Methanaminium, N,N,N-trimethyl-, 2,2-dimethylpropanoate (1:1)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

478-310-4

EC Name:

-

Cas Number:

53803-13-7

Molecular formula:

C9H21NO2

IUPAC Name:

Methanaminium N,N,N-trimethyl-, salt with 2,2-dimethylpropanoic acid

Test material form:

solid: crystalline

Details on test material:

Description: white solid
Batch: 13AP016
Expiry 2015-11-13

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar Han™:RccHan™:WIST strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for 9 days during which time their health status was assessed. A total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed
196 to 229g, the females weighed 144 to 169g, and were approximately seven weeks old.

Animal Care and Husbandry
The animals were housed in groups of five by sex in solid floor polypropylene cages with
stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The
animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad
Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK) was used. Certificates of
analysis of the batches of diet used are given in Annex 5. Mains drinking water was supplied
from polycarbonate bottles attached to the cage. Environmental enrichment was provided in
the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
The diet, drinking water, bedding and environmental enrichment were considered not to
contain any contaminant at a level that might have affected the purpose or integrity of the
study.
The animals were housed in a single air-conditioned room within the Envigo Research
Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at
least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to
give twelve hours continuous light and twelve hours darkness. Environmental conditions
were continuously monitored by a computerized system, and print-outs of hourly
temperatures and humidities are included in the study records. The Study Plan target ranges
for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term
deviations from these targets were considered not to have affected the purpose or integrity of
the study; see deviations from Study Plan.
The animals were randomized and allocated to dose groups on arrival. Subsequent group
mean body weights were checked on Day -1 and animals reallocated where necessary to
ensure similarity between the dose groups. The animals were uniquely identified within the
study, by an ear punch system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily, for up to twenty-eight consecutive days, by gavage
using a stainless steel cannula attached to a disposable plastic syringe. Control animals were
treated in an identical manner with 5 mL/kg of Distilled water.
The volume of test and control item administered to each animal was based on the most
recent scheduled body weight and was adjusted at weekly intervals.

Vehicle:

water

Details on oral exposure:

Test item was dosed as solution in water

Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL)
Control 0 5 0
Low 10 5 2
Intermediate 20 5 4
High 40 5 8

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Intermediate dose

Doses / concentrations 4

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Serial Observations
General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change
immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All
observations were recorded.
Body Weight
Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body
weights were also performed prior to terminal kill.
Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the
study. Food conversion efficiency was calculated retrospectively.
Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water
bottles for any overt changes except during Week 3 where water intake was measured
gravimetrically.
Special Evaluations
Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 26, all animals were observed for
signs of functional/behavioral toxicity. Functional performance tests were also performed on
all animals during Week 4, together with an assessment of sensory reactivity to different
stimuli. Observations were carried out from approximately two hours after dosing on each
occasion.
Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose
built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The
scoring system used is outlined in The Key to Scoring System and Explanation for
Behavioral Assessments and Sensory Reactivity Tests.
Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were
used to assess motor activity. Animals of one sex were tested at each occasion and were
randomly allocated to the activity monitors. The tests were performed at approximately the
same time each occasion (at least two hours after dosing), under similar laboratory
conditions. The evaluation period was one hour for each animal. The time in seconds each
animal was active and mobile was recorded for the overall one hour period and also during
the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail
1979).
Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each
animal was allowed to grip the proximal metal bar of the meter with its forepaws. The
animal was pulled by the base of the tail until its grip was broken. The animal was drawn
along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A
record of the force required to break the grip for each animal was made. Three consecutive
trials were performed for each animal. The assessment was developed from the method
employed by Meyer et al (1979).
Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and
proprioceptive stimuli. This assessment was developed from the methods employed by Irwin
(1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring
System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach
In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all surviving animals
from each test and control group at the end of the study (Day 28). Blood samples were
obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac
puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.
The methods used for hematological and blood chemical investigations are given in Annex 6
and normal ranges are shown in Annex 8.
Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time
(APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11
mol/L).
Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes
containing lithium heparin anti-coagulant:
Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids

Sacrifice and pathology:

Necropsy
On completion of the dosing period all surviving animals were killed by intravenous
overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic
abnormalities were recorded.

Other examinations:

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum
from each animal was stored frozen at approximately -70 °C. No treatment-related effects on
the pituitary-thyroid axis were identified, therefore these samples were discarded.
Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing
period or at the end of the treatment-free period, were dissected free from fat and weighed
before fixation:
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus
Pituitary (post-fixation) Thyroid/Parathyroid (post fixation)
Prostate and Seminal Vesicles Uterus with Cervix
(with coagulating glands and fluids)
Normal ranges for organ weights are given in Annex 9.
Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered
10% formalin, except where stated:
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint) Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and Rectum
pons) Salivary glands (submaxillary)
Caecum Sciatic nerve
Colon Seminal vesicles (with coagulating
Duodenum glands and fluids)
Epididymides  Skin
Esophagus Spinal cord (cervical, mid thoracic
Eyes * and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum Testes ♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Trachea
Lungs (with bronchi)# Urinary bladder
Lymph nodes (mandibular and mesenteric) Uterus & Cervix
Mammary gland Vagina
Muscle (skeletal)
All tissues were dispatched to the histology processing Test Site (Propath UK Ltd. Willow
Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal
Investigator: N Lewis). The tissues shown in bold from all control and 40 mg/kg bw/day
dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5
μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any
macroscopically observed lesions were also processed. In addition, sections of testes from all
Control and 40 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and
examined.
Since there were indications of treatment-related changes, examination was subsequently
extended to include similarly prepared sections of liver (females only) and seminal vesicles
from animals in the low and intermediate groups.
Pathology
Microscopic examination was conducted by the Study Pathologist (Wendy Henderson).
A peer review of the findings observed was conducted by Vasanthi Mowat at Envigo CRS
Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS. A complete
histopathology phase report is presented in Annex 1 and represents the consensus view of
both pathologists.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect
the significance of intergroup differences from control; statistical significance was achieved
at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry,
Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module
as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method.
The homogeneity of variance from mean values was analyzed using Bartlett’s test.
Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with
appropriate covariates. Any transformed data were analyzed to find the lowest treatment
level that showed a significant effect using the Williams Test for parametric data or the
Shirley Test for non-parametric data. If no dose response was found but the data shows nonhomogeneity
of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel
(non-parametric) test to determine significant difference from the control group. Where the
data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test
(parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Instances of increased salivation were evident in animals of either sex treated with 40 mg/kg
bw/day from Day 11 until Days 25 or 27 in females and males respectively. This observation
was noted to a lesser extent in animals of either sex treated with 20 mg/kg bw/day. This type
of observation is commonly seen when dosing an irritant/unpalatable test item/formulation
and is considered not to be toxicologically significant as it does not specifically relate to
systemic toxicity. One female treated with 40 mg/kg bw/day and a further female treated with
10 mg/kg bw/day exhibited signs of noisy respiration which were only present for each
animal for one day only. Noisy respiration was considered to reflect occasional difficulties
with dosing these animals rather than any underlying toxicological effect of the test item.
Decreased respiratory rate, ptosis, lethargy and clonic convulsions were noted in the female
animal treated with 40 mg/kg bw/day prior to being humanely killed on Day 26 of the study.

Mortality:

no mortality observed

Description (incidence):

One female animal treated with 40 mg/kg bw/day was humanely killed on Day 26 of the study due to the severity of the observations noted. There were no further unscheduled deaths during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Overall, there did not appear to be any detrimental effect of treatment with the test item at
any dose level on body weight performance in animals of either sex.
Male animals treated with 20 or 40 mg/kg bw/day exhibited reductions in body weight gain
during the first two weeks of treatment, however, statistical significance was not achieved, a
dose relationship was apparent during the first week but was not apparent during the second
week of treatment. Recovery was evident thereafter such that body weight gains were
comparable to control. Overall body weight gains for these animals were approximately
7.2% and 7% lower for animals treated with 20 or 40 mg/kg bw/day respectively when
compared to control.
No such effects were noted in male animals treated with 10 mg/kg bw/day.
Female animals from the 10 and 40 mg/kg bw/day treatment group showed a reduced body
weight gain in comparison to control animals during Week 1 but this did not attain statistical
significance and a dose relationship was not apparent. Body weight gains for all treatment
groups were then generally comparable to or exceeded control during the remainder of the
treatment period. Overall body weight gains for treated females were comparable to or
exceeded control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically significant effects were detected at any dose level in animals of either sex.
At the end of the dosing period male animals treated with 20 and 40 mg/kg bw/day exhibited
statistically significant increases (p<0.05) in mean corpuscular volume and neutrophils. All
values for mean corpuscular volume were found to be within the historical control data range,
whereas three and four neutrophil values were outside of the historical control data range for
animals treated with 20 and 40 mg/kg bw/day respectively. A dose relationship was not
apparent and as such these findings are considered to be of no toxicological significance.
Male animals from all treatment groups exhibited statistically significant increases (p<0.01)
in reticulocytes. However, two control values were lower than the historical control data
range and the majority of treated males showed values that were within the historical control
data range, as a dose relationship was not apparent these findings are considered to be of no
toxicological significance.
Female animals from all treatment groups exhibited statistically significant increases
(p<0.05) in neutrophils and platelets. However, as a dose relationship was not apparent for
either parameter and the fact that four control values for platelets were lower than the
historical control ranges these findings are considered to be of no toxicological significance.
Female animals treated with 20 mg/kg bw/day exhibited statistically significant increases
(p<0.05) in mean corpuscular volume and females treated with 10 mg/kg bw/day exhibited
statistically significant increases (p<0.01) in hemoglobin, erythrocyte count and hematocrit.
All values for the treated animals were found to be within the historical control data range
and as similar effects were not seen in animals from higher treatment groups, these findings
are considered to be of no toxicological significance.

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant effects were detected at any dose level in animals of either sex.
At the end of the dosing period male animals treated with 20 and 40 mg/kg bw/day exhibited
statistically significant increases (p<0.05) in mean corpuscular volume and neutrophils. All
values for mean corpuscular volume were found to be within the historical control data range,
whereas three and four neutrophil values were outside of the historical control data range for
animals treated with 20 and 40 mg/kg bw/day respectively. A dose relationship was not
apparent and as such these findings are considered to be of no toxicological significance.
Male animals from all treatment groups exhibited statistically significant increases (p<0.01)
in reticulocytes. However, two control values were lower than the historical control data
range and the majority of treated males showed values that were within the historical control
data range, as a dose relationship was not apparent these findings are considered to be of no
toxicological significance.
Female animals from all treatment groups exhibited statistically significant increases
(p<0.05) in neutrophils and platelets. However, as a dose relationship was not apparent for
either parameter and the fact that four control values for platelets were lower than the
historical control ranges these findings are considered to be of no toxicological significance.
Female animals treated with 20 mg/kg bw/day exhibited statistically significant increases
(p<0.05) in mean corpuscular volume and females treated with 10 mg/kg bw/day exhibited
statistically significant increases (p<0.01) in hemoglobin, erythrocyte count and hematocrit.
All values for the treated animals were found to be within the historical control data range
and as similar effects were not seen in animals from higher treatment groups, these findings
are considered to be of no toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Females from all treatment groups exhibited statistically significant increases (p<0.05) in
aspartate aminotransferase. All values were within the historical control data ranges for
females treated with 10 mg/kg bw/day. Two values were found to be higher than the
historical control data ranges in animals treated with 20 mg/kg bw/day. One value was found
to be much higher than the historical control data range in animals treated with 40 mg/kg
bw/day.
Females treated with 40 mg/kg bw/day exhibited statistically significant increases (p<0.01) in
alanine aminotransferase. All individual values for these animals were found to be within the
historical control data ranges.
Even though the majority of individual values for females in relation to aspartate
aminotransferase and alanine aminotransferase were within the historical control data ranges
an association with treatment cannot be discounted due to the effects (considered to be nonadverse)
noted in the liver at histopathological examination.
Females treated with 40 mg/kg bw/day exhibited statistically significant increases (p<0.05) in
bile acids. Two values were found to be outside of the historical data range. Due to the
effects noted in the liver of these animals an association with treatment cannot be discounted.
There were considered to be no further toxicologically significant effects noted in treated
female animals or in male animals at any dose level.
Females treated with 40 mg/kg bw/day exhibited statistically significant decreases (p<0.05)
in sodium. Three control values for sodium were found to be higher than the historical control
data ranges with all values for the animals treated with 40 mg/kg bw/day being within these
data ranges. As such this finding is considered not to be of any toxicological significance.
Male animals treated with 40 mg/kg bw/day exhibited statistically significant reductions in
total protein (p<0.05) and chloride concentrations (p<0.01), these animals also exhibited
statistically significant increases (p<0.05) in alanine aminotransferase. As the majority of
values were found to be within the historical control data range and in the absence of any
histopathological correlates these findings were considered not to be of any toxicological
significance.
Males animals treated with 20 and 40 mg/kg bw/day exhibited statistically significant
reductions (p<0.01) in triglycerides. All values were found to be within the historical control
data range and in the absence of any histopathological correlates these findings were
considered not to be of any toxicological significance.
Males treated with 40 mg/kg bw/day exhibited statistically significant increases (p<0.001) in
bile acids. These values were higher than the historical control data range, however in the
absence of any histopathological correlates these findings were considered not to be of any
toxicological significance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no significant intergroup differences at any dose level.
One male animal from each treatment group exhibited increased salivation during the
behavioural assessments conducted on Day 26, as this observations does not specifically
relate to systemic toxicity of the test item it is considered not to be toxicologically significant.
Functional Performance Tests
There were no treatment-related changes in functional performance at any dose level.
Statistical analysis of the data did not reveal any significant intergroup differences.
Sensory Reactivity Assessments
There were no treatment-related changes detected at any dose level.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Female animals treated with 40 mg/kg bw/day exhibited a statistically significant increase
(p<0.01) in absolute and body weight-related liver weights. Three out of four absolute values
and all relative weights were found to be higher than the historical control data ranges for
these animals. This was correlated with increased cytoplasmic rarefaction noted at
histopathological examination and is likely to be due to alteration in glycogen deposition and
reflects a non-adverse metabolic change.
There were considered to be no further toxicologically significant effects detected in organ
weights which could be specifically related to treatment in treated animals of either sex.
Male animals treated with 40 mg/kg bw/day exhibited a slight reduction in seminal vesicle
weights, however, this failed to achieve statistical significance. It is considered that this could
be related to the reduced body weight noted in the early part of the study. Any treatment
related effect in the reproductive tract is equivocal and therefore these intergroup variations
are considered incidental.
Male animals treated with 40 mg/kg bw/day exhibited a statistically significant decrease
(p<0.05) in absolute and body weight-related heart weights. A statistically significant
increase (p<0.05) in both absolute and body weight-related thymus weights were also noted
in these animals. As the majority of values were within the historical control data ranges and
in the absence of any histopathological correlates these findings were considered to be
incidental of no toxicological significance.
Male animals treated with 20 mg/kg bw/day exhibited a statistically significant decrease
(p<0.05) in absolute and body weight-related thyroid weights. As all values were within the
historical control data ranges for these treated animals and as males treated at 40 mg/kg
bw/day did not show a similar effect and in the absence of any histopathological correlates
these findings were considered to be incidental and of no toxicological significance.
Female animals from all treatment groups exhibited statistically significantly decreases
(p<0.05 - p<-0.01) in absolute and body weight-related brain and thymus weights. As the
majority of values were within the historical control data ranges and in the absence of any
histopathological correlates these findings were considered to be incidental and of no
toxicological significance.
Female animals treated with 20 or 40 mg/kg bw/day exhibited statistically significant
decreases (p<0.05) in absolute and body weight-related adrenal weights. As the majority of
values were within the historical control data ranges and in the absence of any
histopathological correlates these findings were considered to be incidental and of no
toxicological significance.

Description (incidence and severity):

No macroscopic abnormalities considered to be specifically related to treatment were noted in any surviving animal at necropsy.
Macroscopic abnormalities were limited to small epididymides and small prostate and
seminal vesicles in one male treated with 40 mg/kg bw/day and dark discoloration to the
lungs of one male treated with 20 mg/kg bw/day. Due to the isolated nature of these findings
they were considered not to be specifically related to treatment with the test item.
No macroscopic abnormalities were noted in any surviving treated female or in any male
animal treated with 10 mg/kg bw/day.
The female which was humanely killed during the study did not show any macroscopic
abnormalities at necropsy.


The following microscopic change considered to be related to treatment was evident:
Liver
Increased cytoplasmic rarefaction was noted in 2/5 females treated with 40 mg/kg bw/day.
This is likely to be due to alteration in glycogen deposition and reflects a non-adverse
metabolic change. This effect correlated with the weight increase noted in females treated
with 40 mg/kg bw/day.
The following effect was considered to be incidental and relationship to treatment was
considered to be equivocal:
Male Reproductive tract
Reduced secretion in the seminal vesicles occurred in all animals treated with 40 mg/kg
bw/day compared to 2/5 controls, 10 or 20 mg/kg bw/day treated animals. Minimal tubular
degeneration occurred in two males treated with 40 mg/kg bw/day, for one animal this was
unilateral.
Although decreased secretion in the seminal vesicles occurred at a higher incidence in males
treated with 40 mg/kg bw/day, there was no increase in severity in this group and relationship
to the administration of the test item is equivocal.
Minor tubular degeneration was bilateral in only one of the two animals affected which were
treated with 40 mg/kg bw/day and this is seen occasionally as a background finding in young
rodents at this laboratory. There was no indication of a weight change in the testes. As such,
any treatment related effect in the reproductive tract is equivocal and therefore these
intergroup variations are considered incidental.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

mortality

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

The oral (gavage) administration of Methanaminium, N,N,N-trimethyl-, salt with 2,2 -

dimethylpropanoic acid a.k.a. Tetramethylammonium Pivalate Salt. to male and female

Wistar Han™:RccHan™:WIST strain rats at dose levels of up to 40 mg/kg bw/day for a

period of up to twenty-eight consecutive days resulted in the early termination of one female

animal treated with 40 mg/kg bw/day towards the end of the study. Observations noted in

this animal prior to death included; decreased respiratory rate, ptosis, lethargy and clonic

convulsions. Due to this death occurring and the clinical signs apparent it precludes

40 mg/kg bw/day as being considered as a NOAEL for systemic toxicity.

There were considered to be no adverse effects of treatment on body weight gains, food

consumptions, water consumption or the behavioural parameters measured in any treated

animal.

Histopathological examination of the liver (in female animals treated with 40 mg/kg bw/day),

revealed an increased cytoplasmic rarefaction, however, this is likely to be due to alteration in

glycogen deposition and reflects a non-adverse metabolic change. This correlated with the

weight increase in Group 4 females. Blood chemical investigations in females from all

treatment groups exhibited statistically significant increases (p<0.05) in aspartate

aminotransferase. However, all values were within the historical control data ranges for

females treated with 10 mg/kg bw/day. Two values were found to be higher than the

historical control data ranges in animals treated with 20 mg/kg bw/day. One value was found

to be much higher than the historical control data ranges in animals treated with 40 mg/kg

bw/day. Females treated with 40 mg/kg bw/day exhibited statistically significant increases

(p<0.01) in alanine aminotransferase. All individual values for these animals were found to

be within the historical control data ranges. Even though the majority of individual values for

females in relation to aspartate aminotransferase and alanine aminotransferase were within

the historical control data ranges an association with treatment cannot be discounted due to

the effects (considered to be non-adverse) noted in the liver at histopathological examination.

These findings are of note, however, as evidence of a treatment related effect upon metabolic

processes.

Females treated with 40 mg/kg bw/day exhibited statistically significant increases (p<0.05) in

bile acids. Two values were found to be outside of the historical data range. Due to the

effects noted in the liver of these animals an association with treatment cannot be discounted.

A decreased secretion in the seminal vesicles was noted in animals treated with 40 mg/kg

bw/day but occurred at a higher incidence in this treatment group, however, there was no

increase in severity in this group and a relationship to the administration of the test item is

equivocal. Minor tubular degeneration was bilateral in only one of the two male animals

treated with 40 mg/kg bw/day affected and this is seen occasionally as a background finding

in young rodents at this laboratory. There was no indication of a weight change in the testes.

Any treatment related effect in the reproductive tract is equivocal and therefore these

intergroup variations are considered incidental.

Taking into consideration the overall results from this study, 40 mg/kg bw/day could be

assigned a NOAEL for males in terms of systemic toxicity and it is considered that a dose

level of 20 mg/kg bw/day could be assigned a NOAEL for systemic toxicity for females

within the confines of this type of study.

Applicant's summary and conclusion

Conclusions:

The oral (gavage) administration of Methanaminium, N,N,N-trimethyl-, salt with 2,2 -dimethylpropanoic acid a.k.a. Tetramethylammonium Pivalate Salt. to male and female
Wistar Han™:RccHan™:WIST strain rats at dose levels of up to 40 mg/kg bw/day for a period of up to twenty-eight consecutive days resulted in determination of
NOAEL for males in terms of systemic toxicity to be 40 mg/kg bw/day. A dose level of 20 mg/kg bw/day could be assigned a NOAEL for systemic toxicity for females
within the confines of this type of study.

Executive summary:

The oral (gavage) administration of Methanaminium, N,N,N-trimethyl-, salt with 2,2 -dimethylpropanoic acid a.k.a. Tetramethylammonium Pivalate Salt. to male and female

Wistar Han™:RccHan™:WIST strain rats at dose levels of up to 40 mg/kg bw/day for a period of up to twenty-eight consecutive days resulted in determination of

NOAEL for males in terms of systemic toxicity to be 40 mg/kg bw/day. A dose level of 20 mg/kg bw/day could be assigned a NOAEL for systemic toxicity for females

within the confines of this type of study.