(3-methacrylamidopropyl)trimethylammonium chloride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

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

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
RTC code : 13235

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).
The animals were housed in a limited access rodent facility. 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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

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 dosages, selected in consultation with the Sponsor, were 100, 300 and 1000 mg/kg/day.

Details on mating procedure:

Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification occurred.

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’s 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 were treated for a total of 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 3 post partum.

Frequency of treatment:

daily

Details on study schedule:

Parental animals mated 2 weeks after treatment.

Doses / concentrations

Remarks:

Doses / Concentrations:
100 300 1000 mg/kg/day /10 30 100 mg/ml
Basis:
nominal conc.

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels had been selected in consultation with the Sponsor based on information from a previous non GLP Compliant study (RTC Study no.: 90940EXT).

Examinations

Parental animals: Observations and examinations:

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. Measurements were performed using a computer generated random order.

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. Measurements were performed using a computer generated random order.

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.
Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.
Body weight measured weekly during the mating phase is not presented in this report but retained and archived as study raw data.

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 underthe same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kgof 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

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning starting two weeks before pairing until a positive identification of copulation is made. The vaginal smear data was examined to determine the following:

a) anomalies of the oestrous cycle;
b) pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle)

Litter observations:

Parturition and gestation length
A parturition check was performed from Day 20 to Day 25 post coitum. Females which did not give birth after 25 days of post coitum period were sacrificed shortly after. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring are first detected in the cage was considered Day 0 post partum.

Pups identification, weight and observation
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1 and 4 post partum and before necropsy.
Litter observation was performed daily.

Postmortem examinations (parental animals):

Parental animals were killed by exsanguination under isofluorane anaesthesia.

Parental males:
The males were killed after completion of the 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.

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).

The examination was restricted as detailed below:

a) Tissues listed 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.

Postmortem examinations (offspring):

Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Thiopenthal.

All pups found dead in the cage were examined for external and internal abnormalities.
All live pups sacrificed at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection.

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.

Reproductive indices:

Males
Copulatory Index (%) = no. of animals mated/no. of animals paired x 100

Fertility Index (%) = no. of males which induced pregnancy/no. of males paired x 100

Females
Copulatory Index (%) = no. of animals mated/no. of animals paired x 100

Fertility Index (%) = no. of pregnant females/no. of females paired x 100

Males and females
Copulatory Interval = Mean number of days between pairing and mating

Offspring viability indices:

Females
Pre-birth loss was calculated as a percentage from the formula:

(No. of visible implantations - total litter size at birth )/No. of visible implantations x 100


Pup loss at birth was calculated as a percentage from the formula:

(Total litter size - live litter size)/Total litter size x 100

Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:

(Total litter size at birth - live litter size at Day 4)/Total litter size at birth x 100

Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINUCAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMAL)
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.
No intergroup differences were seen in food consumption.

REPRODUCTIVE FUNCTION: OESTRUS CYCLE (PARENTAL ANIMAL)
Oestrous cycles were unaffected by treatment.

REPRODUCTIVE FUNCTION: SPERMATOGENIC CYCLE (PARENTAL ANIMAL)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMAL)
No differences in pre-coital interval or number of copulation plugs were noted between the control and treated animals.
The fertility index was comparable between the control and treated groups.

ORGAN WEIGHT (PARENTAL ANIMAL)
No differences in terminal body weight or organ weights were seen between the controls and treated animals of both sexes.

GROSS PATHOLOGY (PARENTAL ANIMAL)
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 (PARENTAL ANIMAL)
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.

REPRODUCTIVE FUNCTION: SPERMATOGENIC CYCLE (PARENTAL ANIMAL)
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 (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups:
Litter size, pup losses, litter weight and mean pup weight were comparable between groups.

Clinical signs of pups:
Clinical signs of pups were comparable between groups.

Necropsy findings in decedent pups and in pups sacrificed on Day 4 post partum:
No findings related to treatment were seen.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Oestrus cycle – Pre-pairing period - Group summary data

 

 -----------------------------------------------------------------------------------------------------------------------------------

           Group 1              Group 2             Group 3             Group 4

 Animal   Oestrus    Animal   Oestrus   Animal   Oestrus   Animal   Oestrus

 Number   Cycles     Number   Cycles    Number   Cycles    Number   Cycles

 -----------------------------------------------------------------------------------------------------------------------------------

 90950001    4       90950021    2      90950041    3      90950061    4

 90950003    3       90950023    3      90950043    3      90950063    3

 90950005    2       90950025    1      90950045    3      90950065    1

 90950007    3       90950027    1      90950047    3      90950067    1

 90950009    2       90950029    2      90950049    1      90950069    2

 90950011    3       90950031    2      90950051    3      90950071    2

 90950013    4       90950033    3      90950053    3      90950073    2

 90950015    4       90950035    3      90950055    4      90950075    3

 90950017    2       90950037    2      90950057    3      90950077    3

 90950019    2       90950039    3      90950059    3      90950079    2

 

    Means  2.9                  2.2                 2.9                 2.3

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 Note: The number of oestrus cycles is based on the number of non sequential days the dams were in oestrus.

 

Reproductive parameters of males - Summary

 

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                              Group       1          2         3          4

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   Copulatory Index%                    100.0     100.0      100.0     100.0

 

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   Fertility Index%                      80.0      90.0       90.0      80.0

 

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Copulatory Index (%)	=	No. of animals mated	x 100
(males)		No. of animals paired

 

 

Fertility Index (%)	=	No. of males with induced pregnancy	x 100
(males)		No. of animals paired

 

 

  Reproductive parameters of females - Summary

 

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                              Group       1          2         3          4

-----------------------------------------------------------------------------------------------------------------------------------

 

   Copulatory Index%                    100.0     100.0      100.0     100.0

 

-----------------------------------------------------------------------------------------------------------------------------------

 

   Fertility Index%                      80.0      90.0       90.0      80.0

 

-----------------------------------------------------------------------------------------------------------------------------------

 

 

Copulatory Index (%)	=	No. of animals mated	x 100
(females)		No. of animals paired

 

 

Fertility Index (%)	=	No. of pregnant females	x 100
(females)		No. of females paired

 

Applicant's summary and conclusion

Conclusions:

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for reproduction/developmental toxicity screening test could be considered 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 a total of 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 each in the control and high dose groups and one each in the low dose and mid-dose groups.

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

Oestrus cycle, reproductive parameters, pairing combination and mating performance

 No treatment related changes were seen.

 

Implantation, pre-birth loss data and gestation length of females

No treatment-related effects were seen.

 

Litter data and sex ratios

Litter data and sex ratios were unaffected by treatment.

 

Clinical signs of pups

Clinical signs of pups were comparable between the groups

Necropsy findings in decedent pups and in pups sacrificed on Day 4post partum

 No treatment-related effects were seen.

 

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 both general toxicity and reproduction/developmental toxicity could be considered 1000 mg/kg/day for males and females.