kg since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
- Lactation index (number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4, after culling)): not affected by treatment.
- clinical signs: No clinical signs occurred among pups that were considered to be related to treatment.
-Body weights: not affected by treatment
- Sex ratio: not affected by treatment up to 300 mg/kg. At 1000 mg/kg, male/female ratio was 38/62 compared to 50/50 in controls. (Note: there was a large variation in male/female ratio in individual litters in this high dose group.) This result might indicate a sex difference in sensitivity to toxic effects of the test item. If true, the increased number of unaccounted for sites noted at 1000 mg/kg might be the result of selective prenatal loss of male embryos/fetuses, but no definite conclusive could be drawn from the results obtained in this study. An endocrine mediated (antiandrogenic) effect being responsible for the skewed sex ratio, however, was considered unlikely, because there were no changes in any of the endocrine endpoints, i.e. nipple retention, anogenital distance, T4 levels in pups and thyroid gland morphology and reproductive organs in the dams at 1000 mg/kg.
- Anogenital distance: not affected by treatment.
- Areola/nipple retention: not affected by treatment.
- Clinical biochemistry (T4 levels): serum T4 levels in male and female PND 13-15 pups were not affected by treatment.
- Macroscopy: not treatment realted findings

Effect levels

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Key result
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at the highest tested dose
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: : higher number of unaccounted for implantation sites, a higher post natal mortality (up to PND 4), a decreased male/female sex ratio and an effect on growth of the pups

Target system / organ toxicity

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Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
duodenum
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
jejunum
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
The repeated dose toxicity of the test item was investigated in Wistar Han rats according to the OECD Guideline No. 422 (combined 28-day repeated dose toxicity study with the reproduction/ developmental toxicity screening test) and GLP principles. The selected dose were: 0 (only vehicle: propylene glycol), 100, 300 and 1000 mg/kg bw/day. Each dose group had 10 female and 10 male rats. The test item was administred for at least 28 days, once a day, by gavage.
The test item showed parental toxicity at 1000 mg/kg, expressed as reduced body weight gain in males, changes in several red blood cell related haematological parameters and increased liver weights in female rats, and in several biochemistry parameters in both sexes (more pronounced in male than in female rats). Histopathological examination revealed treatment-related changes in the liver and digestive tract in both sexes.
From the results in the reproduction and developmental parameters at 1000 mg/kg, a higher number of unaccounted for implantation sites, a higher post natal mortality (up to PND 4), a decreased male/female sex ratio and an effect on growth of the pups was observed.
Based on these results, the following no-observed-adverse-effect level (NOAEL) of the test item were established:
Parental NOAEL: 300 mg/kg
Reproduction NOAEL: 1000 mg/kg
Developmental NOAEL: 300 mg/kg
Executive summary:

An oral OECD 422 (combined 28-day repeated dose toxicity study with the reproduction/ developmental toxicity screening test) was conducted according to OECD guidelines and GLP principles.

The test item was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, Propylene glycol, alone. 

Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that were not pregnant or had a total litter loss were treated for 43 or 44 days, respectively.

Parental results

One female at 1000 mg/kg was found dead on the day before its scheduled necropsy. The death occurred without clear indications of bad health or signs of toxicity. Moreover, it had delivered a normal healthy litter. Therefore, also taking into account the absence of signs of serious toxicity in the other females of this dose group, this death was considered an accidental finding and not related to treatment.
Mostly slight salivation was noted at dose levels from 100 mg/kg, with a slight dose-related increase in onset and incidence. Taking into account the nature and minor severity of the salivation 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. Breathing difficulties, comprising rales, were noted in a single male and female at 1000 mg/kg on a few occasions. This latter finding was possibly related to treatment, but since it occurred temporarily in two high dose animals only it was considered of no toxicological significance.

Body weights and body weight gain were lower in males at 1000 mg/kg from Day 8 onwards, but without correlating changes in food consumption. Growth and food consumption in females was considered not affected by treatment up to 1000 mg/kg.

Changes in haematology parameters were noted in females at 1000 mg/kg and consisted of slightly decreased red blood cell and reticulocyte counts, with decreased haemoglobin and haematocrit concentrations, and decreased mean corpuscular haemoglobin concentration (MCHC). In addition, slightly increased red blood cell distribution width (RDW) was noted in these females. The combination of these changes might indicate an effect on the formation of red blood cells, i.e. decreased number of red blood cells and reticulocytes (immature red blood cells) and lower haemoglobin and haematocrit values. As a result, the proportion of young and old erythrocytes in the peripheral blood might have been altered, which was supported by an increased RDW value. Knowing that the volume of erythrocytes is increasing with maturation, the decreased values of the red blood cell indices MCHC suggested a larger proportion of old erythrocytes in the peripheral blood. Despite the (slight) effects on red blood cells in the peripheral blood, no alterations were observed in the bone marrow after histopathological examination.

Changes in clinical biochemistry were mainly noted at 1000 mg/kg and more pronounced in males than in females. In males, they included slight to moderate increases in alanine aminotransferase and aspartate aminotransferase activity, and in total bilirubin, creatinine, cholesterol and bile acid levels. In addition, sodium and inorganic phosphate levels were slightly decreased. In females, increased alanine aminotransferase and alkaline phosphatase activity, increased cholesterol levels, and decreased inorganic phosphate levels were observed. At 300 mg/kg, only slight changes in bile acids and inorganic phosphate were observed in males.

Histopathological examination revealed test item-related changes in the liver and parts of the digestive tract. 
In the liver, vacuolation of the Ito cell was observed at 300 and 1000 mg/kg in both sexes and was characterized by a single clear, well delineated cytoplasmic vacuole which caused indentation of the nucleus. Since severities were low and there were no concomitant test item-related morphologic findings of the liver, this finding was considered as non-adverse. As Ito cells represent only 5% of the resident cells in the liver, it was concluded that the vacuolation of the Ito cell was not responsible for the increase in liver weight, which was observed at necropsy for the females at 1000 mg/kg only.
In the digestive tract, lymphangiectasis was observed in the jejunum in females from 100 mg/kg and males from 300 mg/kg, in the duodenum, in females from 300 mg/kg and in the draining mesenteric lymph nodes in females at 1000 mg/kg/day. The lymphangiectasis was not accompanied by inflammation, necrosis or proliferation, nor functional changes in the digestive tract and was therefore regarded as non-adverse. However, lymphangiectasis might be related to the increased levels of cholesterol in plasma.

A tendency towards a dose related increase in mean T4 value in males was observed, with a 33% increase at 1000 mg/kg in comparison with the control value. Since the increase T4 values in high dose males still remained within the historical control range and no microscopic alterations were observed in the thyroid gland of these males the changes in T4 were considered of no toxicological relevance.

No treatment-related toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations and macroscopic examination).

The NOAEL for parental toxicity is 300 mg/kg bw/day

Reproductive results

No reproduction toxicity was observed up to 300 mg/kg.

At 1000 mg/kg, the fertility index was slightly lower (80%, compared to 100% for controls). There were no test item-related morphological findings in the reproductive organs of either sex which could explain the reproductive failure in 2/10 females at 1000 mg/kg. Such an incidence of non-pregnancy is occasionally observed also in control rats of this strain and age, and thus considered as an accidental finding in this study, but a relation with the developmental results cannot be ruled out (see below).

No treatment-related toxicologically significant changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating index, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

NOAEL for reproductive toxicity is 1000 mg/kg bw day

Developmental results

The individual difference between number of implantation sites and number of pups born (the so-called unaccounted for sites) was remarkably high in litters from females treated at 1000 mg/kg, which resulted in a low post-implantation survival index of 74% compared to the normal percentages in control animals ranging from 84%-100%. Whereas the unaccounted for sites in the (ten) control females varied between 0-2, there were four females with 0-1 and four females with 4 or more (up to 9) unaccounted for sites in the high dose group (at 1000 mg/kg). In addition, the total number pups were found dead or found missing from birth (including those found dead at first litter check) until PND 4 (day of culling) was significantly higher than in the control group, i.e. 9 versus 3. Two of the nine decedent pups were the complete litter of a high dose female, which appeared to have a normal number (11) of implantation sites. These two pups had exceptionally low body weights at first litter check and anticipated viability for life was low already at birth. 

When taking into account the lower litter size at 1000 mg/kg, a subtle lower mean body weight at first litter check of the pups in these litters was observed in comparison to controls. Although the growth of the pups in all groups was normal, the difference in body weights of the pups between the control and high dose group had slightly increased on PND 13-15. Another remarkable finding in pups at 1000 mg/kg was a relatively low male/female sex ratio of 38/62, compared to 50/50 in controls. This result might indicate a sex difference in sensitivity to toxic effects of the test item. If true, the increased number of unaccounted for sites noted at 1000 mg/kg might be the result of selective prenatal loss of male embryos/fetuses, but no definite conclusive could be drawn from the results obtained in this study. An endocrine mediated (antiandrogenic) effect being responsible for the skewed sex ratio, however, was considered unlikely, because there were no changes in any of the endocrine endpoints, i.e. nipple retention, anogenital distance, T4 levels in pups and thyroid gland morphology and reproductive organs in the dams at 1000 mg/kg.

The above results might indicate a toxic effect of the test item on the (prenatal) development of the offspring, at a dose level of 1000 mg/kg. In view of this conclusion, it cannot be ruled out that the infertility observed in two females of the same dose group (see reproductive results above) was also related to treatment. Although infertility is occasionally observed among female Wistar Han rats of this age[1], the occurrence of two incidences in the same (high dose) group might not be a coincidence in this case.

Notreatment-related changes were noted at 1000 mg/kg in the other developmental parameters investigated in this study (i.e. gestation, lactation index, duration of gestation, parturition, maternal care, live birth and lactation index and in the early postnatal pup development parameters, clinical signs, anogenital distance, areola/nipple retention (PND 13 males), serum concentration of thyroid hormone T4 (PND 13-15) and macroscopy.

A relatively low post-implantation survival index of 84% was observed at 100 mg/kg, whereas a percentage of 92% was observed at 300 mg/kg (which was comparable to the 94% seen in controls). In the absence of a clear dose response relationship and a relatively high number of unaccounted for implantation sites occurred in 2/10 litters only, the low post-implantation survival index at 100 mg/kg was considered a fortuitous finding and not related to treatment. The changes in all other developmental parameters in the 100 and 300 mg/kg groups were also considered not treatment-related.

NOAEL for developmental toxicity is 300 mg/kg bw/day