Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Remarks:
Draft report, final anticipated Q2 2023
Adequacy of study:
key study
Study period:
13 Sept 2022 - 15 Nov 2022
Test report available April 2023
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Draft report; final report expected Q2 2023
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
An OECD 422 Oral Gavage Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat
Type of information:
experimental study
Remarks:
Draft report, final anticipated Q2 2023
Adequacy of study:
key study
Study period:
13 Sept 2022 - 15 Nov 2022
Test report available April 2023
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Draft report, final anticipated Q2 2023
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
QA statement will be included with final report
Specific details on test material used for the study:
Dihydrate form of the registered substance Trimethylamine N-oxide (CAS 1184-78-7)
Species:
rat
Strain:
Wistar
Remarks:
Han Wistar (Crl:WI(Han))
Details on species / strain selection:
The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS - Source: Charles River UK, Margate, Kent, UK - Females (if applicable) nulliparous and non-pregnant: yes - Age at study initiation: ca 12-13 weeks (males) / 11-12 weeks (females) - Weight at study initiation: ca 250-375 g (males) / 150-250 g (females) - Housing: Group housed (2 or 3 animals of the same sex and same dosing group together). - Diet (e.g. ad libitum): Ad libitum, except during designated procedures - Water (e.g. ad libitum): Freely available to each animal from water bottles (except during designated procedures) - Acclimation period: Up to 3 weeks DETAILS OF FOOD AND WATER QUALITY: Special Diet Services VRF-1; Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study. Public supply tap water; Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that would interfere with the outcome of the study. ENVIRONMENTAL CONDITIONS - Temperature (°C): 19 to 23°C - Humidity (%): 40 to 70% - Air changes (per hr): Ten or more air changes per hour - Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-Q water
Details on mating procedure:
Pairing was on a 1 male to 1 female basis. During the evening (after 5 pm) of Day 15 of dosing, F0 females were housed with their allocated co-group male partner. The animals were paired in ascending numerical order.
Vaginal lavages were taken early each morning from the day of pairing until mating had occurred and the stage of estrous observed in each vaginal lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated as GD 0.
The pairing period for each pair of animals was a maximum of 14 nights.
If evidence of mating was not observed by the end of the pairing period, the female was separated from the male during the morning following the last night of pairing and treated as if mating had occurred during that night. Procedures for that female continued and were scheduled as if it had mated on the last night of pairing. These animals were not recorded as mated or not mated.
For each female the time taken to show a positive mating sign and the number of failed opportunities to mate (estruses passed without a sign of mating) was evaluated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity analyses performed previously in conjunction with Charles River Study No. 431347 demonstrated that the test item is soluble in the vehicle when prepared under the same mixing conditions at concentrations bracketing those used in the present study. Solubility data have been retained in the study records. Sample Allocation: 2 for analysis, 3 for backup Sampling Containers: Appropriate sized volumetric flasks Storage Conditions: Temperature set to maintain between 10 and 25°C, protected from light Acceptance Criteria: For concentration: mean sample concentration results within or equal to ± 10% of theoretical concentration. Each individual sample concentration result within or equal to ± 15%. For homogeneity, relative standard deviation (RSD) of concentrations of ≤ 10% for each group. Stability analyses performed previously in conjunction with Charles River Study No. 431347 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. Stability data have been retained in the study records for 431347
Duration of treatment / exposure:
Males: Once daily for at least 4 weeks overall, starting from 2 weeks prior to mating. Females: Once daily from 2 weeks prior to mating, then continuing until the day prior to termination on PND 13. If a female is found to be in the process of littering, or has recently littered, at the time of scheduled dose administration, the animal may not be dosed on that day and dosing will re-commence the following day. If an animal has littered and is going to be dosed, a body weight must be taken to account for the lower body weight following parturition. Females that failed to produce a litter by their Gestation Day 24,or maintain their litter, will undergo terminal procedures unless otherwise instructed by the Study Director.
Frequency of treatment:
Daily
Details on study schedule:
See duration of treatment/exposure
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
10 mL/kg dose volume
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
10 mL/kg dose volume
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
10 mL/kg dose volume
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The oral gavage route of exposure was selected because this is a potential route of human exposure, during manufacture, handling or use of the test item .
The dose levels were selected on the basis of the results of a previous range finding study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: Daily DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: Males and females: weekly. Mated females: GD 0, 4, 7, 11, 14, 17 and 20 and Lactation Day (LD) 1, 4, 7, 10 and 13 BODY WEIGHT: Yes - Time schedule for examinations: Weekly; from at least Week -1 and throughout the study Weights for females will additionally be presented on GD 0, 4, 7, 11, 14, 17 and 20 and LD 1, 4, 7, 10 and 13. An in-life body weight will also be taken from every animal on the day of scheduled necropsy. FOOD CONSUMPTION: - Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes WATER CONSUMPTION: Yes - Time schedule for examinations: Regular basis throughout study OPHTHALMOSCOPIC EXAMINATION: Not specified HAEMATOLOGY: Yes - Time schedule for collection of blood: At necropsy - Anaesthetic used for blood collection: Yes - Animals fasted: Not specified - How many animals: 5 males and 5 females per group. For males, the first 5 males per group will be tested. For females, the first 5 females per group that have reared their litter to at least Lactation Day 11-12 will be tested - Haematology Parameters: Red blood cell count; Hemoglobin concentration; Hematocrit Mean corpuscular volume; Red Blood Cell Distribution Width; Mean corpuscular hemoglobin concentration; Mean corpuscular hemoglobin; Reticulocyte count (absolute); Platelet count; White blood cell count; Neutrophil count (absolute); Lymphocyte count (absolute); Monocyte count (absolute); Eosinophil count (absolute); Basophil count (absolute); Large unstained cells (absolute); Other cells (as appropriate) - Coagulation Parameters: Activated partial thromboplastin time; Fibrinogen; Prothrombin time; Sample quality CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: at necropsy - Animals fasted: Not specified - How many animals: 5 males and 5 females per group. For males, the first 5 males per group will be tested. For females, the first 5 females per group that have reared their litter to at least Lactation Day 11-12 will be tested - Clinical Chemistry Parameters: Alanine aminotransferase; Aspartate aminotransferase; Alkaline phosphatase; Gamma-glutamyltransferase; Creatine kinase; Total bilirubin; Urea; Creatinine; Calcium Phosphate; Total protein; Albumin; Globulin (calculated); Albumin/globulin ratio; Glucose; Cholesterol; Triglycerides; Sodium; Potassium; Chloride; Sample quality; Total Bile Acids THYROID STIMULATING HORMONE (TSH) AND THYROXINE (T4): Yes - Time of blood sample collection: At necropsy - Animals fasted: No - How many animals: F0 animals (10 rats/sex/group); F1 Unselected pups Pooled litter sample, when possible; F1 pups (1 pup/sex/litter, where possible) URINALYSIS: No NEUROBEHAVIOURAL EXAMINATION: Yes - Time schedule and dose groups for examinations: Day 29 – 5 selected males per group (prior to blood sampling) During Lactation – the first 5 females per group which rear their litter to at least Lactation Day 11 (prior to blood sampling) - Battery of functions tested: sensory activity; excitability; autonomic activity; neuromuscular parameters; sensorimotor parameters; respiration, body temperature IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
Monitoring of Estrous Cycle - Time schedule: 14-day period prior to test item administration and morning of necropsy - Procedure: Vaginal lavages will be taken early each morning and the stages of estrous observed will be recorded. Vaginal smears should be examined on the morning of necropsy to determine the stage of estrous cycle to allow correlation with histopathology of ovaries
Sperm parameters (parental animals):
All Group 1 and Group 4 males, and all males suspected to be infertile or which died before mating, had additional slides of the testes prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 5 micrometers, and stained with PAS/haematoxylin.
Litter observations:
The females were allowed to litter normally. Any observed difficulty or prolongation of parturition was recorded. The day of birth of the litter (day on which first pups are born) was designated PND 0. The duration of gestation in days was calculated.
The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on LD 0. The live pups were counted and examined daily for the presence of milk in the stomach and for any externally visible abnormalities. Each litter was weighed en masse (by sex) on PND 1 and 4.
For females, on LD 4, litter size was standardised to 8 pups per litter (and where possible, comprised of 4 males and 4 females). Any additional pups were selected at random for use in blood-sampling. Where 4 males and 4 females were not available on LD 4, additional pups of the opposite sex were used to ensure there was a total of 8 pups. And where the total number of pups in the litter on LD 4 was 8 or fewer, no adjustment was performed. Extra pups were killed without necropsy according to Test Facility SOPs.
Where practicable, any pups that were found dead or were killed during the postnatal period were sexed and appropriately examined as above. Any externally abnormal decedent pup was preserved, and externally normal pups were discarded.
Deficiencies in maternal care were recorded. This included inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding. White paper tissue was supplied to each mother for incorporation in the nest. This was replaced when it became soiled.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes (see table) HISTOPATHOLOGY: Yes (see table)
Postmortem examinations (offspring):
All extra pups on PND 4 (including those following blood sample collection) were humanely killed and discarded without necropsy examination.

PND 13 animals: organ weights (see table)
Statistics:
Any data collected during the predose period will not tabulated, summarised or statistically analysed. All statistical analyses will be performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions will be summarised and statistically analysed as indicated below according to sex and occasion or by litter. Body Weight Changes Food Consumption Organ Weight Relative to Body Weight: Organ Weight Relative to Brain Weight: . PARENTAL INDICES Pregnancy Rate Pre-Implantation Loss Female Mating Index Female Fertility Index Female Pregnancy Index Male Mating Index Male Fertility Index Male Pregnancy Index DELIVERY/REPRODUCTIVE PARAMETERS Gestation Length Gestation Index Live Birth Index Sex Ratio (% males) Viability Index Lactation Index Post-Implantation Loss/Litter All statistical tests will be conducted at the 5% significance level. All pairwise comparisons will be conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted The pairwise comparisons of interest are listed below: Group 2 vs. Group 1 Group 3 vs. Group 1 Group 4 vs. Group 1 Variables for Inferential Analysis Body Weight; Body Weight Gains; Food Consumption; Haematology Variables; Coagulation Variables; Clinical Chemistry Variables; TSH; T4; FOB Quantitative Variables; Organ Weights; Organ Weight relative to Body Weight; Organ Weight relative to Brain Weight Levene’s test will be used to assess the homogeneity of group variances. The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons will be conducted using Dunnett’s or Dunn’s test, respectively. Datasets with two groups will be compared using a Dunnett’s test (equivalent to t-test in Nevis 2012) or Dunn’s test (equivalent to Wilcoxon Rank-Sum test in Nevis 2012)
Reproductive indices:
Pregnancy Rate = No. of animals pregnant x 100
No. of animals in cohabitation
Pre-Implantation Loss = No. of corpora lutea – no. of implants x 100
No. of corpora lutea
Female Mating Index Number of Females with Evidence of Mating
(or no confirmed mating date and pregnant)
Number of Females Paired
Female Fertility Index Number of Pregnant Females
Number of Females with Evidence of Mating
(or no confirmed mating date and pregnant)
Female Pregnancy Index Number of Pregnant Females
Number of Females Paired
Male Mating Index Number of Males with Evidence of Mating
(or female partner confirmed pregnant)
Number of Males Paired
Male Fertility Index Number of Males Impregnating a Female
Number of Males with Evidence of Mating
(or female partner confirmed pregnant)
Male Pregnancy Index Number of Males Impregnating a Female
Number of Males Paired
Offspring viability indices:
• Gestation Length The gestation length is calculated from GD 0 to the day the first pup is observed.
• Gestation Index Percentage of pregnancies that result in birth of live litters:
Number of Animals with Live Offspring x 100
Number of Animals Pregnant
• Live Birth Index Percentage of pups born alive:
Number of Live Newborn Pups x 100
Number of Newborn Pups
• Sex Ratio (% males) Percentage of male pups per litter:
Number of Live Male Pups x 100
Total Number of Live Pups
• Viability Index Percentage of pups born that survive 4 days postpartum:
Number of Live Pups on Day 4 Postpartum x 100
Number of Live Newborn Pups
• Lactation Index Percentage of pups that survive 13 days postpartum:
Number of Live Pups on Day 13 Postpartum x 100
Number of Live Pups on Day 4 (postculling) Postpartum
• Post-Implantation Loss/Litter Number of Implants – Total Newborn Pups x 100
Number of Implants
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Low levels of dorsal fur staining that were observed in all groups and considered to be incidental background findings commonly observed in this species
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg bw/day, initial weight loss followed by lower body weight gain were observed during the pre-mating period, when compared with control . The slightly lower weight gain continued into early gestation. It was, however, significantly higher than controls between LD 1 to LD 13. The relevance is under discussion between the study director and sponsor.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 150 and 300 mg/kg bw/day slightly lower food consumption was observed for the majority of the gestation period, when compared with controls. However, during early lactation the food consumption at these dose levels was found to be higher than controls. This effect on food consumption in the females was considered to be test item-related. At 50 mg/kg/day there were no effects on female food consumption during the pre-mating period, gestation period or the lactation period at any dose level. There were no effects on male food consumption at any dose level.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles. No intergroup differences were noted.
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related effects on haematologic parameters. Any intergroup differences did not follow a dose-related pattern, were not observed in both sexes and individual values were all within the range observed in the control group
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were several parameters that were significantly different to controls including higher alkaline phosphatase (ALP) and urea in females at 300 mg/kg/day and lower total protein in females at 150 mg/kg/day. There were no effects on organ weights or any histopathology findings that could be correlated, so these intergroup differences were considered to be of no toxicological significance. There were no test item-related effects on the clinical chemistry parameters in the F0 males.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on TSH and T4 in both sexes and pups at PND 13. Any intergroup differences were not observed in both sexes and were considered due to individual variation and not biologically significant.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on the neurobehavioural evaluations for both sexes. Any intergroup differences did not follow a dose-related pattern, were not observed in both sexes or were considered to be due to individual variation. Motor activity findings were variable. Any intergroup differences did not follow a dose-related pattern, were not observed in both sexes or were considered to be due to individual variation.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no test item-related effects on estrous cycles.
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no test item-related effects on reproductive performance, litter size or post-implantation loss. Any intergroup differences did not follow a dose-related pattern.

There were no test item-related effects on natural delivery observations. Any intergroup differences did not follow a dose-related pattern.
Transient and recoverable effects on body weight and/or food consumption in females only at 150 and 300 mg/kg bw/day. No reproductive effects were observed. Conclusions regarding clinical toxicology currently under discussion between study director and sponsor; to be available in Q2 2023
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Conclusion still under discussion with study director and sponsor
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter observations or pup clinical observations.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on pup and litter weights.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on haematologic parameters. Any intergroup differences did not follow a dose-related pattern, were not observed in both sexes and individual values were all within the range observed in the control group.

There were no test-item related effects on coagulation. Any intergroup differences were considered to be due to individual variation and not biologically significant
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were several parameters that were significantly different to controls including higher alkaline phosphatase (ALP) and urea in females at 300 mg/kg/day and lower total protein in females at 150 mg/kg/day. There were no effects on organ weights or any histopathology findings that could be correlated, so these intergroup differences were considered to be of no toxicological significance.
There were no test item-related effects on the clinical chemistry parameters in the F0 males.
Urinalysis findings:
not specified
Sexual maturation:
no effects observed
Description (incidence and severity):
There were no test item-related effects on pup anogenital distance or nipple retention.
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related differences on the thyroid/parathyroid weights.
Gross pathological findings:
not specified
Histopathological findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test item-related effects on the neurobehavioural evaluations for both sexes. Any intergroup differences did not follow a dose-related pattern, were not observed in both sexes or were considered to be due to individual variation.
No effects of toxicological significance were observed.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
The reproductive and developmental NOAEL of Trimethylamine N-oxide Dihydrate is 300 mg/kg bw/day.
Executive summary:

An OECD TG 422 Combined Repeated Dose Toxicity Study with a Reproduction/Developmental Toxicity Screening of Trimethylamine N-oxide Dihydrate by Oral Gavage in Rats was placed by Plant Response Inc. with Charles River Laboratories Edinburgh Limited. 


The following parameters were evaluated in this study: mortality, clinical observations, body weights, food consumption, estrous cycles (F0 animals only), neurobehavioral evaluations, mating performance, duration of gestation, litter observations, litter survival indices, litter and pup weights, pre-weaning physical development of F1 animals, clinical pathology parameters (haematology, coagulation and clinical chemistry), TSH and T4 parameters, organ weights and macroscopic and microscopic examinations.


Administration of Trimethylamine N-oxide dihydrate at 300 mg/kg/day in females was associated with initial body weight loss during the premating period followed by lower body weight gain and lower food consumption throughout gestation. Test item-related effects at 150 mg/kg/day in females was limited to low food consumption during the gestation period only. Recovery of body weight and food consumption effects at both dose levels was observed during the lactation period.


No reproductive or developmental effects were observed.


A reproductive and developmental NOAEL of 300 mg/kg bw/day was concluded

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2023
Report date:
2023

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
GLP compliance:
yes (incl. QA statement)
Remarks:
QA statement will be included with final report
Limit test:
no

Test material

Constituent 1
Reference substance name:
N,N-dimethylmethanamine oxide dihydrate
Cas Number:
62637-93-8
Molecular formula:
C3H13NO3
IUPAC Name:
N,N-dimethylmethanamine oxide dihydrate
Test material form:
solid
Specific details on test material used for the study:
Dihydrate form of the registered substance Trimethylamine N-oxide (CAS 1184-78-7)

Lot No. 0000041916
White solid
Purity 99.0%

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han Wistar (Crl:WI(Han))
Details on species / strain selection:
The Han Wistar rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK, Margate, Kent, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ca 12-13 weeks (males) / 11-12 weeks (females)
- Weight at study initiation: ca 250-375 g (males) / 150-250 g (females)
- Housing: Group housed (2 or 3 animals of the same sex and same dosing group together).
- Diet (e.g. ad libitum): Ad libitum, except during designated procedures
- Water (e.g. ad libitum): Freely available to each animal from water bottles (except during
designated procedures)
- Acclimation period: Up to 3 weeks

DETAILS OF FOOD AND WATER QUALITY: Special Diet Services VRF-1; Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Public supply tap water; Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that would interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-Q water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity analyses performed previously in conjunction with Charles River Study No. 431347 demonstrated that the test item is soluble in the vehicle when prepared under the same mixing conditions at concentrations bracketing those used in the present study. Solubility data have been retained in the study records.
Sample Allocation: 2 for analysis, 3 for backup
Sampling Containers: Appropriate sized volumetric flasks
Storage Conditions: Temperature set to maintain between 10 and 25°C, protected from light
Acceptance Criteria: For concentration: mean sample concentration results within or equal to ± 10% of theoretical concentration. Each individual sample concentration result within or equal to ± 15%. For homogeneity, relative standard deviation (RSD) of concentrations of ≤ 10% for each group.
Stability analyses performed previously in conjunction with Charles River Study No. 431347 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. Stability data have been retained in the study records for 431347
Duration of treatment / exposure:
Males: Once daily for at least 4 weeks overall, starting from 2 weeks prior to mating.
Females: Once daily from 2 weeks prior to mating, then continuing until the day prior to termination on PND 13. If a female is found to be in the process of littering, or has recently littered, at the time of scheduled dose administration, the animal may not be dosed on that day and dosing will re-commence the following day. If an animal has littered and is going to
be dosed, a body weight must be taken to account for the lower body weight following parturition. Females that failed to produce a litter by their Gestation Day 24,or maintain their litter, will undergo terminal procedures unless otherwise instructed by the Study Director.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
10 mL/kg dose volume
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
10 mL/kg dose volume
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
10 mL/kg dose volume
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The oral gavage route of exposure was selected because this is a potential route of human exposure, during manufacture, handling or use of the test item .
The dose levels were selected on the basis of the results of a previous range finding study.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males and females: weekly. Mated females: GD 0, 4, 7, 11, 14, 17 and 20 and Lactation Day (LD) 1, 4, 7, 10 and 13

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly; from at least Week -1 and throughout the study Weights for females will additionally be presented on GD 0, 4, 7, 11, 14, 17 and 20 and LD 1, 4, 7, 10 and 13. An in-life body weight will also be taken from every animal on the day of scheduled necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Regular basis throughout study

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: Yes
- Animals fasted: Not specified
- How many animals: 5 males and 5 females per group. For males, the first 5 males per group will be tested. For females, the first 5 females per group that have reared their litter to at least Lactation Day 11-12 will be tested
- Haematology Parameters: Red blood cell count; Hemoglobin concentration; Hematocrit Mean corpuscular volume; Red Blood Cell Distribution Width; Mean corpuscular hemoglobin concentration; Mean corpuscular hemoglobin; Reticulocyte count (absolute); Platelet count; White blood cell count; Neutrophil count (absolute); Lymphocyte count (absolute); Monocyte count (absolute); Eosinophil count (absolute); Basophil count (absolute); Large unstained cells (absolute); Other cells (as appropriate)
- Coagulation Parameters: Activated partial thromboplastin time; Fibrinogen; Prothrombin time; Sample quality

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Not specified
- How many animals: 5 males and 5 females per group. For males, the first 5 males per group will be tested. For females, the first 5 females per group that have reared their litter to at least Lactation Day 11-12 will be tested
- Clinical Chemistry Parameters: Alanine aminotransferase; Aspartate aminotransferase; Alkaline phosphatase; Gamma-glutamyltransferase; Creatine kinase; Total bilirubin; Urea; Creatinine; Calcium Phosphate; Total protein; Albumin; Globulin (calculated); Albumin/globulin ratio; Glucose; Cholesterol; Triglycerides; Sodium; Potassium; Chloride; Sample quality; Total Bile Acids

THYROID STIMULATING HORMONE (TSH) AND THYROXINE (T4): Yes
- Time of blood sample collection: At necropsy
- Animals fasted: No
- How many animals: F0 animals (10 rats/sex/group); F1 Unselected pups Pooled litter sample, when possible; F1 pups (1 pup/sex/litter, where possible)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule and dose groups for examinations: Day 29 – 5 selected males per group (prior to blood sampling) During Lactation – the first 5 females per group which rear their litter to at least Lactation Day 11 (prior to blood sampling)
- Battery of functions tested: sensory activity; excitability; autonomic activity; neuromuscular parameters; sensorimotor parameters; respiration, body temperature

IMMUNOLOGY: No

OTHER:
Mating Procedure
- Time schedule: Evening of Day 15; maximum of 14 nights for each pair of animals
- Parameters: Day of detection of a copulatory plug in situ and/or of sperm in a vaginal lavage will be designated Gestation Day 0. For each female the time taken to show a positive mating sign and the number of failed opportunities to mate (estruses passed without a sign of mating) will be evaluated

Monitoring of Estrous Cycle
- Time schedule: 14-day period prior to test item administration and morning of necropsy
- Procedure: Vaginal lavages will be taken early each morning and the stages of estrous observed will be recorded. Vaginal smears should be examined on the morning of necropsy to determine the stage of estrous cycle to allow correlation with histopathology of ovaries

Observations of Females with Litters during Lactation
- Parameters: difficulty or prolongation of parturition; day of birth of pups; numbers of live and dead pups; presence of milk in pups’ stomachs; pup weights and external visible abnormalities; any deficiencies in material care
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)
Statistics:
Any data collected during the predose period will not tabulated, summarised or statistically analysed. All statistical analyses will be performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions will be summarised and statistically analysed as indicated below according to sex and occasion or by litter.

Body Weight Changes
Food Consumption
Organ Weight Relative to Body Weight:
Organ Weight Relative to Brain Weight:
.
PARENTAL INDICES

Pregnancy Rate
Pre-Implantation Loss
Female Mating Index
Female Fertility Index
Female Pregnancy Index
Male Mating Index
Male Fertility Index
Male Pregnancy Index

DELIVERY/REPRODUCTIVE PARAMETERS
Gestation Length
Gestation Index
Live Birth Index
Sex Ratio (% males)
Viability Index
Lactation Index
Post-Implantation Loss/Litter

All statistical tests will be conducted at the 5% significance level. All pairwise comparisons will be conducted using two sided tests and will be reported at the 1% and 5% levels, unless
otherwise noted
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Variables for Inferential Analysis
Body Weight; Body Weight Gains; Food Consumption; Haematology Variables; Coagulation Variables; Clinical Chemistry Variables; TSH; T4; FOB Quantitative Variables; Organ Weights; Organ Weight relative to Body Weight; Organ Weight relative to Brain Weight

Levene’s test will be used to assess the homogeneity of group variances.
The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons will be conducted using Dunnett’s or Dunn’s test, respectively. Datasets with two groups will be compared using a Dunnett’s test (equivalent to t-test in Nevis 2012) or Dunn’s test (equivalent to Wilcoxon Rank-Sum test in Nevis 2012)

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Low levels of dorsal fur staining that were observed in all groups and considered to be incidental background findings commonly observed in this species
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg bw/day, initial weight loss followed by lower body weight gain were observed during the pre-mating period, when compared with control . The slightly lower weight gain continued into early gestation. It was, however, significantly higher than controls between LD 1 to LD 13. The relevance is under discussion between the study director and sponsor.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 150 and 300 mg/kg bw/day slightly lower food consumption was observed for the majority of the gestation period, when compared with controls. However, during early lactation the food consumption at these dose levels was found to be higher than controls. This effect on food consumption in the females was considered to be test item-related.
At 50 mg/kg/day there were no effects on female food consumption during the pre-mating period, gestation period or the lactation period at any dose level.
There were no effects on male food consumption at any dose level.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles. No intergroup differences were noted.
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related effects on haematologic parameters. Any intergroup differences did not follow a dose-related pattern, were not observed in both sexes and individual values were all within the range observed in the control group
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were several parameters that were significantly different to controls including higher alkaline phosphatase (ALP) and urea in females at 300 mg/kg/day and lower total protein in females at 150 mg/kg/day. There were no effects on organ weights or any histopathology findings that could be correlated, so these intergroup differences were considered to be of no toxicological significance.
There were no test item-related effects on the clinical chemistry parameters in the F0 males.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on TSH and T4 in both sexes and pups at PND 13. Any intergroup differences were not observed in both sexes and were considered due to individual variation and not biologically significant.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on the neurobehavioural evaluations for both sexes. Any intergroup differences did not follow a dose-related pattern, were not observed in both sexes or were considered to be due to individual variation.

Motor activity findings were variable. Any intergroup differences did not follow a dose-related pattern, were not observed in both sexes or were considered to be due to individual variation.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item related organ weight differences.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross findings.
All gross findings observed were of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals and, therefore, were considered not to be test item-related.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings.
Details on results:
Transient and recoverable effects on body weight and/or food consumption in females only at 150 and 300 mg/kg bw/day. No other test-item related effects were observed. Conclusions currently under discussion between study director and sponsor; to be available in Q2 2023. Reproductive and developmental parameters discussed under 7.8

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Conclusion still under discussion with study director and sponsor

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Conclusions currently under discussion between study director and sponsor; to be available in Q2 2023
Executive summary:

An OECD TG 422 Combined Repeated Dose Toxicity Study with a Reproduction/Developmental Toxicity Screening of Trimethylamine N-oxide Dihydrate by Oral Gavage in Rats was placed by Plant Response Inc. with Charles River Laboratories Edinburgh Limited. 


The following parameters were evaluated in this study: mortality, clinical observations, body weights, food consumption, estrous cycles (F0 animals only), neurobehavioral evaluations, mating performance, duration of gestation, litter observations, litter survival indices, litter and pup weights, pre-weaning physical development of F1 animals, clinical pathology parameters (haematology, coagulation and clinical chemistry), TSH and T4 parameters, organ weights and macroscopic and microscopic examinations.


Administration of Trimethylamine N-oxide dihydrate at 300 mg/kg/day in females was associated with initial body weight loss during the premating period followed by lower body weight gain and lower food consumption throughout gestation. Test item-related effects at 150 mg/kg/day in females was limited to low food consumption during the gestation period only. Recovery of body weight and food consumption effects at both dose levels was observed during the lactation period.


Reproductive and developmental observations are discussed under section 7.8.


Conclusions currently under discussion between study director and sponsor; to be available in Q2 2023