(3-methacrylamidopropyl)trimethylammonium chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 June - 10 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: compliance to GLP and testing guideline, coherence between data, results and conclusions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(3-methacrylamidopropyl)trimethylammonium chloride

EC Number:

257-182-1

EC Name:

(3-methacrylamidopropyl)trimethylammonium chloride

Cas Number:

51410-72-1

Molecular formula:

C10 H21 N2 O. Cl

IUPAC Name:

trimethyl-[3-(2-methylprop-2-enoylamino)propyl]azanium chloride

Test material form:

other: liquid

Details on test material:

Identity : 3-Trimethylammonium propyl methacrylamide chloride (50% solution in water)
Alternative names : VISIOMER® MAPTAC; VISIOMER® MAPTAC 50% in water;
N-Trimethylammoniumpropylmethacrylamid-chlorid
CAS number : 51410-72-1
EINECS number : 257-182-1
Batch Number : 7201235T05
Date of expiry : 19 September 2012
Appearance : Undefined clear free-flowing yellow liquid
Storage conditions : Room temperature, protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 90 Sprague Dawley [Crl:CD(SD)BR] rats (45 males and 45 virgin females), 6 to 7 weeks old and weighing 176 to 200 g for males and 151 to 175 g for females, were ordered from Charles River Italia S.p.A., Calco (Lecco), Italy.
After arrival the weight range for each sex was determined (194-210 g for males and 168-196 g for females) and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian.
An acclimatisation period of approximately 3 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

Animal room controls were set to maintain temperature and relative humidity at 22°C +/- 2°C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

In-life data from 15 June to 10 August 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same dose volume.

The required amount of 3-Trimethylammonium propyl methacrylamide chloride (50% solution in water) was dissolved in the vehicle (purified water) at the concentrations of 10, 30 and 100 mg/mL. The formulations were prepared daily and the concentrations were calculated and expressed in terms of test item corrected for active ingredient purity (50 %).

Dose levels, selected in consultation with the Sponsor based on information from a previous non GLP Compliant study (RTC Study no.: 90940EXT), were 100, 300 and 1000 mg/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation is satisfactory.
The stability was found to be 24 hours at room temperature in the concentration range of 10 to 100 mg/mL.
Samples of the formulations prepared on Weeks 1 and 6 (when all females were present) were also analysed to check the concentration. The overall results of the analyses were within the limits of acceptance stated in RTC SOPs for concentration of solutions (95-105%).
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study no. 90930) in the range from 1 to 200 mg/mL. The software used for this activity was the Empower® Pro build No. 2154.

Duration of treatment / exposure:

Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.


Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.

Frequency of treatment:

daily

No. of animals per sex per dose:

10 per sex per dose

Details on study design:

The test item was administered orally, by gavage, to parental animals at 10 mL/kg body weight. The oral route was selected as it is a possible route of exposure of the test item in man.
The dosages, selected in consultation with the Sponsor, were 100, 300 and 1000 mg/kg/day.

Examinations

Observations and examinations performed and frequency:

Mortality
Throughout the study, all animals were checked in each working day early in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs
Once before commencement of treatment (data not presented) and at least once daily during the study, each animal was observed and any clinical signs recorded.

Clinical observations (Functional Observation Battery Tests)
Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern).
All observations were recorded for individual animals.

Grip strength and sensory reactivity to stimuli
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength.

Motor activity assessment (MA)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device.

Food consumption
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on Day 4 post partum starting from Day 1 post partum.

Body weight
Males were weighed weekly from allocation to termination. Males were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter and at termination.
Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until pairing and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.

Clinical pathology investigations
As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group, under condition of food deprivation.
The blood samples collected were divided into tubes as follows:

EDTA anticoagulant: for haematological investigations
Heparin anticoagulant: for biochemical tests
Citrate anticoagulant: for coagulation tests

The measurements performed on blood samples are listed below:

Haematology

Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count - Neutrophils
- Lymphocytes
- Eosinophils
- Basophils
- Monocytes
- Large unstained cells
Platelets

Coagulation tests

Prothrombin time

Clinical chemistry

Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

Urinalysis (Only males)

At the same time interval of the clinical pathology investigations, individual overnight urine samples were also collected from the same animals under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.

The measurements performed on urine samples are listed below:

Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood

The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:

Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components

Sacrifice and pathology:

Parental animals were killed by exsanguination under isofluorane anaesthesia.

Parental males:
The males were killed after completion of mating period after 31 days of treatment.

Parental females:
The females with live pups were killed on Day 4 post partum.
The females which did not give birth 25 days after positive identification of mating were killed shortly after.

Necropsy

The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices).
Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Females:
All females were examined also for the following:

a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea (pregnant animals).

Uteri of females with no visible implantations were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.

Organ weights

From all animals completing the scheduled test period, the organs were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved

Samples of all the tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination

After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.

In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

The examination was restricted as detailed below:

a) Tissues specified in Annex 1 of protocol, from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
b) All abnormalities in all groups.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study.
A total of 6 females were found not pregnant at necropsy and distributed in the groups as follows:
Two in the control group (animal nos. 90950011 and 90950015);
One in the low dose group (animal no. 90950029);
One in the mid-dose group (animal no. 90950045);
Two in the high dose group (animal nos . 90950061 and 90950073).
The number of females with live pups on Day 4 post partum was: 8 in each of the control and high dose groups, 9 in each of the low and mid-dose groups.

Clinical signs
No relevant signs were observed in males and females throughout the study.

NEUROBEHAVIOUR
Clinical observations (Functional Observation Battery Tests) and Neurotoxicity assessment (removal of animals from the home cage and open arena)
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

Motor activity and sensory reaction to stimuli
No relevant differences were noted in all parameters investigated between control and treated groups.

BODY WEIGHT AND WEIGHT GAIN
No differences of toxicological significance were seen. The reduction in body weight gain noted on Day 15 of the mating phase in high dose males was considered incidental.

FOOD CONSUMPTION
No intergroup differences were seen in food consumption.

HAEMATOLOGY
No changes of toxicological significance were recorded.
The difference of platelets between control and treated animals was due to the low value of one control animal (no. 90950020).
The statistically significant difference of mean corpuscular volume recorded between control and animals dosed with 100 mg/kg/day (6%) was considered unrelated to treatment.

No changes were recorded at the coagulation tests.

CLINICAL CHEMISTRY
Males dosed with 1000 mg/kg/day showed statistically significant increase of protein (9%), albumin (7%), phosphorus (12%), calcium (6%) and decrement of chloride (2%). Those receiving 300 mg/kg/day showed increase of phosphorus (13%) and decrease of glucose (27%).
Females dosed with 300 and/or 1000 mg/kg/day showed decreases of aspartate aminotransferase (up to 23%) and urea (22%).
The above changes were of low severity or not dose-related, therefore considered of no toxicological relevance.
Bile acids showed a 7-fold increment in males receiving 1000 mg/kg/day. However, since only 2 animals (nos. 90950072 and 90950076) showed high values and due to the lack of other relevant findings (e.g. liver markers), this alteration was considered of no toxicological significance.

URINALYSIS
No changes were recorded in urinalysis parameters.

ORGAN WEIGHTS
No differences in terminal body weight or organ weights were seen between the controls and treated animals of both sexes.

GROSS PATHOLOGY
No treatment-related changes were noted.
All observed changes had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under our experimental conditions.

HISTOPATHOLOGY
No treatment-related changes were noted.
All observed changes had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under our experimental conditions.

Spermatogenic cycle
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
A single control male (no. 90950012) showed severe bilateral tubular atrophy. Such lesion was considered an expression of spontaneous pathology.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for general toxicity is determined to 1000 mg/kg/day for males and females.

Executive summary:

Study design

The effects of3-Trimethylammonium propyl methacrylamide chloride (50% solution in water)after repeated dosing, as well as any affects and on reproductive function such as gonadal function, mating behaviour, conception, parturition and development of offspring up to Day 4post partumwere evaluated in rats.

The test item, dissolved inpurified water, was administered by oral gavage to 3 groups of 10 males and 10 females each as indicated below. A similar constituted control group (Group 1) received the vehicle alone during the treatment period.

 

Group Number	Treatment (mg/kg/day)	Level	Number of animals
1 2 3 4	0 100 300 1000	Control Low Medium High	10 males + 10 females 10 males + 10 females 10 males + 10 females 10 males + 10 females

Males were treated for 31 days including 2 weeks prior to pairing and continuously thereafter, up to the day before necropsy.

Females were dosed throughout the study including 2 weeks before pairing, thereafter during pairing, gestation and lactation periods until Day 3post partum.

The following parameters were evaluated in parental animals: body weight, clinical signs(including neurotoxicity assessment, motor activity and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology, clinical chemistry and urinalysis only males), litter data, macroscopic observations, organ weights and histopathological examination.

 

Mortality and fate of females

No mortality occurred in the study.

A total of 6 females were found not pregnant at necropsy: two in each of the control and high dose groups and one in each of the low dose and mid-dose groups.

The number of females with live pups on Day 4post partumwere: 8 in each of the control and in the high dose groups, 9 in each of the low and mid-dose groups.

 

Clinical signs and clinical observations (Functional ObservationBatteryTests)

Clinical signs and functional observation battery tests were unaffected by treatment.

 

Body weight and body weight gain

Body weight and body weight gain were unaffected by treatment.

 

Food consumption

No effects on food consumption were observed.

 

Motor activity and sensory reaction to stimuli

No relevant differences were noted in all parameters investigated between control and treated groups.

Haematology

No changes of toxicological significance were observed in haematological parameters and coagulation tests.

 

Clinical chemistry

No toxicological relevant changes were noted, even if an increased value of bile acids was observed only in single males treated at 1000 mg/kg/day.   

 

Urinalysis

No changes were recorded.

 

Terminal body weight and organ weights

No differences in terminal body weight or organ weights were seen between the controls and treated animals of both sexes.

Macroscopic and microscopic observations

No treatment-related changes were noted at macroscopic and microscopic examinations.

Evaluation of the spermatogenic cycle did not show differences between the groups. Regular layering in the germinal epithelium was noted.

 

Conclusion

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for general toxicity is determined to 1000 mg/kg/day for males and females.