Trimethylamine, N-oxide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Remarks:

QA statement will be included with final report

Limit test:

no

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%

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

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

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

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)

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

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

Critical effects observed:

no

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

The potential toxicity of trimethylamine N‑oxide dihydrate in the rat after oral (gavage) administration was assessed.

Three test groups and one control group, each containing 10 males and 10 females were used. Males were treated for 2 weeks prior to mating, until necropsy after at least 4 weeks of treatment. Females were treated for 2 weeks prior to mating, then through mating, gestation and until the day before necropsy. Dose levels were 50, 150, and 300 mg/kg bw/day

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 bw/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.

There were no test item-related effects on paternal toxicity, embryo-fetal development, pup development or neurobehavior.

In conclusion, administration of Trimethylamine N‑oxide dihydrate by once daily oral gavage in Han Wistar rats was associated with transient and recoverable effects on body weight and/or food consumption in females only at 150 and 300 mg/kg bw/day.

Justification for classification or non-classification

Based on the transient and reversable nature of the effects, trimethylamine N-oxide does not meet the classification criteria of EC 1272/2008 (as amended) for specific organ toxicity following repeated exposure.