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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 July 2020 - 23 March 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 July 2020 - 23 March 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
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:
yes
Remarks:
The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the 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:
yes
Remarks:
The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.
Principles of method if other than guideline:
An OECD genotoxicity test was integrated into this toxicity study as follows:
- Principle of test: OECD TG474 Mammalian Erythrocyte Micronucleus test in vivo (2016)
- Short description of test conditions: Animals are exposed to the test substance by an appropriate route (gavage). Bone marrow is collected, prepared and stained. Each treated and control group must include at least 5 analysable animals per sex.
- Parameters analysed / observed: Preparations are analyzed for the presence of micronuclei, which detects damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts, by analysis of erythrocytes
The results of this test are detailed within Genetic Toxicity (section 7.6)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This study plan was reviewed and agreed within the framework of the project license, approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat - Crl: WI(Han) - 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 and micronucleus (bone marrow) 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 (if applicable) nulliparous and non-pregnant: not reported but may be assumed
- Age at study initiation: Males 11-12 weeks old; Females 13-14 weeks old
- Weight at study initiation: Females 191-243g
- Fasting period before study: not reported
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
Positive control males were group housed (up to 5 animals) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Makrolon 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.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room in which the animals were kept was documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): 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.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for 6 days prior to start of the pretest period.

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21˚C
- Humidity (%): 48-74%
- Air changes (per hr): 10 or more, with 100% fresh air (no recirculation)
- Photoperiod (hrs dark / hrs light): 12/12h

IN-LIFE DATES: From: 04 August 2020 To: 07 October 2020
Route of administration:
oral: gavage
Vehicle:
DMSO
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Dimethyl sulfoxide (DMSO). Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw data of these trials will be retained by the Test Facility.

FORMULATION PREPARATION
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 daily as a solution and dosed within 6 hours after adding the vehicle to the test item.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item
The dose volume for each animal was based on the most recent body weight measurement.

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as day 0 post-coitum.
- A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
- Further matings after unsuccessful attempt: no
- After successful mating each pregnant female was caged (how): individually housed in Makrolon plastic cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Within a separate study (test facility No. 20257162), development and validation of an analytical method for the quantitative analysis of Fatty acids, C18 (unsaturated), reaction products with diethylenetriamine in propylene glycol including trial formulation analysis to confirm the accuracy, homogeneity, stability and resuspension was undertaken.

The analytical method was validated for the analysis of Fatty acids, C18 (unsaturated), reaction products with diethylenetriamine in propylene glycol for the following parameters: i) Specificity, ii) Calibration curve, iii) Accuracy and Repeatability, iv) Limit of quantification, v) Stability of analytical system and end solutions.
Stability of stock solutions in methanol was demonstrated in Test Facility Study No. 491557 to be at least 4 days at room temperature. The stability of stock solutions was therefore not determined during this study.

Formulation Analysis: The concentrations analyzed in the formulations at 1 mg/mL and 200 mg/mL were in agreement with target concentrations (i.e. mean accuracies between 90 and 110%). It was observed that dissolving formulation samples in methanol took time.
The formulations were homogeneous (i.e. coefficient of variation ≤ 5%).
Formulations were stable when stored at room temperature under normal laboratory light conditions for at least 24 hours, in a refrigerator (2-8°C) for at least 8 days, and in a freezer (≤ -15°C) for at least 21 days (3 weeks).
Resuspension homogeneity was demonstrated (i.e. coefficient of variation ≤ 5%).

Within the main study, dose formulations samples were collected on Week 1 of treatment (day 1) and week 4 of treatment (day 22). The method development 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. Samples were tested from all groups for concentration, and from groups 2 & 4 (50mg/kg/day and 650mg/kg/day) for homogeneity.

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. The concentrations analyzed in the formulations of Groups 2, 3 and 4 (50, 150 & 650mg/kg/day, respectively) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulations (vehicle control).

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%. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).


Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals for a minimum of 28 days as follows: i) 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. ii) Females that delivered were treated for 50-64 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 days after delivery, up to and including the day before scheduled necropsy. iii) Females which failed to deliver were treated for 41-44 days.
Frequency of treatment:
Once by daily oral gavage 7 days a week
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Vehicle control DMSO)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2 (low)
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 3 (intermediate)
Dose / conc.:
650 mg/kg bw/day (nominal)
Remarks:
Group 4 (high)
No. of animals per sex per dose:
10 male, 10 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The oral route of administration selected because this is a possible route of human exposure during manufacture, handling or use of the test item. The dose levels were selected based on the results of a 10 Day Dose Range Finder with oral administration of N-Phenyl-diethanolamine, reaction products with formaldehyde in rats (see accompanying entry), and in an attempt to produce graded responses to the test item. Dose level spacing was selected in consultation with the Sponsor, and in agreement with OECD 422 testing requirements.

- Rationale for animal assignment (if not random): N/A
- Fasting period before blood sampling for clinical biochemistry: F0 males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0 females were not fasted overnight.
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A
- Other: N/A
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, morning & end of working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, 30 minutes ± 15 minutes post-dose.

BODY WEIGHT: Yes
- Time schedule for examinations: individually on the first day of treatment (prior to dosing), and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: N/A

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

OTHER:

PATHOLOGY:
Samples for clinical pathology evaluation were collected as per table No. 01

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day of necropsy between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane
- Animals fasted: F0 males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0 females were not fasted overnight.
- How many animals: 5/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day of necropsy between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane
- Animals fasted: F0 males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0 females were not fasted overnight.
- How many animals: 5/sex/group

PLASMA/SERUM HORMONES/LIPIDS: Yes - serum for T4 (thyroxine) & TSH (thyroid stimulating hormone)
- Time of blood sample collection: F0 - Day of necropsy between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane. F1 - pups/litter PND 4 (via decapitation) and PND 14-16 (by aorta puncture under anaesthesia using isoflurane as part of necropsy).
- Animals fasted: F0 males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0 females were not fasted overnight.
- How many animals: All F0 animals (Initial analysis only conducted for male animals). F1 2 pups/litter PND 4 and PND 14-16.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 5 males/dose during Week 4 of treatment and the 5 females/dose during the last week of lactation (i.e. PND 6-13). Tests were performed after dosing, after completion of clinical observations.
- Dose groups that were examined: All dose groups.
- Battery of functions tested: sensory activity (hearing ability, pupillary reflex) / grip strength / motor activity / static righting reflex

COAGULATION:
- Time schedule for collection of blood: Day of necropsy between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane
- Animals fasted: F0 males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0 females were not fasted overnight.
- How many animals: 5/sex/group
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in male parental generation: testis weight, epididymis weight, testes histopathology (progression of stages of the spermatogenic cycle, cell associations, and proportions expected to be present during spermatogenesis along with assessment of interstitial and supporting cell types (Leydig cells, macrophages, vasculature, and rete testis)).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, sex ratio of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, clinical observations (physical/behavioural), anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development; gross evaluation of external genitalia.

GROSS EXAMINATION OF DEAD PUPS:
Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally); possible cause of death was evaluated for pups born or found dead where possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

OTHER: Clinical Pathology
Samples for clinical pathology evaluation are detailed in table 01. On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup. If the target volume of 0.4 mL was not reached by pooling two pups, a third surplus pup of the same litter was added.
On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female, if possible). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anesthesia using isoflurane as part of the necropsy procedure.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were sacrificed following completion of the mating period (after a minimum of 28 days of administration).
- Maternal animals: All surviving animals were sacrificed at PND14-16, or post-coitum day 25-26 for females which failed to deliver.
- Table 02 outlines terminal procedures.

GROSS NECROPSY
All animals (excluding positive control animals) were subjected to a full post-mortem examination, with special attention being paid to the reproductive organs. Table 03 outlines macroscopic examinations undertaken.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.
The numbers of former implantation sites were recorded for all paired females.
In case no macroscopically visible implantation sites were present, non-gravid 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organs and tissues were weighed and prepared for histology as outlined in table 04, 05 and 06.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 14-16 days of age.

- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
The reviewer considers the analyses used (detailed in table 05) to be appropriate.
Reproductive indices:
Mating index; precoital time; fertility index; gestation index; duration of gestation.
Indices calculations are outlined within table 06.
Offspring viability indices:
Post-implantation survival index; live birth index; % live males/females at 1st litter check; viability index; lactation index.
Indices calculations are outlined within table 06.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 650 mg/kg/day, lethargy and flat posture were recorded for 8 out of 10 males and all females, and for each of these animals these signs occurred on a few days during treatment. Most of these animals also showed hunched posture, rales, piloerection, uncoordinated movements and/or ptosis on a few days during treatment.
At 150 mg/kg/day, flat posture was noted for one male and two females on treatment Days 12 and/or 13 only. One female at 150 mg/kg/day showed hunched posture, labored respiration, rales and/or piloerection during the last three days of treatment.
One female each at 50 and 150 mg/kg/day showed piloerection on two or three consecutive days during the 6th treatment week. As the incidence was similar or even less than encountered in the control group where two females showed piloerection (including hunched posture), these were considered not to be related to treatment with the test item.
Salivation seen after dosing among animals of the 50, 150 and 650 mg/kg/day dose groups with a dose-related trend during the treatment period 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 rather than a sign of systemic toxicity and is a common occurrence in oral gavage studies.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Clinical observations outlined in full within Appendix 1 (summary) and 2 (individual) in attached study results file.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight (gram) is summarised in table 09. Bodyweight gain is summarised in table 10. At 650 mg/kg/day, mean body weight gain of males was statistically significantly lower throughout the treatment period (mean weight gain at the end of the treatment period was 0.5x of control). Five of these males (Nos. 32, 33, 37, 38 and 39) showed a temporary and minimal weight loss during the first, second and/or third treatment week, ranging between 2 and 6% absolute weight loss over the respective period. Overall weight gain of most of these animals over the treatment period was also slightly lower with 6 to 7% vs 10 to 16% for the other animals within this group. Other male body weight variations at this dose level occurred within the range encountered for control animals.
At 650 mg/kg/day, mean body weight gain of females was lower throughout the lactation period (0.67x of control at the end of lactation; not statistically significant). In particular, Female (No. 79) showed no to minimal weight gain over the lactation period (1% over lactation Days 1 to 13). One other female at this dose level showed weight loss over lactation Days 1 to 7 (up to 13% of lactation Day 1 body weight).Overall, body weights and body weight gain at 50 and 150 mg/kg/day were considered not affected by treatment with the test item.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption is summarised in table 11, bodyweight consumption corrected for bodyweight is summarised in table 12. At 650 mg/kg/day, a lower food consumption (before and after correction for body weight) was recorded for males during the first three weeks of treatment (up to 0.85x of control). Overall mean food intake after correction for body weight was however similar to the control mean.
For females at 650 mg/kg/day, slightly lower mean food intake was only noted over Days 7 to 13 of lactation. This was ascribed to a reduced food intake of one female (No. 79). It is notable that this female only delivered two pups, and as a result presumably had a lower food consumption requirement. As this observation was isolated to one animal, the overall lowered group mean food intake was therefore considered not to be related to treatment with the test item.
Absolute and relative food consumption at 50 and 150 mg/kg/day was considered not affected by treatment with the test item.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematology is summarised in table 13. At 650 mg/kg/day, the following (statistically significant) changes in hematological parameters were recorded: i) Higher white blood cell counts (WBC) in males and females (1.46x and 1.34x of control, respectively); ii) Higher neutrophil counts (NEUT) in males and females (2.13x and 1.33x of control, respectively; not statistically significant for females); iii) Higher lymphocyte counts (LYMPH) in males and females (each 1.34x of control; not statistically significant for females); iv) Lower red blood cell counts (RBC) in males (0.94x of control); v) Higher reticulocyte counts (RETIC) in males (1.33x of control); vi) Lower hemoglobin (HGB) in males (0.94x of control); and vii) Lower mean corpuscular hemoglobin concentration (MCHC) in males (0.97x of control).
At 150 and 650 mg/kg/day, lower mean corpuscular hemoglobin (MCH) was recorded for females (each 0.95x of control), but in absence of correlating changes in red blood cell parameters or histopathological changes, and absence of a clear dose-related trend, this was considered not to be related to treatment with the test item.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical biochemistry is summarised in table 14. At 650 mg/kg/day, the following (statistically significant) changes in clinical biochemistry parameters were recorded: i) Higher total bilirubin (TBIL) in males (1.59x of control); ii) Higher urea in males (1.38x of control); iii) Higher bile acid (BILEAC) in males (2.22x of control, not statistically significant); iv) Lower total bilirubin (TBIL) in females (0.76x of control, not statistically significant).
At 50 and 150 mg/kg/day, clinical biochemistry parameters were considered not affected by treatment with the test item.
Endocrine findings:
no effects observed
Description (incidence and severity):
Serum levels of T4 in F0 males were considered unaffected by treatment with the test item up to 650 mg/kg/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observations are summarised in table 15. At 650 mg/kg/day, mean ambulation counts of males appeared higher (1.28x of control, not statistically significant). This was ascribed to higher activity of two out of five males (Nos. 31 and 33).
For females at 50 and 650 mg/kg/day, mean total movements and ambulations appeared lower than control means (not statistically significant). However, control means for both parameters exceeded the historical control range while means of these parameters at 50 and
650 mg/kg/day were well within this range. In addition, the expected decreasing trend in motor activity over the duration of the test period (habituation profile) of control females was less apparent, in particular for total movements. It could not be ascertained what factors may have attributed to this. Motor activity habituation profile of control males followed the expected pattern. In absence of a dose-related trend, these variations over the dose groups were considered not to be related to treatment with the test item.
At 50 and 150 mg/kg/day, motor activity was considered not to be affected by treatment with the test item. All male groups (including the 650 mg/kg/day group) showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Hearing ability, pupillary reflex, static righting reflex were normal in all examined animals up to 650 mg/kg/day. Grip strength was considered not affected by treatment with the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A summary of relevant histopathological (microscopic) findings is in tables 18 & 19. Test item-related microscopic findings after treatment with the test item were noted in the stomach and spleen of males and females and in kidneys and liver of males. Stomach-forestomach: Ulcerative inflammation (up to massive degree) and transmural necrosis (slight or marked degree) was observed in the forestomach of males and females at 650 mg/kg/day. The ulcers were large and were accompanied by inflammation (mostly in the submucosa and/or at the serosal side) and large necrotic areas throughout all layers of the forestomach (transmural).

Stomach-glandular stomach: In the glandular stomach of two males and a single female at 650 mg/kg/day, an ulcer or erosion (slight degree) was observed and submucosal inflammation was noted to be slightly above background (minimal inflammation is considered background). In a few males (a single male at 50 mg/kg/day and three males at 650 mg/kg/day) hemorrhages were observed which correlated at 650 mg/kg/day with reddish focus/foci in the glandular stomach.

Spleen: A dose-dependent increase in incidence and severity in extramedullary hematopoiesis was observed in males starting at 50 mg/kg/day, up to slight degree. Pigmentation was increased in severity in males and females treated at 650 mg/kg/day, up to slight degree. Liver: A dose-dependent increased incidence of minimal hepatocellular centrilobular hypertrophy was observed in males starting at 150 mg/kg/day.

Kidney: A combination of tubular findings was observed in kidneys of males treated at 150 and 650 mg/kg/day consisting of increased incidence and severity of tubular basophilia up to marked degree, presence of granular casts up to slight degree and degeneration of tubular epithelial cells up to moderate degree.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.
During premating, all females had regular cycles of 4 or 5 days. During pretest, estrous cycle regularity could not be determined for one control female (No. 49) and for one female at 50 mg/kg/day (No. 51). Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these occurrences did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
Reproductive performance is summarised in table 20. No effects were observed in any relevant indices. Mating Index: not affected by treatment with the test item. All females showed evidence of mating, resulting in a mating index of 100% for all dose groups.
Precoital Time: not affected by treatment with the test item. Most females showed evidence of mating within 4 to 5 days, except for one control female (No. 48) which showed evidence of mating after 13 days. As this concerned a control animal, this was unrelated to treatment with the test item.
Number of Implantation Sites: not affected by treatment with the test item. At 650 mg/kg/day, the number of implantations was slightly lower than the control mean (10.3 at 650 mg/kg/day vs 11.8 in the control group (not statistically significant), and 11.8 and 11.6 implantation sites at 50 and 150 mg/kg/day, respectively). This was attributed to a low number of implantations for Female No. 79 (2 implantations). The mean number of implantation sites without the value for Female No. 79 was 11.4, i.e. comparable to the control mean; based on this no clear dose-related reduction in number of implantations was apparent. Therefore, it was considered that this single case of a lower number of implantation sites did not represent a test item-related effect. One control female (No. 50) had a single implantation site only (without offspring). As this occurred in the control group, this was unrelated to treatment with the test item.
Fertility Index: not affected by treatment with the test item. The fertility indices were 100% for the control, 50 and 150 mg/kg/day groups, and 90% for the 650 mg/kg/day group. One female at 650 mg/kg/day (No. 80) was not pregnant. Since all other females of this dose group were pregnant and in the absence of any supportive reproductive/developmental toxicity, this incidental case was considered not to be related to treatment with the test item.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
presumably yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment with the test item.
The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
One pup at 50 mg/kg/day, two pups at 150 mg/kg/day and one pup at 650 mg/kg/day 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. Table 20 details live birth index.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment with the test item.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment with the test item up to 650 mg/kg/day had no effect on areola/nipple retention. No nipples were observed for any of the examined male pups at PND 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 650 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
650 mg/kg bw/day (nominal)
Treatment related:
no

The following section includes all tables considered to be relevant to the study summary. Further raw data tables are provided as attached background material.

 

Table 09: Summary bodyweights (gram) 

BODY WEIGHTS MALES

(GRAM) SUMMARY

 

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

PRE MATING

 

 

 

 

 

 

DAY 1

MEAN

312

330 *

325

318

WEEK 1

ST.DEV

14.6

11.5

17.4

8.1

 

N

10

10

10

10

DAY 8

MEAN

338

341

340

321 *

WEEK 2

ST.DEV

11.6

8.6

18.7

12.3

 

N

10

10

10

10

MATING PERIOD

 

 

 

 

 

 

DAY 1

MEAN

359

364

361

339 *

WEEK 1

ST.DEV

12.8

12.5

21.6

17.0

 

N

10

10

10

10

DAY 8

MEAN

369

373

373

344 *

WEEK 2

ST.DEV

11.7

15.0

25.9

19.0

 

N

10

10

10

10

DAY 15

MEAN

381

383

383

354 **

WEEK 3

ST.DEV

12.9

15.5

27.4

15.6

 

N

10

10

10

10

 

BODY WEIGHTS FEMALES

(GRAM) SUMMARY

 

 

 

 

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

PRE MATING

 

 

 

 

 

 

 

DAY 1

 

MEAN

 

220

 

217

 

216

 

225

WEEK 1

ST.DEV

9.6

13.6

12.4

13.4

 

N

10

10

10

10

DAY 8

MEAN

221

216

220

227

WEEK 2

ST.DEV

8.3

14.6

10.1

12.5

 

N

10

10

10

10

MATING PERIOD

 

 

 

 

 

 

 

DAY 1

 

MEAN

 

227

 

225

 

227

 

231

WEEK 1

ST.DEV

10.4

13.2

8.0

12.9

 

N

10

10

10

10

DAY 8

WEEK 2

MEAN

247

--- 1

 

0 x

 

0 x

 

ST.DEV

N

DAY 15

WEEK 3

MEAN

 

---

---

0 x

---

---

0 x

 

ST.DEV

 

DAY 22

WEEK 4

MEAN

 

---

---

0 x

---

---

0 x

 

ST.DEV

N

FEMALES F0-GENERATION

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG/DAY

GROUP 3

150

MG/KG/DAY

GROUP 4

650

MG/KG/DAY

POST COITUM

 

 

 

 

 

 

DAY 0

MEAN

232

227

223

231

 

ST.DEV.

10.8

16.8

7.0

12.9

 

N

9

9

9

9

DAY 4

MEAN

244

238

239

243

 

ST.DEV.

8.1

17.2

7.7

12.4

 

N

9

9

9

9

DAY 7

MEAN

249

245

247

250

 

ST.DEV.

8.5

17.7

8.4

11.1

 

N

9

9

9

9

DAY 11

MEAN

263

258

263

264

 

ST.DEV.

6.2

21.3

10.1

12.5

 

N

9

9

9

9

DAY 14

MEAN

273

270

274

272

 

ST.DEV.

8.5

19.7

13.1

13.6

 

N

9

9

9

9

DAY 17

MEAN

298

293

297

294

 

ST.DEV.

7.5

20.5

15.7

19.3

 

N

9

9

9

9

DAY 20

MEAN

337

329

337

328

 

ST.DEV.

8.3

21.5

24.3

31.0

 

N

9

9

9

9

LACTATION

 

 

 

 

 

 

DAY 1

MEAN

262

254

263

266

 

ST.DEV.

8.0

21.3

14.7

12.1

 

N

7

10

10

9

DAY 4

MEAN

277

267

276

272

 

ST.DEV.

5.3

21.3

14.0

12.8

 

N

9

10

10

9

DAY 7

MEAN

278

275

285

274

 

ST.DEV.

14.8

19.5

15.0

13.5

 

N

8

10

10

8

DAY 13

MEAN

302

295

297

293

 

ST.DEV.

7.8

26.6

20.0

16.3

 

N

9

10

10

9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values (see attached additional study reporting)

Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post‑coitum and lactation phase.

 

Table 10: Bodyweight gain (%) summary

BODY WEIGHT GAIN (%) SUMMARY MALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

PRE MATING

DAY 1

 

MEAN

 

0

 

0

 

0

 

0

WEEK 1

ST.DEV

0.0

0.0

0.0

0.0

 

N

10

10

10

10

DAY 8

MEAN

8

3 **

5 **

1 **

WEEK 2

ST.DEV

2.7

2.0

1.5

2.6

 

N

10

10

10

10

 

MATING PERIOD

DAY 1

 

MEAN

 

15

 

10 **

 

11 **

 

6 **

WEEK 1

ST.DEV

2.7

2.2

1.2

4.0

 

N

10

10

10

10

DAY 8

MEAN

18

13 **

15

8 **

WEEK 2

ST.DEV

3.4

3.2

2.3

5.0

 

N

10

10

10

10

DAY 15

MEAN

22

16 **

18 *

11 **

WEEK 3

ST.DEV

4.1

3.3

2.7

3.6

 

N

10

10

10

10

 

BODY WEIGHT GAIN (%)SUMMARY FEMALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

PRE MATING

DAY 1

 

MEAN

 

0

 

0

 

0

 

0

WEEK 1

ST.DEV

0.0

0.0

0.0

0.0

 

N

10

10

10

10

DAY 8

MEAN

1

0

2

1

WEEK 2

ST.DEV

3.3

1.5

3.6

2.5

 

N

10

10

10

10

 

MATING PERIOD

DAY 1

 

MEAN

 

4

 

4

 

5

 

3

WEEK 1

ST.DEV

3.4

2.9

4.1

3.4

 

N

10

10

10

10

DAY 8

WEEK 2

MEAN ST.DEV N

13

--- 1

---

---

0 x

---

---

0 x

 

DAY 15

WEEK 3

MEAN ST.DEV N

 

---

---

0 x

---

---

0 x

 

DAY 22

WEEK 4

MEAN ST.DEV N

 

---

---

0 x

---

---

0 x

 

BODY WEIGHT GAIN (%)SUMMARY FEMALES

F0-GENERATION

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG/DAY

GROUP 3

150

MG/KG/DAY

GROUP 4

650

MG/KG/DAY

 

POST COITUM

DAY 0

 

MEAN

 

0

 

0

 

0

 

0

 

ST.DEV.

0.0

0.0

0.0

0.0

 

N

9

9

9

9

DAY 4

MEAN

5

5

7

5

 

ST.DEV.

2.8

1.6

1.9

1.9

 

N

9

9

9

9

DAY 7

MEAN

7

8

11 **

8

 

ST.DEV.

3.2

1.5

2.7

1.7

 

N

9

9

9

9

DAY 11

MEAN

14

13

18 *

14

 

ST.DEV.

3.9

2.7

2.9

2.5

 

N

9

9

9

9

DAY 14

MEAN

18

19

23 **

18

 

ST.DEV.

2.9

2.4

4.0

3.2

 

N

9

9

9

9

DAY 17

MEAN

28

29

33

27

 

ST.DEV.

5.2

2.9

5.3

5.1

 

N

9

9

9

9

DAY 20

MEAN

45

45

51

42

 

ST.DEV.

7.5

5.1

8.3

9.5

 

N

9

9

9

9

 

LACTATION

DAY 1

 

MEAN

 

0

 

0

 

0

 

0

 

ST.DEV.

0.0

0.0

0.0

0.0

 

N

7

10

10

9

DAY 4

MEAN

5

5

5

2

 

ST.DEV.

2.9

5.4

2.9

3.7

 

N

7

10

10

9

DAY 7

MEAN

5

9

8

3

 

ST.DEV.

6.7

6.4

3.4

7.5

 

N

6

10

10

8

DAY 13

MEAN

15

16

13

10

 

ST.DEV.

5.1

7.6

5.7

5.2

 

N

7

10

10

9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values (see attached additional study reporting)

Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post‑coitum and lactation phase

 

Table 11: Absolute Food consumption (g/animal/day) summary

2.    FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY

3.    MALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

PRE MATING

DAYS 1-8

 

MEAN

 

25

 

25

 

25

 

22

WEEKS 1-2

ST.DEV

0.2

0.5

0.5

0.6

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

24

24

24

22

WEEKS 2-3

ST.DEV

0.0

0.0

1.4

1.5

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER PRE MATI...

 

MEAN

 

24

 

24

 

25

 

22

MATING PERIOD

DAYS 1-8

 

MEAN

 

26

 

27

 

26

 

22

WEEKS 1-2

ST.DEV

1.5

1.9

1.9

1.1

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

26

25

26

25

WEEKS 2-3

ST.DEV

0.1

0.7

1.0

0.5

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER MATING P...

 

MEAN

 

26

 

26

 

26

 

24

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY

FEMALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

PRE MATING

DAYS 1-8

 

MEAN

 

17

 

17

 

18

 

17

WEEKS 1-2

ST.DEV

0.6

0.1

0.2

2.3

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

17

16

17

17

WEEKS 2-3

ST.DEV

0.2

0.4

0.1

1.2

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER PRE MATI...

 

MEAN

 

17

 

17

 

17

 

17

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY FEMALES

F0-GENERATION

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG/DAY

GROUP 3

150

MG/KG/DAY

GROUP 4

650

MG/KG/DAY

 

POST COITUM

DAYS 0-4

 

MEAN

 

18

 

18

 

20

 

19

 

ST.DEV.

1.4

3.1

2.0

1.9

 

N

9

9

9

9

DAYS 4-7

MEAN

21

21

22

21

 

ST.DEV.

1.5

2.8

2.3

1.5

 

N

9

9

9

9

DAYS 7-11

MEAN

22

22

24

22

 

ST.DEV.

1.6

3.4

2.9

1.9

 

N

9

9

9

9

DAYS 11-14

MEAN

23

22

24

23

 

ST.DEV.

2.2

2.5

2.7

1.1

 

N

9

9

9

9

DAYS 14-17

MEAN

23

22

24

23

 

ST.DEV.

1.9

2.9

2.4

2.4

 

N

9

9

9

9

DAYS 17-20

MEAN

24

24

25

25

 

ST.DEV.

1.5

2.7

2.9

1.7

 

N

9

9

9

9

 

MEAN OF MEANS

 

 

22

 

22

 

23

 

22

LACTATION

DAYS 1-4

 

MEAN

 

32

 

30

 

32

 

32

 

ST.DEV.

3.2

4.4

5.2

9.4

 

N

9

10

10

9

DAYS 4-7

MEAN

47

42

43

40

 

ST.DEV.

9.1

5.4

4.5

9.6

 

N

9

10

10

8

DAYS 7-13

MEAN

54

55

53

47

 

ST.DEV.

6.5

7.4

6.3

9.6

 

N

9

10

10

9

 

MEAN OF MEANS

 

 

44

 

42

 

43

 

40

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values (see attached additional study reporting)

Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post‑coitum and lactation phase

 

 

 

Table 12: Relative Food consumption (g/animal/day) summary

RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY)SUMMARY MALES

 

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

PRE MATING

DAYS 1-8

 

MEAN

 

73

 

72

 

74

 

68

WEEKS 1-2

ST.DEV

0.3

2.5

0.4

0.7

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

71

71

72

70

WEEKS 2-3

ST.DEV

0.2

0.9

2.5

3.2

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER PRE MATI...

 

MEAN

 

72

 

72

 

73

 

69

MATING PERIOD

DAYS 1-8

 

MEAN

 

70

 

73

 

71

 

65

WEEKS 1-2

ST.DEV

4.7

6.9

1.7

1.1

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

67

65

69

69

WEEKS 2-3

ST.DEV

1.3

0.6

0.7

0.5

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER MATING PERIOD

 

MEAN

 

69

 

69

 

70

 

67

RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY)

FEMALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

PRE MATING

DAYS 1-8

 

MEAN

 

77

 

77

 

80

 

73

WEEKS 1-2

ST.DEV

3.1

0.9

0.0

9.1

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

75

76

78

75

WEEKS 2-3

ST.DEV

1.3

2.1

1.2

4.2

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER PRE-MATING PERIOD

 

MEAN

 

76

 

77

 

79

 

74

RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY)

F0-GENERATION

 

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG/DAY

GROUP 3

150

MG/KG/DAY

GROUP 4

650

MG/KG/DAY

 

 

POST COITUM

DAYS 0-4

 

MEAN

 

74

 

77

 

83

 

77

 

 

ST.DEV.

7.0

9.1

6.9

7.4

 

 

N

9

9

9

9

 

DAYS 4-7

MEAN

84

85

89

86

 

 

ST.DEV.

6.8

8.1

7.3

6.3

 

 

N

9

9

9

9

 

DAYS 7-11

MEAN

83

84

89

85

 

 

ST.DEV.

7.1

9.2

8.2

6.7

 

 

N

9

9

9

9

 

DAYS 11-14

MEAN

83

82

89

86

 

 

ST.DEV.

8.8

7.4

7.5

4.8

 

 

N

9

9

9

9

 

DAYS 14-17

MEAN

77

76

81

78

 

 

ST.DEV.

6.7

5.6

5.3

7.2

 

 

N

9

9

9

9

 

DAYS 17-20

MEAN

73

73

76

77

 

 

ST.DEV.

3.7

6.6

5.5

5.7

 

 

N

9

9

9

9

 

 

MEAN OF MEANS

 

 

79

 

80

 

84

 

81

 

RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY)

LACTATION

 

DAYS 1-4

 

MEAN

 

115

 

111

 

116

 

116

 

 

ST.DEV.

12.4

12.4

15.6

35.5

 

 

N

9

10

10

9

 

DAYS 4-7

MEAN

164

152

150

144

 

 

ST.DEV.

38.8

15.6

13.2

38.7

 

 

N

8

10

10

7

 

DAYS 7-13

MEAN

179

187

180

161

 

 

ST.DEV.

20.6

17.0

16.5

27.9

 

 

N

9

10

10

9

 

 

MEAN OF MEANS

 

 

153

 

150

 

149

 

140

 

                       

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values (see attached additional study reporting)

Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post‑coitum and lactation phase

 

Table 13: Summary of hematology values: F0 Generation

MALES: day 30 relative to start

Sex: Male

Reporting Hematology

WBC (10^9/L)

[G]

NEUT (10^9/L)

[G]

LYMPH (10^9/L)

[G1]

MONO (10^9/L)

[G]

EOS (10^9/L)

[G]

BASO (10^9/L)

[G]

LUC (10^9/L)

[G1]

RBC (10^12/L)

[G]

RETIC (10^9/L)

[G]

RDWG (%)

[G]

HGB

 

(g/L)

 

[G]

HCT (L/L)

[G]

0

Mean

6.302

0.946

5.148

0.100

0.068

0.014

0.030

8.780

231.88

13.10

155.0

0.4572

mg/kg/day

SD

0.511

0.264

0.290

0.040

0.013

0.005

0.010

0.278

21.44

0.23

3.0

0.0101

Group 1

N

5

5

5

5

5

5

5

5

5

5

5

5

50

Mean

6.482

0.820

5.468

0.090

0.056

0.010

0.032

8.836

202.46

12.94

156.0

0.4580

mg/kg/day

SD

1.672

0.137

1.709

0.032

0.009

0.000

0.027

0.281

32.66

0.72

3.7

0.0087

Group 2

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

1.03

0.87

1.06

0.90

0.82

0.71

1.07

1.01

0.87

0.99

1.01

1.00

150

Mean

7.796

1.342

6.230

0.092

0.086

0.012

0.028

8.652

246.02

13.06

151.8

0.4504

mg/kg/day

SD

1.224

0.390

1.487

0.026

0.030

0.004

0.008

0.372

26.96

0.27

5.9

0.0197

Group 3

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

1.24

1.42

1.21

0.92

1.26

0.86

0.93

0.99

1.06

1.00

0.98

0.99

650

Mean

9.230 *

2.014 *

6.904

0.142

0.104

0.020

0.048

8.266 *

307.64 **

13.14

146.4 *

0.4450

mg/kg/day

SD

2.107

0.870

1.215

0.048

0.067

0.007

0.011

0.303

48.43

0.87

5.2

0.0184

Group 4

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

1.46

2.13

1.34

1.42

1.53

1.43

1.60

0.94

1.33

1.00

0.94

0.97

 

Sex: Male

Reporting Hematology

MCV (fL)

[G]

MCH

 

(pg)

 

[G]

MCHC

 

(g/L)

 

[G]

PLT (10^9/L)

[G1]

0

Mean

52.10

17.68

338.8

725.8

mg/kg/day

SD

1.70

0.44

3.0

90.5

Group 1

N

5

5

5

5

50

Mean

51.86

17.64

340.8

754.4

mg/kg/day

SD

1.17

0.59

4.9

68.4

Group 2

N

5

5

5

5

 

tCtrl

1.00

1.00

1.01

1.04

150

Mean

52.08

17.54

336.8

757.0

mg/kg/day

SD

0.98

0.15

4.1

27.6

Group 3

N

5

5

5

5

 

tCtrl

1.00

0.99

0.99

1.04

650

Mean

53.84

17.76

329.4 **

869.4

mg/kg/day

SD

1.11

0.45

3.8

121.1

Group 4

N

5

5

5

5

 

tCtrl

1.03

1.00

0.97

1.20

[G] - Anova & Dunnett: * = p ≤ 0.05

[G1] - Kruskal-Wallis & Dunn

 

FEMALES: Day 51 relative to start date

Sex: Female

Reporting Hematology

WBC (10^9/L)

[G]

NEUT (10^9/L)

[G]

LYMPH (10^9/L)

[G]

MONO (10^9/L)

[G]

EOS (10^9/L)

[G]

BASO (10^9/L)

[G]

LUC (10^9/L)

[G]

RBC (10^12/L)

[G]

RETIC (10^9/L)

[G]

RDWG (%)

[G]

HGB

 

(g/L)

 

[G]

HCT(L/L)

[G1]

0

Mean

5.062

1.598

3.278

0.092

0.072

0.008

0.020

6.824

261.04

13.02

140.2

0.4076

mg/kg/day

SD

0.749

0.317

0.518

0.025

0.026

0.004

0.000

0.117

47.87

0.44

3.2

0.0228

Group 1

N

5

5

5

5

5

5

5

5

5

5

5

5

50

Mean

5.536

1.662

3.594

0.124

0.130

0.004

0.020

6.936

238.30

12.90

136.2

0.3978

mg/kg/day

SD

0.901

0.264

0.810

0.040

0.053

0.005

0.022

0.289

37.34

1.02

1.8

0.0090

Group 2

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

1.09

1.04

1.10

1.35

1.81

0.50

1.00

1.02

0.91

0.99

0.97

0.98

150

Mean

4.770

1.262

3.314

0.096

0.072

0.004

0.024

6.958

210.54

12.72

135.6

0.4012

mg/kg/day

SD

0.780

0.457

0.579

0.027

0.029

0.005

0.026

0.245

59.83

1.02

3.6

0.0080

Group 3

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

0.94

0.79

1.01

1.04

1.00

0.50

1.20

1.02

0.81

0.98

0.97

0.98

650

Mean

6.804 *

2.118

4.382

0.154

0.106

0.012

0.028

7.012

248.92

13.56

136.2

0.3986

mg/kg/day

SD

1.393

0.958

0.636

0.072

0.069

0.004

0.015

0.356

53.85

1.06

1.5

0.0082

Group 4

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

1.34

1.33

1.34

1.67

1.47

1.50

1.40

1.03

0.95

1.04

0.97

0.98

 

Sex: Female

Reporting Hematology

MCV (fL)

[G]

MCH

 

(pg)

 

[G]

MCHC

 

(g/L)

 

[G]

PLT (10^9/L)

[G]

0

Mean

59.74

20.54

344.6

703.2

mg/kg/day

SD

3.39

0.42

13.5

72.5

Group 1

N

5

5

5

5

50

Mean

57.40

19.66

342.6

725.2

mg/kg/day

SD

2.11

0.70

5.7

75.1

Group 2

N

5

5

5

5

 

tCtrl

0.96

0.96

0.99

1.03

150

Mean

57.72

19.50 *

337.8

717.0

mg/kg/day

SD

2.03

0.47

4.9

128.4

Group 3

N

5

5

5

5

 

tCtrl

0.97

0.95

0.98

1.02

650

Mean

56.90

19.44 *

341.6

737.8

mg/kg/day

SD

2.36

0.84

4.6

108.5

Group 4

N

5

5

5

5

 

tCtrl

0.95

0.95

0.99

1.05

 

[G] - Anova & Dunnett: * = p ≤ 0.05

[G1] - Kruskal-Wallis & Dunn

 

 

Table 14: Summary of clinical chemistry values: F0 Generation

MALES: day 30 relative to start

Sex: Male

Reporting Biochemistry

ALT (U/L)

[G]

AST (U/L)

[G]

ALP (U/L)

[G]

TPROT

 

(g/L)

 

[G]

ALB

 

(g/L)

 

[G]

BILEAC

(umol/L) [G1]

TBIL

(umol/L) [G]

UREA

(mmol/L) [G]

CREAT

(umol/L) [G]

GLUC

(mmol/L) [G]

CHOL

(mmol/L) [G1]

NA

(mmol/L) [G]

0

Mean

53.7

87.8

94.9

61.64

37.80

21.52

2.36

5.83

27.7

8.704

2.118

143.4

mg/kg/day

SD

6.5

10.5

14.1

1.63

1.05

17.56

0.33

0.74

3.6

0.621

0.249

0.5

Group 1

N

5

5

5

5

5

5

5

5

5

5

5

5

50

Mean

47.2

81.1

106.2

62.06

38.22

10.98

2.30

6.08

26.5

8.460

1.860

143.4

mg/kg/day

SD

5.7

3.3

25.4

1.69

0.82

4.21

0.22

0.98

2.6

1.376

0.154

0.5

Group 2

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

0.88

0.92

1.12

1.01

1.01

0.51

0.97

1.04

0.96

0.97

0.88

1.00

150

Mean

58.8

89.3

100.7

60.60

37.36

37.91

2.92

7.09

26.6

9.936

1.924

142.2

mg/kg/day

SD

15.3

12.8

4.6

2.43

1.35

22.33

0.40

1.20

2.9

2.316

0.494

1.1

Group 3

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

1.09

1.02

1.06

0.98

0.99

1.76

1.24

1.22

0.96

1.14

0.91

0.99

650

Mean

61.5

86.1

83.3

60.58

37.72

47.67

3.76 **

8.06 *

28.4

9.516

2.448

142.0

mg/kg/day

SD

10.9

7.4

13.8

1.36

1.19

22.84

0.51

1.27

3.5

1.792

0.541

1.6

Group 4

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

1.15

0.98

0.88

0.98

1.00

2.22

1.59

1.38

1.03

1.09

1.16

0.99

 

Sex: Male

Reporting Biochemistry

Reporting S

K

(mmol/L) [G]

CL

(mmol/L) [G]

CA

(mmol/L) [G]

PHOS

(mmol/L) [G]

T4

(ng/mL) [G]

0

Mean

3.91

106.2

2.608

1.958

38.60

mg/kg/day

SD

0.31

1.3

0.072

0.135

6.99

Group 1

N

5

5

5

5

10

50

Mean

4.04

105.8

2.646

1.840

39.68

mg/kg/day

SD

0.12

0.8

0.076

0.222

5.79

Group 2

N

5

5

5

5

10

 

tCtrl

1.03

1.00

1.01

0.94

1.03

150

Mean

3.83

105.0

2.584

1.872

44.89

mg/kg/day

SD

0.14

2.0

0.103

0.120

11.30

Group 3

N

5

5

5

5

10

 

tCtrl

0.98

0.99

0.99

0.96

1.16

650

Mean

4.19

105.4

2.588

1.852

34.52

mg/kg/day

SD

0.15

1.3

0.095

0.194

4.50

Group 4

N

5

5

5

5

10

 

tCtrl

1.07

0.99

0.99

0.95

0.89

[G] - Anova & Dunnett: * = p ≤ 0.05

[G1] - Kruskal-Wallis & Dunn

 

FEMALES: Day 51 relative to start date

Sex: Female

Reporting Biochemistry

ALT (U/L)

[G]

AST (U/L)

[G]

ALP (U/L)

[G]

TPROT

 

(g/L)

 

[G]

ALB

 

(g/L)

 

[G]

BILEAC

(umol/L) [G]

TBIL

(umol/L) [G]

UREA

(mmol/L) [G]

CREAT

(umol/L) [G]

GLUC

(mmol/L) [G]

CHOL

(mmol/L) [G]

NA

(mmol/L) [G]

0

Mean

129.9

95.7

253.3

56.74

34.96

11.35

2.30

9.72

22.4

7.384

2.134

141.0

mg/kg/day

SD

15.6

5.7

93.1

1.27

1.08

3.20

0.89

0.65

2.7

1.628

0.310

1.2

Group 1

N

5

5

5

5

5

5

5

5

5

5

5

5

50

Mean

138.0

98.5

197.9

54.60

33.74

11.96

2.52

9.69

21.0

8.904

2.160

140.2

mg/kg/day

SD

25.7

7.8

90.2

2.04

0.76

4.30

0.77

0.17

2.2

0.876

0.249

1.3

Group 2

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

1.06

1.03

0.78

0.96

0.97

1.05

1.10

1.00

0.94

1.21

1.01

0.99

150

Mean

122.4

97.2

231.3

55.10

33.98

8.16

2.14

9.72

24.8

8.160

1.866

140.8

mg/kg/day

SD

20.8

11.3

107.9

2.90

1.98

2.61

0.46

0.93

4.1

1.484

0.418

1.3

Group 3

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

0.94

1.02

0.91

0.97

0.97

0.72

0.93

1.00

1.11

1.11

0.87

1.00

650

Mean

120.9

96.2

234.0

55.60

33.80

11.06

1.74

9.13

21.1

8.692

2.230

141.2

mg/kg/day

SD

21.2

14.6

120.3

2.71

1.48

2.02

0.44

0.81

3.9

0.741

0.197

1.1

Group 4

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

0.93

1.00

0.92

0.98

0.97

0.97

0.76

0.94

0.94

1.18

1.04

1.00

 

Sex: Female

Reporting Biochemistry

K

(mmol/L) [G]

CL

(mmol/L) [G]

CA

(mmol/L) [G]

PHOS

(mmol/L) [G1]

0

Mean

4.54

105.8

2.442

1.270

mg/kg/day

SD

0.19

1.3

0.054

0.491

Group 1

N

5

5

5

5

50

Mean

4.91

106.0

2.450

1.080

mg/kg/day

SD

0.51

2.1

0.060

0.305

Group 2

N

5

5

5

5

 

tCtrl

1.08

1.00

1.00

0.85

150

Mean

4.85

106.4

2.456

1.534

mg/kg/day

SD

0.31

1.5

0.076

0.309

Group 3

N

5

5

5

5

 

tCtrl

1.07

1.01

1.01

1.21

650

Mean

4.72

105.8

2.504

1.496

mg/kg/day

SD

0.54

0.4

0.053

0.138

Group 4

N

5

5

5

5

 

tCtrl

1.04

1.00

1.03

1.18

[G] - Anova & Dunnett: * = p ≤ 0.05

[G1] - Kruskal-Wallis & Dunn

 

 

 

Table 15: Functional Observations & Motor Activity Summary

FUNCTIONAL OBSERVATIONS SUMMARY MALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

END OF TREATMENT

HEARING

 

MEDIAN

 

0

 

0

 

0

 

0

SCORE 0/1

N

5

5

5

5

PUPIL L

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

PUPIL R

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

STATIC R

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

GRIP FORE

MEAN

1249

1149

1201

1112

GRAM

ST.DEV

287

258

115

91

 

N

5

5

5

5

GRIP HIND

MEAN

560

534

537

492

GRAM

ST.DEV

108

64

68

89

 

N

5

5

5

5

FUNCTIONAL OBSERVATIONS SUMMARY FEMALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

END OF TREATMENT

HEARING

 

MEDIAN

 

0

 

0

 

0

 

0

SCORE 0/1

N

5

5

5

5

PUPIL L

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

PUPIL R

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

STATIC R

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

GRIP FORE

MEAN

959

787

889

895

GRAM

ST.DEV

181

93

219

171

 

N

5

5

5

5

GRIP HIND

MEAN

330

312

317

341

GRAM

ST.DEV

49

61

54

57

 

N

5

5

5

5

 

MOTOR ACTIVITY SUMMARY MALES

Total

Movements

GROUP 1

CONTROL

 

50 MG/KG/DAY

GROUP 3

150 MG/KG/DAY

GROUP 4 650 MG/KG/DAY

MEAN

3185

3370

2753

3155

N

5

5

5

5

STDEV

850

594

492

1153

* indicates a p-value <0.05, ** indicates a p-value <0.01

MEAN and STDEV values are calculated per group, from each animal's total Total Movements over all intervals

 

MOTOR ACTIVITY SUMMARY MALES

Ambulations

GROUP 1

CONTROL

 

50 MG/KG/DAY

GROUP 3

150 MG/KG/DAY

GROUP 4650MG/KG/DAY

MEAN

570

617

469

731

N

5

5

5

5

STDEV

158

158

141

450

* indicates a p-value <0.05, ** indicates a p-value <0.01

MEAN and STDEV values are calculated per group, from each animal's total Ambulations over all intervals

MOTOR ACTIVITY SUMMARY FEMALES

Total

Movements

GROUP 1

CONTROL

 

50 MG/KG/DAY

GROUP 3

150 MG/KG/DAY

GROUP 4 650 MG/KG/DAY

MEAN

5712

4124

4755

3559

N

5

5

5

5

STDEV

1708

979

1492

1435

* indicates a p-value <0.05, ** indicates a p-value <0.01

MEAN and STDEV values are calculated per group, from each animal's total Total Movements over all intervals

 

MOTOR ACTIVITY SUMMARY FEMALES

Ambulations

GROUP 1

CONTROL

 

50 MG/KG/DAY

GROUP 3

150 MG/KG/DAY

GROUP 4650MG/KG/DAY

MEAN

1459

969

1307

817

N

5

5

5

5

STDEV

408

302

618

330

* indicates a p-value <0.05, ** indicates a p-value <0.01

MEAN and STDEV values are calculated per group, from each animal's total Ambulations over all intervals

 

Table 16: Absolute Organ Weights (gram) - Summary of selected findings

ORGAN WEIGHTS (GRAM) SUMMARY MALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

END OF TREATMENT

BODY W.

 

MEAN

 

356

 

359

 

357

 

330 **

(GRAM)

ST.DEV

13

14

26

17

 

N

10

10

10

10

BRAIN

MEAN

2.07

2.06

2.07

2.11

(GRAM)

ST.DEV

0.07

0.04

0.09

0.09

 

N

5

5

5

5

LIVER

MEAN

8.38

8.37

8.41

9.46 *

(GRAM)

ST.DEV

0.36

0.84

0.56

0.47

 

N

5

5

5

5

KIDNEYS

MEAN

2.52

2.53

2.42

2.84

(GRAM)

ST.DEV

0.07

0.15

0.14

0.39

 

N

5

5

5

5

SPLEEN

MEAN

0.558

0.554

0.600

0.650 *

(GRAM)

ST.DEV

0.055

0.073

0.036

0.024

 

N

5

5

5

5

ORGAN WEIGHTS (GRAM) SUMMARY FEMALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

END OF TREATMENT

BODY W.

 

MEAN

 

294

 

286

 

293

 

320

(GRAM)

ST.DEV

23

24

17

107

 

N

10

10

10

10

BRAIN

MEAN

1.92

1.92

1.96

1.92

(GRAM)

ST.DEV

0.07

0.05

0.07

0.02

 

N

5

5

5

5

LIVER

MEAN

12.81

11.33

11.91

12.74

(GRAM)

ST.DEV

0.75

1.61

1.04

1.18

 

N

5

5

5

5

KIDNEYS

MEAN

2.12

1.94

2.01

2.13

(GRAM)

ST.DEV

0.09

0.16

0.14

0.18

 

N

5

5

5

5

SPLEEN

MEAN

0.523

0.455

0.502

0.529

(GRAM)

ST.DEV

0.021

0.072

0.066

0.072

 

N

5

5

5

5

                 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

 

Table 17: Relative Organ Weights (%) - Summary of selected findings

 

ORGAN/BODY WEIGHT RATIOS (%) SUMMARY MALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

END OF TREATMENT

BODY W.

 

MEAN

 

356

 

359

 

357

 

330 **

(GRAM)

ST.DEV

13

14

26

17

 

N

10

10

10

10

BRAIN

MEAN

0.57

0.58

0.60

0.62 **

(%)

ST.DEV

0.02

0.01

0.01

0.03

 

N

5

5

5

5

LIVER

MEAN

2.32

2.35

2.43

2.79 **

(%)

ST.DEV

0.10

0.17

0.06

0.08

 

N

5

5

5

5

KIDNEYS

MEAN

0.70

0.71

0.70

0.84 *

(%)

ST.DEV

0.03

0.04

0.03

0.15

 

N

5

5

5

5

SPLEEN

MEAN

0.155

0.156

0.174

0.192 **

(%)

ST.DEV

0.014

0.019

0.018

0.010

 

N

5

5

5

5

ORGAN/BODY WEIGHT RATIOS (%)SUMMARY FEMALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

END OF TREATMENT

BODY W.

 

MEAN

 

294

 

286

 

293

 

320

(GRAM)

ST.DEV

23

24

17

107

 

N

10

10

10

10

BRAIN

MEAN

0.64

0.71

0.68

0.66

(%)

ST.DEV

0.03

0.06

0.03

0.04

 

N

5

5

5

5

LIVER

MEAN

4.31

4.13

4.11

4.39

(%)

ST.DEV

0.24

0.36

0.18

0.30

 

N

5

5

5

5

KIDNEYS

MEAN

0.71

0.71

0.69

0.73

(%)

ST.DEV

0.03

0.05

0.05

0.04

 

N

5

5

5

5

SPLEEN

MEAN

0.176

0.166

0.174

0.182

(%)

ST.DEV

0.006

0.018

0.021

0.020

 

N

5

5

5

5

                 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 18: Summary Test Item-Related Microscopic Stomach and Spleen Findings – Scheduled Euthanasia Animals

 

Males

Females

Dose level (mg/kg/day / ppm):

0

50

150

650

0

50

150

650

 

 

 

 

 

 

 

 

 

STOMACH a

5

5

5

10

5

5

5

7

     Ulcerative inflammation, forestomach

 

 

 

 

 

 

 

 

         Moderate

-

-

-

1

-

-

-

-

         Marked

-

-

-

3

-

-

-

-

         Massive

-

-

-

4

-

-

-

4

    Necrosis, transmural, forestomach

 

 

 

 

 

 

 

 

         Slight

-

-

-

1

-

-

-

-

         Marked

-

-

-

7

-

-

-

4

    Ulcer/Erosion, glandular stomach

 

 

 

 

 

 

 

 

       Slight

-

-

-

2

-

-

-

1

    Inflammation, glandular stomach

 

 

 

 

 

 

 

 

       Minimal

-

2

-

3

-

-

-

-

       Slight

-

-

-

2

-

-

-

1

    Hemorrhage, glandular stomach

 

 

 

 

 

 

 

 

       Minimal

-

1

-

2

-

-

-

-

       Slight

-

-

-

1

-

-

-

-

 

 

 

 

 

 

 

 

 

SPLEEN a

5

5

5

5

5

5

5

5

     Extramedullary hematopoiesis

 

 

 

 

 

 

 

 

       Minimal

1

3

2

1

3

2

2

2

      Slight

-

1

2

4

2

-

1

-

      Moderate

-

-

-

-

-

-

-

1

     Pigmentation

 

 

 

 

 

 

 

 

         Minimal

4

3

4

2

4

4

2

-

       Slight

-

-

-

3

1

1

3

5

 

Table 19: Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals (Males)

 

 

Males

Dose level (mg/kg/day / ppm):

0

50

150

650

 

 

 

 

 

LIVER a

5

5

5

5

     Hepatocellular hypertrophy

 

 

 

 

         Minimal

-

-

2

4

 

 

 

 

 

KIDNEYS a

5

5

5

5

    Basophilia, tubular

 

 

 

 

         Minimal

1

4

4

-

       Slight

-

-

1

2

         Moderate

-

-

-

1

       Marked

-

-

-

2

     Cast, granular

 

 

 

 

         Minimal

-

-

-

2

       Slight

-

-

-

1

     Degeneration, tubular

 

 

 

 

         Minimal

-

-

1

-

       Slight

-

-

-

2

       Moderate

-

-

-

1

a  =  Number of tissues examined from each group.

 

Table 20: Reproduction data summary

 

 

 

GROUP 1

Control

 

GROUP 2 50mg/kg/day

 

GROUP 3 150mg/kg/day

 

GROUP 4 650mg/kg/day

 

 

Females paired

10

10

10

10

 

Females mated

10

10

10

10

 

Pregnant females

10

10

10

9

 

Females with implantations only

1

1

0

0

 

Females with living pups on Day 1

9

10

10

9

 

Mating index (%)

100

100

100

100

 

Fertility index (%)

100

100

100

90

 

Gestation index (%)

90

100

100

100

 

 

PRECOITAL TIME: F0-GENERATION - POST COITUM

 

DAY OF THE PAIRING PERIOD

GROUP 1

Control

GROUP 2 50mg/kg/day

GROUP 3 150mg/kg/day

GROUP 4 650mg/kg/day

 

 

NUMBER OF FEMALES MATED

 

 

 

 

 

1

2

3

4

3

 

2

3

1

2

1

 

3

3

4

4

3

 

4

1

2

-

2

 

5

-

-

-

1

 

13

1

-

-

-

 

MEDIAN PRECOITAL TIME

3

3

2

3

 

MEAN PRECOITAL TIME

3.4

2.5

2.0

2.7

 

N

10

10

10

10

 

 

SUMMARY - IMPLANTATION SITES FEMALES (at necropsy)

 

 

GROUP 1

Control

GROUP 2 50mg/kg/day

GROUP 3 150mg/kg/day

GROUP 4 650mg/kg/day

Implantations

MEAN

11.8

11.8

11.6

10.3

 

ST.DEV

4.2

3.0

3.6

4.7

 

N

10

10

10

9

 

Conclusions:
Based on the results of this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Level (NOAEL) of N-Phenyl-diethanolamine, reaction products with formaldehyde were established:
Reproduction NOAEL: at least 650 mg/kg/day
Executive summary:

The objectives of this study were to determine the potential toxic effects of N-Phenyl-diethanolamine, reaction products with formaldehyde when given orally by gavage for a minimum of 28 days to Wistar Han rats.

The dose levels in this study were selected to be 0, 50, 150 and 650 mg/kg/day, based on the results of the Dose Range Finder.

 

Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle, clinical pathology, measurement of thyroid hormone T4 (F0‑males), gross necropsy findings, organ weights and histopathologic examinations. 

Formulation analyses confirmed that formulations of test item in dimethyl sulfoxide were prepared accurately and homogenously.

 

 

Parental results

Treatment resulted in adverse stomach lesions at 650 mg/kg/day and in adverse kidney lesions at 150 and 650 mg/kg/day as outlined below.

 

Adverse and severe stomach lesions at 650 mg/kg/day in males and females consisted of degenerative and inflammatory changes at high severities. These consisted of large ulcerations with moderate to massive inflammation and areas of necrosis affecting all layers of the forestomach and of ulcer/erosions at slight degree in the glandular stomach of two males and a single female. The forestomach lesions correlated to macroscopic lesions consisting of black and hard crateriform retractions and thickening of the limiting ridge. In addition, the stomach lesions correlated with higher white blood cell counts and lymphocyte counts (males and females) and neutrophils (males) in this dose group. The stomach findings were considered to be a local test item effect due to irritating properties of the test item.

 

The ulcerative inflammation and transmural necrosis in the forestomach were considered to have resulted in blood loss in the gastric lumen with subsequent reuptake of blood by the intestines, as evidenced by morphological lesions in the spleen and haematological changes at 650 mg/kg/day. These splenic and haematological changes consisted of increased pigmentation of the spleen in males and females, extramedullary haematopoiesis in the spleen of males, higher spleen weight (16% compared to controls) and hematological changes consisting of lower red blood cell counts, higher reticulocyte counts and lower hemoglobin (with subsequent lower mean corpuscular hemoglobin concentration) in males.

 

The aforementioned stomach lesions were also considered to be related to observed clinical signs at 650 mg/kg/day (i.e. lethargy, flat/hunched posture, rales, piloerection, uncoordinated movements and/or ptosis) as recorded for most animals during treatment. Likewise, effects on the stomach were considered to be related to lower mean body weight gain (0.5x of control) recorded for males throughout the treatment period and for females throughout the lactation period. Only for males this lower body weight gain was accompanied by lower food intake during the first three weeks of treatment (up to 0.85x of control).

 

At 50 and 150 mg/kg/day, incidence and severity of extramedullary hematopoiesis in the spleen was also increased, but this was not associated with hematological changes or stomach lesions, and as such this finding was considered not adverse at these dose levels.

 

Treatment-related, but non-adverse findings in the glandular stomach at 650 mg/kg/day consisted of slightly increased submucosal inflammation and slight haemorrhages. These findings correlated at necropsy to reddish foci in the glandular stomach in half of the examined males. Since these histopathological lesions can be found as background findings at low incidences and did not exceed a slight degree, these were considered non-adverse.   

 

Adverse lesions were also recorded in the male kidney at 150 and 650 mg/kg/day, and consisted of degenerative tubular changes, including an increase in tubular basophilia (up to marked degree) combined with tubular granular casts and degeneration of tubular epithelial cells. This finding is most likely related to the increased urea levels in males treated at 650 mg/kg/day. Based on the degenerative nature, these findings were considered adverse.

 

Other treatment-related but non-adverse changes at 150 and 650 mg/kg/day were recorded for clinical appearance, liver morphology, motor activity and clinical pathology as described below.

 

Treatment-related clinical signs were confined to flat posture for a few animals on one or two treatment days at 150 mg/kg/day. One female showed hunched posture, labored respiration, rales and/or piloerection during the last three days of treatment but delivered a normal offspring. Since these clinical signs occurred incidentally and were not accompanied by stomach lesions or changes in food intake, these were considered not adverse. They were considered to be related to treatment with the test item, considering that similar signs were recorded at the next higher dose level.

 

A non-adverse, dose-dependent increased incidence of minimal hepatocellular centrilobular hypertrophy was observed in males at 150 and 650 mg/kg/day and was associated with higher liver weights at 650 mg/kg/day (13% compared to controls). Since these findings occurred without any degenerative findings these were considered non-adverse.

 

Higher motor activity (mean ambulation counts) was recorded for males at 650 mg/kg/day (1.28x of control), which was ascribed to higher activity of two out of five males. Mean total movements were considered not affected by treatment with the test item. Since this response was recorded for two animals only and occurred in the absence of corroborative changes in examined parameters, this was considered not to be adverse, and possibly did not reflect a test item-related response.

 

Next to changes in clinical pathology parameters that were associated with stomach lesions as described above, other changes in clinical biochemistry parameters at 650 mg/kg/day included higher total bilirubin and bile acid in males and lower total bilirubin in females. These changes may in part be related to the observed histopathological lesions as described above. A lower activated partial thromboplastin time was recorded for males at 150 and 650 mg/kg/day (0.80x and 0.78x of control, respectively). Since an increase in this clotting parameter is expected in case of target organ toxicity, this was considered not to be an adverse change.

 

The bone marrow micronucleus assay showed that the test item was not clastogenic or aneugenic. No increase in mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test item compared to the vehicle treated animals.

 

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, functional observations (grip strength, hearing ability, pupillary reflex and static righting reflex), male T4 thyroid hormone levels).

 

Reproductive results

No reproductive toxicity was recorded up to the highest dose level tested (650 mg/kg/day).

No treatment-related changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

 

Conclusions

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Level (NOAEL) of N-Phenyl-diethanolamine, reaction products with formaldehyde was established:

 

Reproduction NOAEL: at least 650 mg/kg/day

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
17 July 2020 - 27 July 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. The purpose of this study was the assessment of systemic toxic potential of the test item in a 10 day dietary study in the Crl:WI(Han) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Dose Range Finding test for the full OECD TG422 study
Deviations:
yes
Remarks:
Any deviations did not affect the outcome of the study.
Principles of method if other than guideline:
- Principle of test: OECD TG422 (dose range finding test)
- Short description of test conditions: The purpose of this study was the assessment of systemic toxic potential of the test item in a 10 day dietary study in the Crl:WI(Han) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).
- Parameters analysed / observed: Clinical observations, mortality, body weights and food consumption were monitored in order to determine systemic toxic potential of the test item.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han rats Crl: WI(Han) rats were chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. The laboratory has general and reproduction/developmental and micronucleus (bone marrow) 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 (if applicable) nulliparous and non-pregnant: not reported
- Age at study initiation: 13-14 weeks old
- Weight at study initiation: males 297-352g, females 191-207g
- Fasting period before study: not reported
- Housing: On arrival and following assignment to groups at random at the discretion of the biotechnician, animals were group housed (up to 3 animals of the same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm). At study assignment, each animal was identified using a chip.
- Diet (e.g. ad libitum): 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 (e.g. ad libitum): 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.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for 6 days prior to start of the pretest period.

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21˚C
- Humidity (%): 48-74%
- Air changes (per hr): 10 or more, with 100% fresh air (no recirculation)
- Photoperiod (hrs dark / hrs light): 12/12h

IN-LIFE DATES: From: 17 July 2020 To: 27 July 2020
Route of administration:
oral: gavage
Details on route of administration:
The test item with vehicle were administered to the appropriate animals by once daily oral gavage for 10 consecutive days.
Vehicle:
DMSO
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Method of preparation not specified in the dose range finding study. However, in the main study, 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 daily as a solution and dosed within 6 hours after adding the vehicle to the test item.

DIET PREPARATION - N/A

VEHICLE
- Justification for use and choice of vehicle (if other than water):Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: not reported
- Amount of vehicle (if gavage): 1.2mL/kg bodyweight
- Lot/batch no. (if required): Batch#SJ9M0088
- Purity: UVCB
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
N/A
Duration of treatment / exposure:
10 days
Frequency of treatment:
1x daily
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3 females per dose.
Control animals:
no
Details on study design:
The dose levels were selected based on the results of an acute oral toxicity study in rats (LD50 > 2000 mg/kg, Charles River Study No. 20194890). In the current dose range finding study, doses of 500 and 1000 mg/kg bodyweight/day were selected.
Positive control:
N/A
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was checked twice daily throughout the study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least daily from Days 1-10, at 0-15 minutes, 1 hour (± 15 minutes) and 3 hours (± 30 minutes) after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: On Day 1 prior to dosing and on Days 5 and 10.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes, qualitatively Over Days 1-5 and 5-10.

WATER CONSUMPTION: Yes
- Time schedule for examinations:
Daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes macroscopic examination undertaken on sacrifice
All animals were subjected to an external, thoracic and abdominal examination on Day 11 (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy. No organs were fixed and histopathological examination was not performed.

HISTOPATHOLOGY: No
Other examinations:
N/A
Statistics:
Statistical analysis was not performed
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Following administration of 500mg/ml/day, Salivation was observed in 3/3 animals, directly after dosing on Days 3, 5, 6, 7, 8, 9 and/or 10. No other clinical signs were noted.

Following administration of 1000mg/kg/day, Hunched posture was noted in 3/3 animals, most or all time points on Days 1 to 8. Lethargy was observed in 2/3 animals, 1 or 3 hours after dosing on Days 1, 3 and/or 5. In addition, flat posture (1/3 animals, 1 hour after dosing on Day 3), uncoordinated movements (2/3 animals, seen incidentally but at all time points on Days 2, 3, 4, 5 and/or 6), and piloerection (2/3 animals, once 1 hour after dosing on Day 1 for one animal and multiple times at all different time points on Days 2 to 6 and Days 8 and 9 for another animal), hypothermia (1/3 animals, 3 hours after dosing on Day 3), deep respiration and ptosis (1/3 animals at 1 hour after dosing on Day 1), and salivation (3/3 animals, directly after dosing on Days 2 to 10) were observed.

Outcomes are summarised within table 2.
Mortality:
no mortality observed
Description (incidence):
N/A
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Following administration of 500mg/kg/day, slight body weight loss (2%) was measured between days 5 and 10 for 1 of 3 animals.
Following administration of 1000mg/kg/day, slight body weight loss (3%) was measured between days 1 and 5 for 1 of 3 animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Following administration of 500mg/kg/day, no food consumption changes were noted.
Following administration of 1000mg/kg/day, slightly lower food consumption was noted over days 1-5.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
not examined
Description (incidence and severity):
N/A
Haematological findings:
not examined
Description (incidence and severity):
N/A
Clinical biochemistry findings:
not examined
Description (incidence and severity):
N/A
Endocrine findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
not examined
Description (incidence and severity):
N/A
Behaviour (functional findings):
not examined
Description (incidence and severity):
N/A
Immunological findings:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No test item related effects were recorded in liver and kidney weight for animals dosed at 500mg/kg/day
Following administration of 1000mg/kg/day, kidney weight was considered not affected by treatment with the test item, but increased liver weight was recorded.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Following administration of 500mg/kg/day, the following macroscopic findings were noted: Red foci on glandular mucosa of the stomach (2/3 animals), thickened limiting ridge of the stomach (3/3 animals), pale discolouration of the glandular mucosa (1/3 animals).

Following administration of 1000mg/kg/day, the following macroscopic findings were noted: Black and/or red foci on glandular mucosa of the stomach and forestomach, thickening of the stomach wall or limiting ridge and irregular surface of the glandular mucosa and forestomach (3/3 animals), and hardened stomach wall with enlarged liver and spleen (1/3 animals).
Neuropathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
N/A
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
N/A
Other effects:
not examined
Description (incidence and severity):
N/A

Table 2: Results DRF

Parameter

500 mg/kg/day

1000 mg/kg/day

Mortality

No mortality.

No mortality.

Clinical appearance

Salivation (3/3 animals, directly after dosing on Days 3, 5, 6, 7, 8, 9 and/or 10).

Hunched posture (3/3 animals, most or all time points on Days 1 to 8), lethargy (2/3 animals, 1 or 3 hours after dosing on Days 1, 3 and/or 5), flat posture (1/3 animals, 1 hour after dosing on Day 3), uncoordinated movements (2/3 animals, seen incidentally but at all time points on Days 2, 3, 4, 5 and/or 6), piloerection (2/3 animals,

once 1 hour after dosing on Day 1 for one animal and multiple times at all different time points on Days 2 to 6 and Days 8 and 9 for another animal), hypothermia (1/3 animals, 3 hours after dosing on Day 3), deep respiration and ptosis (1/3 animals at 1 hour after dosing on Day 1), salivation (3/3 animals, directly after dosing on Days 2 to 10).

Body weight

Slight body weight loss (2%) between Days 5 and 10 for 1/3 animals.

Slight body weight loss (3%) between Days 1 and 5 for 1/3 animals.

Food consumption

Considered not affected by treatment with the test item.

Slightly lower over Days 1-5.

Macroscopic examination

Red foci on glandular mucosa of the stomach (2/3 animals), thickened limiting ridge of the stomach (3/3 animals), pale discolouration of the glandular mucosa (1/3 animals)

Black and/or red foci on glandular mucosa of the stomach and forestomach, thickening of the stomach wall or limiting ridge and irregular surface of the glandular mucosa and forestomach (3/3 animals), and hardened stomach wall with enlarged liver and spleen (1/3 animals).

Organ weights

Liver and kidney weight considered not affected by treatment with the test item.

Increased liver weight.

Kidney weight considered not affected by treatment with the test item.

Conclusions:
Once daily oral gavage administration of 500 or 1000 mg/kg/day N-Phenyl- diethanolamine, reaction products with formaldehyde to female rats resulted in some evidence of adverse effects. These were minimal at 500mg/kg/day and more notable at 1000mg/kg/day. Based on the results of this dose range finder, selected dose levels for the Main study were 50, 150 and 650 mg/kg/day.
Executive summary:

In order to provide the basis for the selection of dose levels for a subsequent OECD 422 study, N-Phenyl- diethanolamine, reaction products with formaldehyde was adminstered to the rat once daily via oral gavage for up to 10 days. In dosing phase one, 3 female rats were administered 500 mg/kg/day test item for 10 days. In phase two, 3 different female rats were administered 1000 mg/kg/day test item for 10 days. 

Clinical observations for animals administered 500 mg/kg/day was limited to salivation directly after dosing. These animals showed slight body weight loss (2%) but no changes to food or water consumption. Clinical observations for animals administered 1000 mg/kg/day included one or more of the following: hunched posture, lethargy, flat posture, uncoordinated movements, piloerection, hypothermia, deep respiration, ptosis and salivation (directly after dosing). Together, these are indicative of systemic toxicity. In addition, these animals showed slight body weight loss (3%) and reduced food consumption (days 1-5).

All animals survived to their scheduled sacrifice. Macroscopic observations were recorded in both dose groups. For animals administered 500 mg/kg/day, red foci on glandular mucosa of the stomach, alongside a thickened limiting ridge of the stomach and pale discolouration of the glandular mucosa were noted in some animals. For all animals administered 1000 mg/kg/day, black and/or red foci on glandular mucosa of the stomach and forestomach, thickening of the stomach wall or limiting ridge and irregular surface of the glandular mucosa and forestomach were noted. In some animals, there was also hardened stomach wall (1/3 animals) and enlarged liver/spleen (1/3 animals). Examination of liver and kidney weight revealed increased liver weight in animals of the 1000 mg/kg/day dosing group.

In conclusion, once daily oral gavage administration of 500 or 1000 mg/kg/day N-Phenyl- diethanolamine, reaction products with formaldehyde to female rats resulted in no mortality. Test article-related effects including clinical signs of systemic toxicity, macroscopic findings (in particular relating to the gastrointestinal tract) and slight body weight loss and reduced food consumption were present. Based on the findings of this 10-day repeated dose study, dose level of 50, 150 and 650 mg/kg/day are suggested for the following OECD 422 study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guidelineopen allclose all
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:
yes
Remarks:
The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.
Qualifier:
according to guideline
Guideline:
other: • OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2016
Deviations:
yes
Remarks:
The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.
Principles of method if other than guideline:
An OECD genotoxicity test was integrated into this toxicity study as follows:
- Principle of test: OECD TG474 Mammalian Erythrocyte Micronucleus test in vivo (2016)
- Short description of test conditions: Animals are exposed to the test substance by an appropriate route (gavage). Bone marrow is collected, prepared and stained. Each treated and control group must include at least 5 analysable animals per sex.
- Parameters analysed / observed: Preparations are analyzed for the presence of micronuclei, which detects damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts, by analysis of erythrocytes
The results of this test are detailed within Genetic Toxicity (section 7.6)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Test material form:
liquid
Details on test material:
Light orange liquid
Storage at room temperature protected from light
Expiry date 15 December 2021

Test animals

Species:
rat
Strain:
Wistar
Remarks:
The Wistar Han rat - Crl: WI(Han) - 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 and micronucleus (bone ma
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: not reported but may be assumed
- Age at study initiation: Males 11-12 weeks old; Females 13-14 weeks old
- Weight at study initiation: Females 191-243g
- Fasting period before study: not reported
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
Positive control males were group housed (up to 5 animals) in polycarbonate cages (Makrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Makrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Makrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Makrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Makrolon 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.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The room in which the animals were kept was documented in the study records.
Animals were separated during designated procedures/activities.
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): 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.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for 6 days prior to start of the pretest period.

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21˚C
- Humidity (%): 48-74%
- Air changes (per hr): 10 or more, with 100% fresh air (no recirculation)
- Photoperiod (hrs dark / hrs light): 12/12h

IN-LIFE DATES: From: 04 August 2020 To: 07 October 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
DMSO
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Dimethyl sulfoxide (DMSO). Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study and these preparations were not used for dosing. Raw data of these trials will be retained by the Test Facility.

FORMULATION PREPARATION
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 daily as a solution and dosed within 6 hours after adding the vehicle to the test item.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item
The dose volume for each animal was based on the most recent body weight measurement.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Within a separate study (test facility No. 20257162), development and validation of an analytical method for the quantitative analysis of Fatty acids, C18 (unsaturated), reaction products with diethylenetriamine in propylene glycol including trial formulation analysis to confirm the accuracy, homogeneity, stability and resuspension was undertaken.

The analytical method was validated for the analysis of Fatty acids, C18 (unsaturated), reaction products with diethylenetriamine in propylene glycol for the following parameters: i) Specificity, ii) Calibration curve, iii) Accuracy and Repeatability, iv) Limit of quantification, v) Stability of analytical system and end solutions.
Stability of stock solutions in methanol was demonstrated in Test Facility Study No. 491557 to be at least 4 days at room temperature. The stability of stock solutions was therefore not determined during this study.

Formulation Analysis: The concentrations analyzed in the formulations at 1 mg/mL and 200 mg/mL were in agreement with target concentrations (i.e. mean accuracies between 90 and 110%). It was observed that dissolving formulation samples in methanol took time.
The formulations were homogeneous (i.e. coefficient of variation ≤ 5%).
Formulations were stable when stored at room temperature under normal laboratory light conditions for at least 24 hours, in a refrigerator (2-8°C) for at least 8 days, and in a freezer (≤ -15°C) for at least 21 days (3 weeks).
Resuspension homogeneity was demonstrated (i.e. coefficient of variation ≤ 5%).

Within the main study, dose formulations samples were collected on Week 1 of treatment (day 1) and week 4 of treatment (day 22). The method development 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. Samples were tested from all groups for concentration, and from groups 2 & 4 (50mg/kg/day and 650mg/kg/day) for homogeneity.

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. The concentrations analyzed in the formulations of Groups 2, 3 and 4 (50, 150 & 650mg/kg/day, respectively) were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the Group 1 formulations (vehicle control).

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%. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as day 0 post-coitum.
- A maximum of 14 days was allowed for mating, after which females who have not shown evidence of mating were separated from their males.
- Further matings after unsuccessful attempt: no
- After successful mating each pregnant female was caged (how): individually housed in Makrolon plastic cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
The test item and vehicle were administered to the appropriate animals for a minimum of 28 days as follows: i) 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. ii) Females that delivered were treated for 50-64 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 days after delivery, up to and including the day before scheduled necropsy. iii) Females which failed to deliver were treated for 41-44 days.
Frequency of treatment:
Once by daily oral gavage 7 days a week
Duration of test:
Total in life period (detail of exposure within 'duration of treatment'): 5-8 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (DMSO vehicle control)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2 (low)
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 3 (intermediate)
Dose / conc.:
650 mg/kg bw/day (nominal)
Remarks:
Group 4 (high)
No. of animals per sex per dose:
10 male, 10 female
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The oral route of administration selected because this is a possible route of human exposure during manufacture, handling or use of the test item. The dose levels were selected based on the results of a 10 Day Dose Range Finder with oral administration of N-Phenyl-diethanolamine, reaction products with formaldehyde in rats (see accompanying entry), and in an attempt to produce graded responses to the test item. Dose level spacing was selected in consultation with the Sponsor, and in agreement with OECD 422 testing requirements.

- Rationale for animal assignment (if not random): N/A
- Fasting period before blood sampling for clinical biochemistry: F0 males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0 females were not fasted overnight.
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A
- Other: N/A

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, morning & end of working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, 30 minutes ± 15 minutes post-dose.

BODY WEIGHT: Yes
- Time schedule for examinations: individually on the first day of treatment (prior to dosing), and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: N/A

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

OTHER:

PATHOLOGY:
Samples for clinical pathology evaluation were collected as per the samples & processing for clinical pathology detailed in tables 01 & 02.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day of necropsy between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane
- Animals fasted: F0 females were not fasted overnight.
- How many animals: 5/sex/group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day of necropsy between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane
- Animals fasted: F0 females were not fasted overnight.
- How many animals: 5/sex/group

PLASMA/SERUM HORMONES/LIPIDS: Yes - serum for T4 (thyroxine) & TSH (thyroid stimulating hormone)
- Time of blood sample collection: F0 - Day of necropsy between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane. F1 - pups/litter PND 4 (via decapitation) and PND 14-16 (by aorta puncture under anaesthesia using isoflurane as part of necropsy).
- Animals fasted: F0 females were not fasted overnight.
- How many animals: All F0 animals (Initial analysis only conducted for male animals). F1 2 pups/litter PND 4 and PND 14-16.

NEUROBEHAVIOURAL EXAMINATION: No (F0 males were examined within the TG422 protocol but not F0 females)

COAGULATION:
- Time schedule for collection of blood: Day of necropsy between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane
- Animals fasted: F0 females were not fasted overnight.
- How many animals: 5/sex/group

ESTROUS CYCLICITY:
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Partial
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other: All reproductive tissues including ovaries (paired) and uterus were examined in control and high dose group as part of a wider pathological examination (outlined within table 02).
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
- Anogenital distance of all live rodent pups: Yes: all per litter
Statistics:
The reviewer considers the analyses used (detailed in table 03) to be appropriate.
Indices:
Mating index; precoital time; fertility index; gestation index; duration of gestation, post-implantation survival index, live birth index, %males/females alive at 1st litter check; viability index; lactation index.
Indices calculations are outlined within table 04.
Historical control data:
Laboratory historical control data was available to the Study Director.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 650 mg/kg/day, lethargy and flat posture were recorded for all females, and for each of these animals these signs occurred on a few days during treatment. Most of these animals also showed hunched posture, rales, piloerection, uncoordinated movements and/or ptosis on a few days during treatment.
At 150 mg/kg/day, flat posture was noted for two females on treatment Days 12 and/or 13 only. One female at 150 mg/kg/day showed hunched posture, labored respiration, rales and/or piloerection during the last three days of treatment.
One female each at 50 and 150 mg/kg/day showed piloerection on two or three consecutive days during the 6th treatment week. As the incidence was similar or even less than encountered in the control group where two females showed piloerection (including hunched posture), these were considered not to be related to treatment with the test item.
Salivation seen after dosing among animals of the 50, 150 and 650 mg/kg/day dose groups with a dose-related trend during the treatment period 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 rather than a sign of systemic toxicity and is a common occurrence in oral gavage studies.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Clinical observations outlined in full within Appendix 1 (summary) and 2 (individual) in attached study results file.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 650 mg/kg/day, mean body weight gain of females was lower throughout the lactation period (0.67x of control at the end of lactation; not statistically significant). In particular, Female (No. 79) showed no to minimal weight gain over the lactation period (1% over lactation Days 1 to 13). One other female at this dose level showed weight loss over lactation Days 1 to 7 (up to 13% of lactation Day 1 body weight).Overall, body weights and body weight gain at 50 and 150 mg/kg/day were considered not affected by treatment with the test item. Bodyweight (gram) is summarised in table 05. Bodyweight gain is summarised in table 06.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
For females at 650 mg/kg/day, slightly lower mean food intake was only noted over Days 7 to 13 of lactation. This was ascribed to a reduced food intake of one female (No. 79). It is notable that this female only delivered two pups, and as a result presumably had a lower food consumption requirement. As this observation was isolated to one animal, the overall lowered group mean food intake was therefore considered not to be related to treatment with the test item.
Absolute and relative food consumption at 50 and 150 mg/kg/day was considered not affected by treatment with the test item.
Food consumption is summarised in table 07, bodyweight consumption corrected for bodyweight is summarised in table 08.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 650 mg/kg/day, the following (statistically significant) changes in hematological parameters were recorded: i) Higher white blood cell counts (WBC) (1.34x control); ii) Higher neutrophil counts (NEUT) (1.33x of control; not statistically significant); iii) Higher lymphocyte counts (LYMPH) (not statistically significant for females).
At 150 and 650 mg/kg/day, lower mean corpuscular hemoglobin (MCH) was recorded for females (each 0.95x of control), but in absence of correlating changes in red blood cell parameters or histopathological changes, and absence of a clear dose-related trend, this was considered not to be related to treatment with the test item. Hematology is summarised in table 09.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Clinical biochemistry was not significantly altered in maternal animals (statistically significant findings were reported for paternal F0 animals). Summarised in table 10.
Endocrine findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related organ weight changes in females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were present in the stomach females treated at 650 mg/kg/day. These findings consisted of: i) Crateriform retractions (black and hard) in the forestomach in 4/10 females and thickened limiting ridge in 4/10 females. The microscopic correlate was ulcerative inflammation of the forestomach with transmural necrosis.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with the test item were noted in the stomach and spleen of females. Stomach-forestomach: Ulcerative inflammation (up to massive degree) and transmural necrosis (slight or marked degree) was observed in the forestomach of females at 650 mg/kg/day. The ulcers were large and were accompanied by inflammation (mostly in the submucosa and/or at the serosal side) and large necrotic areas throughout all layers of the forestomach (transmural).

Stomach-glandular stomach: In the glandular stomach of a single female at 650 mg/kg/day, an ulcer or erosion (slight degree) was observed and submucosal inflammation was noted to be slightly above background (minimal inflammation is considered background).

Spleen: Pigmentation was increased in severity in females treated at 650 mg/kg/day, up to slight degree.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

A summary of relevant histopathological (microscopic) findings is in table 13.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
ESTROUS CYCLE: Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The number of implantation sites was considered not to be affected by treatment with the test item.
At 650 mg/kg/day, the number of implantations was slightly lower than the control mean (not statistically significant). This was attributed to a low number of implantations for Female No. 79 (2 implantations). The mean number of implantation sites without the value for Female No. 79 was 11.4, i.e. comparable to the control mean; based on this no clear dose-related reduction in number of implantations was apparent. Therefore, it was considered that this single case of a lower number of implantation sites did not represent a test item-related effect.
One control female (No. 50) had a single implantation site only (without offspring). As this occurred in the control group, this was unrelated to treatment with the test item.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were considered not to be affected by treatment with the test item.
Except for one control female (No. 50), all pregnant females had live offspring. The gestation indices were 90% the control group and 100% for the 50, 150 and 650 mg/kg/day groups. The failed pregnancy of control Female No. 50 was unrelated to treatment with the test item since this concerned a control group female.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of premature birth.
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
REPRODUCTIVE PERFORMANCE: No effects were observed in any relevant indices (table 14).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 650 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment with the test item.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment with the test item.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Live litter sizes were considered not affected by treatment with the test item.
At 650 mg/kg/day, the mean number of living pups per litter at first litter check was slightly lower (9.3 pups vs 12.0 pups in the control group (not statistically significant), and 10.5 and 10.7 pups at 50 and 150 mg/kg/day, respectively). This was essentially attributed to a low number of living pups for Female No. 79 (2 pups; this female also had 2 implantation sites). The mean number of living pups per litter without the value for Female No. 79 was 10.3, based on which no dose-related reduction in litter size was apparent. Therefore, it was considered that this single case of a lower litter size did not represent a test item-related effect.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was considered not to be affected by treatment with the test item.
Post-implantation survival index was 92, 90, 94 and 91% for the control, 50, 150 and 650 mg/kg/day groups, respectively.
External malformations:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment with the test item.
The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item.
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 650 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

The following section includes all tables considered to be relevant to the study summary. Further raw data tables are provided as attached background material.

 

Table 05: Summary bodyweights (gram) 

BODY WEIGHTS FEMALES

(GRAM) SUMMARY

 

 

 

 

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

PRE MATING

 

DAY 1

 

MEAN

 

220

 

217

 

216

 

225

WEEK 1

ST.DEV

9.6

13.6

12.4

13.4

 

N

10

10

10

10

DAY 8

MEAN

221

216

220

227

WEEK 2

ST.DEV

8.3

14.6

10.1

12.5

 

N

10

10

10

10

MATING PERIOD

 

DAY 1

 

MEAN

 

227

 

225

 

227

 

231

WEEK 1

ST.DEV

10.4

13.2

8.0

12.9

 

N

10

10

10

10

DAY 8

WEEK 2

MEAN

247

--- 1

 

0 x

 

0 x

 

ST.DEV

N

DAY 15

WEEK 3

MEAN

 

---

---

0 x

---

---

0 x

 

ST.DEV

 

DAY 22

WEEK 4

MEAN

 

---

---

0 x

---

---

0 x

 

ST.DEV

N

FEMALES F0-GENERATION

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG/DAY

GROUP 3

150

MG/KG/DAY

GROUP 4

650

MG/KG/DAY

POST COITUM

 

DAY 0

MEAN

232

227

223

231

 

ST.DEV.

10.8

16.8

7.0

12.9

 

N

9

9

9

9

DAY 4

MEAN

244

238

239

243

 

ST.DEV.

8.1

17.2

7.7

12.4

 

N

9

9

9

9

DAY 7

MEAN

249

245

247

250

 

ST.DEV.

8.5

17.7

8.4

11.1

 

N

9

9

9

9

DAY 11

MEAN

263

258

263

264

 

ST.DEV.

6.2

21.3

10.1

12.5

 

N

9

9

9

9

DAY 14

MEAN

273

270

274

272

 

ST.DEV.

8.5

19.7

13.1

13.6

 

N

9

9

9

9

DAY 17

MEAN

298

293

297

294

 

ST.DEV.

7.5

20.5

15.7

19.3

 

N

9

9

9

9

DAY 20

MEAN

337

329

337

328

 

ST.DEV.

8.3

21.5

24.3

31.0

 

N

9

9

9

9

LACTATION

 

 

 

 

 

DAY 1

MEAN

262

254

263

266

 

ST.DEV.

8.0

21.3

14.7

12.1

 

N

7

10

10

9

DAY 4

MEAN

277

267

276

272

 

ST.DEV.

5.3

21.3

14.0

12.8

 

N

9

10

10

9

DAY 7

MEAN

278

275

285

274

 

ST.DEV.

14.8

19.5

15.0

13.5

 

N

8

10

10

8

DAY 13

MEAN

302

295

297

293

 

ST.DEV.

7.8

26.6

20.0

16.3

 

N

9

10

10

9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values (see attached additional study reporting)

Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post‑coitum and lactation phase.

 

Table 06: Bodyweight gain (%) summary

 

BODY WEIGHT GAIN (%)SUMMARY FEMALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

PRE MATING

DAY 1

 

MEAN

 

0

 

0

 

0

 

0

WEEK 1

ST.DEV

0.0

0.0

0.0

0.0

 

N

10

10

10

10

DAY 8

MEAN

1

0

2

1

WEEK 2

ST.DEV

3.3

1.5

3.6

2.5

 

N

10

10

10

10

 

MATING PERIOD

DAY 1

 

MEAN

 

4

 

4

 

5

 

3

WEEK 1

ST.DEV

3.4

2.9

4.1

3.4

 

N

10

10

10

10

DAY 8

WEEK 2

MEAN ST.DEV N

13

--- 1

---

---

0 x

---

---

0 x

 

DAY 15

WEEK 3

MEAN ST.DEV N

 

---

---

0 x

---

---

0 x

 

DAY 22

WEEK 4

MEAN ST.DEV N

 

---

---

0 x

---

---

0       x

 

BODY WEIGHT GAIN (%)SUMMARY FEMALES

F0-GENERATION

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG/DAY

GROUP 3

150

MG/KG/DAY

GROUP 4

650

MG/KG/DAY

 

POST COITUM

DAY 0

 

MEAN

 

0

 

0

 

0

 

0

 

ST.DEV.

0.0

0.0

0.0

0.0

 

N

9

9

9

9

DAY 4

MEAN

5

5

7

5

 

ST.DEV.

2.8

1.6

1.9

1.9

 

N

9

9

9

9

DAY 7

MEAN

7

8

11 **

8

 

ST.DEV.

3.2

1.5

2.7

1.7

 

N

9

9

9

9

DAY 11

MEAN

14

13

18 *

14

 

ST.DEV.

3.9

2.7

2.9

2.5

 

N

9

9

9

9

DAY 14

MEAN

18

19

23 **

18

 

ST.DEV.

2.9

2.4

4.0

3.2

 

N

9

9

9

9

DAY 17

MEAN

28

29

33

27

 

ST.DEV.

5.2

2.9

5.3

5.1

 

N

9

9

9

9

DAY 20

MEAN

45

45

51

42

 

ST.DEV.

7.5

5.1

8.3

9.5

 

N

9

9

9

9

 

LACTATION

DAY 1

 

MEAN

 

0

 

0

 

0

 

0

 

ST.DEV.

0.0

0.0

0.0

0.0

 

N

7

10

10

9

DAY 4

MEAN

5

5

5

2

 

ST.DEV.

2.9

5.4

2.9

3.7

 

N

7

10

10

9

DAY 7

MEAN

5

9

8

3

 

ST.DEV.

6.7

6.4

3.4

7.5

 

N

6

10

10

8

DAY 13

MEAN

15

16

13

10

 

ST.DEV.

5.1

7.6

5.7

5.2

 

N

7

10

10

9

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values (see attached additional study reporting)

Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post‑coitum and lactation phase

 

Table 07: Absolute Food consumption (g/animal/day) summary

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY

FEMALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

PRE MATING

DAYS 1-8

 

MEAN

 

17

 

17

 

18

 

17

WEEKS 1-2

ST.DEV

0.6

0.1

0.2

2.3

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

17

16

17

17

WEEKS 2-3

ST.DEV

0.2

0.4

0.1

1.2

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER PRE MATI...

 

MEAN

 

17

 

17

 

17

 

17

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY FEMALES

F0-GENERATION

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG/DAY

GROUP 3

150

MG/KG/DAY

GROUP 4

650

MG/KG/DAY

 

POST COITUM

DAYS 0-4

 

MEAN

 

18

 

18

 

20

 

19

 

ST.DEV.

1.4

3.1

2.0

1.9

 

N

9

9

9

9

DAYS 4-7

MEAN

21

21

22

21

 

ST.DEV.

1.5

2.8

2.3

1.5

 

N

9

9

9

9

DAYS 7-11

MEAN

22

22

24

22

 

ST.DEV.

1.6

3.4

2.9

1.9

 

N

9

9

9

9

DAYS 11-14

MEAN

23

22

24

23

 

ST.DEV.

2.2

2.5

2.7

1.1

 

N

9

9

9

9

DAYS 14-17

MEAN

23

22

24

23

 

ST.DEV.

1.9

2.9

2.4

2.4

 

N

9

9

9

9

DAYS 17-20

MEAN

24

24

25

25

 

ST.DEV.

1.5

2.7

2.9

1.7

 

N

9

9

9

9

 

MEAN OF MEANS

 

 

22

 

22

 

23

 

22

LACTATION

DAYS 1-4

 

MEAN

 

32

 

30

 

32

 

32

 

ST.DEV.

3.2

4.4

5.2

9.4

 

N

9

10

10

9

DAYS 4-7

MEAN

47

42

43

40

 

ST.DEV.

9.1

5.4

4.5

9.6

 

N

9

10

10

8

DAYS 7-13

MEAN

54

55

53

47

 

ST.DEV.

6.5

7.4

6.3

9.6

 

N

9

10

10

9

 

MEAN OF MEANS

 

 

44

 

42

 

43

 

40

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values (see attached additional study reporting)

Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post‑coitum and lactation phase

 

Table 08: Relative Food consumption (g/animal/day) summary

RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY)

F0-GENERATION

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG/DAY

GROUP 3

150

MG/KG/DAY

GROUP 4

650

MG/KG/DAY

 

POST COITUM

DAYS 0-4

 

MEAN

 

74

 

77

 

83

 

77

 

ST.DEV.

7.0

9.1

6.9

7.4

 

N

9

9

9

9

DAYS 4-7

MEAN

84

85

89

86

 

ST.DEV.

6.8

8.1

7.3

6.3

 

N

9

9

9

9

DAYS 7-11

MEAN

83

84

89

85

 

ST.DEV.

7.1

9.2

8.2

6.7

 

N

9

9

9

9

DAYS 11-14

MEAN

83

82

89

86

 

ST.DEV.

8.8

7.4

7.5

4.8

 

N

9

9

9

9

DAYS 14-17

MEAN

77

76

81

78

 

ST.DEV.

6.7

5.6

5.3

7.2

 

N

9

9

9

9

DAYS 17-20

MEAN

73

73

76

77

 

ST.DEV.

3.7

6.6

5.5

5.7

 

N

9

9

9

9

 

MEAN OF MEANS

 

 

79

 

80

 

84

 

81

RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY)

LACTATION

DAYS 1-4

 

MEAN

 

115

 

111

 

116

 

116

 

ST.DEV.

12.4

12.4

15.6

35.5

 

N

9

10

10

9

DAYS 4-7

MEAN

164

152

150

144

 

ST.DEV.

38.8

15.6

13.2

38.7

 

N

8

10

10

7

DAYS 7-13

MEAN

179

187

180

161

 

ST.DEV.

20.6

17.0

16.5

27.9

 

N

9

10

10

9

 

MEAN OF MEANS

 

 

153

 

150

 

149

 

140

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

x Explanations for excluded data are listed in the tables of the individual values (see attached additional study reporting)

Note to clinical signs tables: For males, “Repro period” represents the mating phase. For females, “Repro period” represents the mating, post‑coitum and lactation phase

 

Table 09: Summary of hematology values: F0 Generation

FEMALES: Day 51 relative to start date

Sex: Female

Reporting Hematology

WBC (10^9/L)

[G]

NEUT (10^9/L)

[G]

LYMPH (10^9/L)

[G]

MONO (10^9/L)

[G]

EOS (10^9/L)

[G]

BASO (10^9/L)

[G]

LUC (10^9/L)

[G]

RBC (10^12/L)

[G]

RETIC (10^9/L)

[G]

RDWG (%)

[G]

HGB

 

(g/L)

 

[G]

HCT(L/L)

[G1]

0

Mean

5.062

1.598

3.278

0.092

0.072

0.008

0.020

6.824

261.04

13.02

140.2

0.4076

mg/kg/day

SD

0.749

0.317

0.518

0.025

0.026

0.004

0.000

0.117

47.87

0.44

3.2

0.0228

Group 1

N

5

5

5

5

5

5

5

5

5

5

5

5

50

Mean

5.536

1.662

3.594

0.124

0.130

0.004

0.020

6.936

238.30

12.90

136.2

0.3978

mg/kg/day

SD

0.901

0.264

0.810

0.040

0.053

0.005

0.022

0.289

37.34

1.02

1.8

0.0090

Group 2

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

1.09

1.04

1.10

1.35

1.81

0.50

1.00

1.02

0.91

0.99

0.97

0.98

150

Mean

4.770

1.262

3.314

0.096

0.072

0.004

0.024

6.958

210.54

12.72

135.6

0.4012

mg/kg/day

SD

0.780

0.457

0.579

0.027

0.029

0.005

0.026

0.245

59.83

1.02

3.6

0.0080

Group 3

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

0.94

0.79

1.01

1.04

1.00

0.50

1.20

1.02

0.81

0.98

0.97

0.98

650

Mean

6.804 *

2.118

4.382

0.154

0.106

0.012

0.028

7.012

248.92

13.56

136.2

0.3986

mg/kg/day

SD

1.393

0.958

0.636

0.072

0.069

0.004

0.015

0.356

53.85

1.06

1.5

0.0082

Group 4

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

1.34

1.33

1.34

1.67

1.47

1.50

1.40

1.03

0.95

1.04

0.97

0.98

 

Sex: Female

Reporting Hematology

MCV (fL)

[G]

MCH

 

(pg)

 

[G]

MCHC

 

(g/L)

 

[G]

PLT (10^9/L)

[G]

0

Mean

59.74

20.54

344.6

703.2

mg/kg/day

SD

3.39

0.42

13.5

72.5

Group 1

N

5

5

5

5

50

Mean

57.40

19.66

342.6

725.2

mg/kg/day

SD

2.11

0.70

5.7

75.1

Group 2

N

5

5

5

5

 

tCtrl

0.96

0.96

0.99

1.03

150

Mean

57.72

19.50 *

337.8

717.0

mg/kg/day

SD

2.03

0.47

4.9

128.4

Group 3

N

5

5

5

5

 

tCtrl

0.97

0.95

0.98

1.02

650

Mean

56.90

19.44 *

341.6

737.8

mg/kg/day

SD

2.36

0.84

4.6

108.5

Group 4

N

5

5

5

5

 

tCtrl

0.95

0.95

0.99

1.05

 

[G] - Anova & Dunnett: * = p ≤ 0.05

[G1] - Kruskal-Wallis & Dunn

 

Table 10: Summary of clinical chemistry values: F0 Generation

FEMALES: Day 51 relative to start date

Sex: Female

Reporting Biochemistry

ALT (U/L)

[G]

AST (U/L)

[G]

ALP (U/L)

[G]

TPROT

 

(g/L)

 

[G]

ALB

 

(g/L)

 

[G]

BILEAC

(umol/L) [G]

TBIL

(umol/L) [G]

UREA

(mmol/L) [G]

CREAT

(umol/L) [G]

GLUC

(mmol/L) [G]

CHOL

(mmol/L) [G]

NA

(mmol/L) [G]

0

Mean

129.9

95.7

253.3

56.74

34.96

11.35

2.30

9.72

22.4

7.384

2.134

141.0

mg/kg/day

SD

15.6

5.7

93.1

1.27

1.08

3.20

0.89

0.65

2.7

1.628

0.310

1.2

Group 1

N

5

5

5

5

5

5

5

5

5

5

5

5

50

Mean

138.0

98.5

197.9

54.60

33.74

11.96

2.52

9.69

21.0

8.904

2.160

140.2

mg/kg/day

SD

25.7

7.8

90.2

2.04

0.76

4.30

0.77

0.17

2.2

0.876

0.249

1.3

Group 2

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

1.06

1.03

0.78

0.96

0.97

1.05

1.10

1.00

0.94

1.21

1.01

0.99

150

Mean

122.4

97.2

231.3

55.10

33.98

8.16

2.14

9.72

24.8

8.160

1.866

140.8

mg/kg/day

SD

20.8

11.3

107.9

2.90

1.98

2.61

0.46

0.93

4.1

1.484

0.418

1.3

Group 3

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

0.94

1.02

0.91

0.97

0.97

0.72

0.93

1.00

1.11

1.11

0.87

1.00

650

Mean

120.9

96.2

234.0

55.60

33.80

11.06

1.74

9.13

21.1

8.692

2.230

141.2

mg/kg/day

SD

21.2

14.6

120.3

2.71

1.48

2.02

0.44

0.81

3.9

0.741

0.197

1.1

Group 4

N

5

5

5

5

5

5

5

5

5

5

5

5

 

tCtrl

0.93

1.00

0.92

0.98

0.97

0.97

0.76

0.94

0.94

1.18

1.04

1.00

 

Sex: Female

Reporting Biochemistry

K

(mmol/L) [G]

CL

(mmol/L) [G]

CA

(mmol/L) [G]

PHOS

(mmol/L) [G1]

0

Mean

4.54

105.8

2.442

1.270

mg/kg/day

SD

0.19

1.3

0.054

0.491

Group 1

N

5

5

5

5

50

Mean

4.91

106.0

2.450

1.080

mg/kg/day

SD

0.51

2.1

0.060

0.305

Group 2

N

5

5

5

5

 

tCtrl

1.08

1.00

1.00

0.85

150

Mean

4.85

106.4

2.456

1.534

mg/kg/day

SD

0.31

1.5

0.076

0.309

Group 3

N

5

5

5

5

 

tCtrl

1.07

1.01

1.01

1.21

650

Mean

4.72

105.8

2.504

1.496

mg/kg/day

SD

0.54

0.4

0.053

0.138

Group 4

N

5

5

5

5

 

tCtrl

1.04

1.00

1.03

1.18

[G] - Anova & Dunnett: * = p ≤ 0.05

[G1] - Kruskal-Wallis & Dunn

 

Table 11: Absolute Organ Weights (gram) - Summary of selected findings

ORGAN WEIGHTS (GRAM) SUMMARY FEMALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

END OF TREATMENT

BODY W.

 

MEAN

 

294

 

286

 

293

 

320

(GRAM)

ST.DEV

23

24

17

107

 

N

10

10

10

10

BRAIN

MEAN

1.92

1.92

1.96

1.92

(GRAM)

ST.DEV

0.07

0.05

0.07

0.02

 

N

5

5

5

5

LIVER

MEAN

12.81

11.33

11.91

12.74

(GRAM)

ST.DEV

0.75

1.61

1.04

1.18

 

N

5

5

5

5

KIDNEYS

MEAN

2.12

1.94

2.01

2.13

(GRAM)

ST.DEV

0.09

0.16

0.14

0.18

 

N

5

5

5

5

SPLEEN

MEAN

0.523

0.455

0.502

0.529

(GRAM)

ST.DEV

0.021

0.072

0.066

0.072

 

N

5

5

5

5

                 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 12: Relative Organ Weights (%) - Summary of selected findings

ORGAN/BODY WEIGHT RATIOS (%)SUMMARY FEMALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG/DAY

 

GROUP 3

150

MG/KG/DAY

 

GROUP 4

650

MG/KG/DAY

 

END OF TREATMENT

BODY W.

 

MEAN

 

294

 

286

 

293

 

320

(GRAM)

ST.DEV

23

24

17

107

 

N

10

10

10

10

BRAIN

MEAN

0.64

0.71

0.68

0.66

(%)

ST.DEV

0.03

0.06

0.03

0.04

 

N

5

5

5

5

LIVER

MEAN

4.31

4.13

4.11

4.39

(%)

ST.DEV

0.24

0.36

0.18

0.30

 

N

5

5

5

5

KIDNEYS

MEAN

0.71

0.71

0.69

0.73

(%)

ST.DEV

0.03

0.05

0.05

0.04

 

N

5

5

5

5

SPLEEN

MEAN

0.176

0.166

0.174

0.182

(%)

ST.DEV

0.006

0.018

0.021

0.020

 

N

5

5

5

5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

Table 13: Summary Test Item-Related Microscopic Stomach and Spleen Findings – Scheduled Euthanasia Animals

 

Females

Dose level (mg/kg/day / ppm):

0

50

150

650

 

 

 

 

 

STOMACH a

5

5

5

7

     Ulcerative inflammation, forestomach

 

 

 

 

         Moderate

-

-

-

-

         Marked

-

-

-

-

         Massive

-

-

-

4

    Necrosis, transmural, forestomach

 

 

 

 

         Slight

-

-

-

-

         Marked

-

-

-

4

    Ulcer/Erosion, glandular stomach

 

 

 

 

       Slight

-

-

-

1

    Inflammation, glandular stomach

 

 

 

 

       Minimal

-

-

-

-

       Slight

-

-

-

1

    Hemorrhage, glandular stomach

 

 

 

 

       Minimal

-

-

-

-

       Slight

-

-

-

-

 

 

 

 

 

SPLEEN a

5

5

5

5

     Extramedullary hematopoiesis

 

 

 

 

       Minimal

3

2

2

2

      Slight

2

-

1

-

      Moderate

-

-

-

1

     Pigmentation

 

 

 

 

         Minimal

4

4

2

-

       Slight

1

1

3

5

 

Table 14: Reproduction data summary

 

 

 

GROUP 1

Control

 

GROUP 2 50mg/kg/day

 

GROUP 3 150mg/kg/day

 

GROUP 4 650mg/kg/day

 

 

Females paired

10

10

10

10

 

Females mated

10

10

10

10

 

Pregnant females

10

10

10

9

 

Females with implantations only

1

1

0

0

 

Females with living pups on Day 1

9

10

10

9

 

Mating index (%)

100

100

100

100

 

Fertility index (%)

100

100

100

90

 

Gestation index (%)

90

100

100

100

 

 

PRECOITAL TIME: F0-GENERATION - POST COITUM

 

DAY OF THE PAIRING PERIOD

GROUP 1

Control

GROUP 2 50mg/kg/day

GROUP 3 150mg/kg/day

GROUP 4 650mg/kg/day

 

 

NUMBER OF FEMALES MATED

 

 

 

 

 

1

2

3

4

3

 

2

3

1

2

1

 

3

3

4

4

3

 

4

1

2

-

2

 

5

-

-

-

1

 

13

1

-

-

-

 

MEDIAN PRECOITAL TIME

3

3

2

3

 

MEAN PRECOITAL TIME

3.4

2.5

2.0

2.7

 

N

10

10

10

10

 

 

SUMMARY - IMPLANTATION SITES FEMALES (at necropsy)

 

 

GROUP 1

Control

GROUP 2 50mg/kg/day

GROUP 3 150mg/kg/day

GROUP 4 650mg/kg/day

Implantations

MEAN

11.8

11.8

11.6

10.3

 

ST.DEV

4.2

3.0

3.6

4.7

 

N

10

10

10

9

 

Table 15: Developmental data summary

 

 

 

GROUP 1

Control

 

GROUP 2 50mg/kg/day

 

GROUP 3 150mg/kg/day

 

GROUP 4 650mg/kg/day

Total Litters

9

10

10

9

Duration of Gestation

MEAN (+)

21.2

21.3

21.0

21.3

ST.DEV.

0.4

0.5

0.0

0.5

N

9

10

10

9

DEAD PUPS AT FIRST LITTER CHECK

LITTERS AFFECTED (#)

0

1

2

1

TOTAL

0

1

2

1

MEAN (+)

0.0

0.1

0.2

0.1

ST.DEV.

0.0

0.3

0.4

0.3

N

9

10

10

9

 

LIVING PUPS AT FIRST LITTER CHECK

% OF MALES / FEMALES (#)

52 / 48

53 / 47

62 / 38

58 / 42

TOTAL

108

105

107

84

MEAN (+)

12.0

10.5

10.7

9.3

ST.DEV.

1.9

2.7

3.4

4.2

N

9

10

10

9

POSTNATAL LOSS

% OF LIVING PUPS

0.0

1.9

0.0

0.0

LITTERS AFFECTED (#)

0

1

0

0

TOTAL (#)

0

2

0

0

MEAN (+)

0.0

0.2

0.0

0.0

ST.DEV.

0.0

0.6

0.0

0.0

N

9

10

10

9

CULLED PUPS

TOTAL

36

26

32

23

LIVING PUPS DAY 4 P.P.

TOTAL

72

77

75

61

MEAN (+)

8.0

7.7

7.5

6.8

ST.DEV.

0.0

0.7

1.0

2.1

N

9

10

10

9

BREEDING LOSS DAYS 5 - 13 P.P.

 

% OF LIVING PUPS AT DAY 4 P.P.

 

0.0

 

0.0

 

0.0

 

0.0

LITTERS AFFECTED (#)

0

0

0

0

TOTAL (#)

0

0

0

0

MEAN (+)

0.0

0.0

0.0

0.0

ST.DEV.

0.0

0.0

0.0

0.0

N

9

10

10

9

LIVING PUPS DAY 13 P.P.

% OF MALES / FEMALES (#)

54 / 46

52 / 48

53 / 47

52 / 48

TOTAL

72

77

75

61

MEAN (+)

8.0

7.7

7.5

6.8

ST.DEV.

0.0

0.7

1.0

2.1

N

9

10

10

9

 

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

 

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Level (NOAEL) of N-Phenyl-diethanolamine, reaction products with formaldehyde were established:

Developmental NOAEL: at least 650 mg/kg/day
Executive summary:

The objectives of this study were to determine the potential toxic effects of N-Phenyl-diethanolamine, reaction products with formaldehyde when given orally by gavage for a minimum of 28 days to Wistar Han rats.

The dose levels in this study were selected to be 0, 50, 150 and 650 mg/kg/day, based on the results of the Dose Range Finder (Charles River Study No. 20194898).

The study design is outlined within table 1. 

 

Chemical analyses of formulations were conducted twice during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle, clinical pathology, measurement of thyroid hormone T4 (F0‑males), gross necropsy findings, organ weights and histopathologic examinations. 

Formulation analyses confirmed that formulations of test item in dimethyl sulfoxide were prepared accurately and homogenously.

 

 

Parental results

Treatment resulted in adverse stomach lesions at 650 mg/kg/day and in adverse kidney lesions (in parental F0 males) at 150 and 650 mg/kg/day as outlined below.

 

Adverse and severe stomach lesions at 650 mg/kg/day in males and females consisted of degenerative and inflammatory changes at high severities. These consisted of large ulcerations with moderate to massive inflammation and areas of necrosis affecting all layers of the forestomach and of ulcer/erosions at slight degree in the glandular stomach of two males and a single female. The forestomach lesions correlated to macroscopic lesions consisting of black and hard crateriform retractions and thickening of the limiting ridge. In addition, the stomach lesions correlated with higher white blood cell counts and lymphocyte counts (males and females) and neutrophils (males) in this dose group. The stomach findings were considered to be a local test item effect due to irritating properties of the test item.

 

The ulcerative inflammation and transmural necrosis in the forestomach were considered to have resulted in blood loss in the gastric lumen with subsequent reuptake of blood by the intestines, as evidenced by morphological lesions in the spleen and haematological changes at 650 mg/kg/day. These splenic and haematological changes consisted of increased pigmentation of the spleen in males and females, extramedullary haematopoiesis in the spleen of males, higher spleen weight (16% compared to controls) and hematological changes consisting of lower red blood cell counts, higher reticulocyte counts and lower hemoglobin (with subsequent lower mean corpuscular hemoglobin concentration) in males.

 

The aforementioned stomach lesions were also considered to be related to observed clinical signs at 650 mg/kg/day (i.e. lethargy, flat/hunched posture, rales, piloerection, uncoordinated movements and/or ptosis) as recorded for most animals during treatment. Likewise, effects on the stomach were considered to be related to lower mean body weight gain (0.5x of control) recorded for males throughout the treatment period and for females throughout the lactation period. Only for males this lower body weight gain was accompanied by lower food intake during the first three weeks of treatment (up to 0.85x of control).

 

At 50 and 150 mg/kg/day, incidence and severity of extramedullary hematopoiesis in the spleen was also increased, but this was not associated with hematological changes or stomach lesions, and as such this finding was considered not adverse at these dose levels.

 

Treatment-related, but non-adverse findings in the glandular stomach at 650 mg/kg/day consisted of slightly increased submucosal inflammation and slight haemorrhages. These findings correlated at necropsy to reddish foci in the glandular stomach in half of the examined males. Since these histopathological lesions can be found as background findings at low incidences and did not exceed a slight degree, these were considered non-adverse.   

 

Adverse lesions were also recorded in the male kidney at 150 and 650 mg/kg/day, and consisted of degenerative tubular changes, including an increase in tubular basophilia (up to marked degree) combined with tubular granular casts and degeneration of tubular epithelial cells. This finding is most likely related to the increased urea levels in males treated at 650 mg/kg/day. Based on the degenerative nature, these findings were considered adverse.

 

Other treatment-related but non-adverse changes at 150 and 650 mg/kg/day were recorded for clinical appearance, liver morphology, motor activity and clinical pathology as described below.

 

Treatment-related clinical signs were confined to flat posture for a few animals on one or two treatment days at 150 mg/kg/day. One female showed hunched posture, labored respiration, rales and/or piloerection during the last three days of treatment but delivered a normal offspring. Since these clinical signs occurred incidentally and were not accompanied by stomach lesions or changes in food intake, these were considered not adverse. They were considered to be related to treatment with the test item, considering that similar signs were recorded at the next higher dose level.

 

A non-adverse, dose-dependent increased incidence of minimal hepatocellular centrilobular hypertrophy was observed in males at 150 and 650 mg/kg/day and was associated with higher liver weights at 650 mg/kg/day (13% compared to controls). Since these findings occurred without any degenerative findings these were considered non-adverse.

 

Higher motor activity (mean ambulation counts) was recorded for males at 650 mg/kg/day (1.28x of control), which was ascribed to higher activity of two out of five males. Mean total movements were considered not affected by treatment with the test item. Since this response was recorded for two animals only and occurred in the absence of corroborative changes in examined parameters, this was considered not to be adverse, and possibly did not reflect a test item-related response.

 

Next to changes in clinical pathology parameters that were associated with stomach lesions as described above, other changes in clinical biochemistry parameters at 650 mg/kg/day included higher total bilirubin and bile acid in males and lower total bilirubin in females. These changes may in part be related to the observed histopathological lesions as described above. A lower activated partial thromboplastin time was recorded for males at 150 and 650 mg/kg/day (0.80x and 0.78x of control, respectively). Since an increase in this clotting parameter is expected in case of target organ toxicity, this was considered not to be an adverse change.

 

The bone marrow micronucleus assay showed that the test item was not clastogenic or aneugenic. No increase in mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test item compared to the vehicle treated animals.

 

No treatment-related changes were noted in any of the remaining parameters investigated in this study (i.e. mortality, functional observations (grip strength, hearing ability, pupillary reflex and static righting reflex), male T4 thyroid hormone levels).

 

Developmental results

No developmental toxicity was recorded up to the highest dose level tested (650 mg/kg/day).

No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

 

Conclusions

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Level (NOAEL) of N-Phenyl-diethanolamine, reaction products with formaldehyde was established:

Developmental NOAEL: at least 650 mg/kg/day. 

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