N-Phenyl-diethanolamine, reaction products with formaldehyde

Developmental toxicity / teratogenicity

Administrative data

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-reference

Cross-reference 1

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

Cross-reference 2

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 guideline

Test guideline 1

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.

Test guideline 2

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

Test material information

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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (DMSO vehicle control)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 2 (low)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Group 3 (intermediate)

Doses / concentrations 4

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.