Diallyldimethylammonium 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:

supporting study

Study period:

The study was performed between 28 August 2009 and 7 December 2009. The in-life phase of the study was conducted between 23 September 2009 (first day of treatment) and 7 November 2009 (final day of necropsy).

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15/9/2009, date of signature: 26/11/2009

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diallyldimethylammonium chloride

EC Number:

230-993-8

EC Name:

Diallyldimethylammonium chloride

Cas Number:

7398-69-8

Molecular formula:

C8H16N.Cl

IUPAC Name:

diallyldimethylammonium chloride

Details on test material:

Identification: Diallyl Dimethyl Ammonium chloride
CAS number: 7398-69-8
EINECS number: 230-993-8
Description: Clear colourless liquid
Purity: 63.52%
Batch number: GV9H0728
Date received : 25 August 2009
Storage conditions : room temperature in the dark
Expiry date : 05 August 2011

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A sufficient number of male and female Wistar Han™:HsdRccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: (P) approximately 12 wks
- Weight at study initiation: (P) Males: 329 - 376 g; Females: 175 - 221 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): The animals were allowed free access to food. A pelleted diet was used throughout the treatment period.
- Water (e.g. ad libitum): The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 13 days during which their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature controls were set to achieve target values of 21 ± 2°C.
- Humidity (%): The relative humidity controls were set to achieve target values of 55 ± 15%.
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): Low intensity fluorescent lighting was controlled to give tweleve hours continous light and twelve hours darkness.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of the study the test material was prepared at the appropriate concentrations as a solution in distilled water.
The stability and homogeneity of the test material formulations were determined. Results show the formulations to be stable for at least fourteen
days. Formulations were prepared daily and stored at approximately +4ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Prior to preparing the test material formulations a vehicle determination was carried out to ensure the suitability of the vehicle.
- Amount of vehicle (if gavage): 5 ml/kg/day

Details on mating procedure:

- M/F ratio per cage: one male:one female basis with each dose group (during mating phase).
- Length of cohabitation: For a period of 14 days. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
- Proof of pregnancy: Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation)
- After successful mating each pregnant female was caged (how): Females were housed individually during the period of gestation and lactation in soolid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Any other deviations from standard protocol: None.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of test material formulations were taken and analysed for concentration of Diallyl Dimethyl Ammonium chloride.

The concentration of Diallyl Dimethyl Ammonium chloride in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

The results indicated that the prepared formulations were within 7% of the nominal concentration.

Duration of treatment / exposure:

Up to forty-five consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females).

Frequency of treatment:

The test material was administered daily.

Details on study schedule:

Chronological Sequence of Study
i) Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.
iii) Following evidence of mating (designated as Day 0 post coitum) , the males were returned to their original cages and females were transferred to individual cages.
iv) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and landmark developmental signs were also performed during this period.
v) The male dose groups were killed and examined macroscopically on Day 43.
vi) At Day 5 post partum, all surviving females and surviving offspring were killed and examined macroscopically.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Vehicle alone(distilled water)
Basis:
other: Control animals were treated with 5 ml/kg/day of Distilled water.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg/day (incorporating a correction factor for 63.52% purity)
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
500 mg/kg/day (incorporating a correction factor for 63.52% purity)
Basis:
nominal conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 mg/kg/day (incorporating a correction factor for 63.52% purity)
Basis:
nominal conc.

No. of animals per sex per dose:

Groups of 10 male and 10 female animals per dose group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on results included in a chemical satey report on the substance
- Other: The test material was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at regular intervals.

Positive control:

No positive control animals.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, soon after dosing and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends (except for females during parturition where applicable). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination. Surviving females were weighed weekly until mating was evident. Bodyweights were then recorded on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION: Yes
- During the maturation period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14–20. For surviving females with live litters, food consumption was recorded during the lactation period (Days 1 – 4).

Weekly food efficiency (bodyweight gain/food intake) was calculated retrospectively for males and for females during the pre-mating phase.


WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

OTHER:
Pregnancy and Parturition
Each pregnant female was observed at approximately 0830, 1230 and 1630 hours and around the period of expected parturition. Observations were carried out at approximately 0830 and 1230 hours at weekends and public holidays. The following was recorded for each female:
i) Date of pairing
ii) Date of mating
iii) Date and time of observed start of parturition
iv) Date and time of observed completion of parturition

Oestrous cyclicity (parental animals):

A vaginal smear was prepared for each female and the stage of he oestrous cycle or the presence of sperm was recorded.

Sperm parameters (parental animals):

Parameters examined inmale parental generations: testis and epididymis spermatozal content.

Litter observations:

STANDARDISATION OF LITTERS
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded and offspring were individually identified within each litter by a tattoo on Day 1 post partum.
For each surviving litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily and reported on Day 1 and 4 post partum
iii) Sex of offspring on Days 1 and 4 post partum
iv) Clinical condition of offspring from birth to Day 5 post partum
v) Individual offspring and litter weights on Days 1 and 4 post partum

Physical Development:
All live offspring were assessed for surface righting reflex on Day 1 post partum.

GROSS EXAMINATION OF DEAD PUPS:
Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43.
- Maternal animals: Surviving adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.

GROSS NECROPSY
- All adult animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
- For all females the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights: The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Epididymides
Testes

Histopathology: Samples of the following tissues were preserved from all animals from each dose group, in buffered 10% formalin, except where indicated:
Coagulating gland
Prostate
Epididymides *
Seminal vesicles
Ovaries
Testes *
Mammary tissue (females only)
Uterus/Cervix
Pituitary
Vagina
* preserved in Bouin’s fluid and then transferred to 70% Industrial Methylated Spirits (IMS) after approximately forty-eight hours

Postmortem examinations (offspring):

SACRIFICE
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.

GROSS NECROPSY
Offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Statistics:

The following parameters were subjected to statistical analysis:
- Bodyweight and bodyweight change
- Food consumption for females during gestation and lactation
- Litter data
- Sex ratio
- Implantation losses and viability indices
- Offspring bodyweight and bodyweight change
- Offspring surface righting
- Adult absolute and bodyweight relative organ weights (Males)

Reproductive indices:

Mating Performance and Fertility:
The following parameters were calculated from the individual data during the mating period of the parental generation:

i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = Number of animals mated/Number of animals paired x 100
Pregnancy Index (%) = Number of pregnant females/Number of animals mated x 100

Gestation and Parturition Data:
The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.

i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii) Parturition Index
The following was calculated for each group:
Parturition Index (%) = Number of females delivering live offspring/Number of pregnant females x 100

Offspring viability indices:

Litter Responses:
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
% pre-implantation loss = [(Number of Corpora Lutea - Number of implantation sites)/Number of pregnant females] x 100

% post-implantation loss = [(Number of implantation site - Total number of offspring born)/Number of implantation sites] x 100

ii) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = Number of offspring alive on Day 1/Number of offspring born x 100
Viability Index 1 (%) = Number of offspring alive on Day 4/Number of offspring alive on Day 1 x 100

iii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Day 1 and 4 post partum, using the following formula:
Number of male offspring / Total number of offspring x 100

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:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One female treated with 1000 mg/kg/day was found dead on Day 3 of lactation. There were no further unscheduled deaths.

No toxicologically significant clinical findings were observed throughout the study.

Up to 1 hour post dosing on Day 6 of treatment one 1000 mg/kg/day male was observed with occasional body tremors, lethargy and a decreased respiratory rate. The animal had recovered by 5 hours post dosing. These findings may be attributed to transient irritation caused by a small amount of test material entering the respiratory tract during the removal of the dosing needle. In the absence of any histopathological correlates these observations are considered to be of no toxicological significance. In addition, episodes of increased salivation were evident in the 1000 mg/kg/day dose group. Two 1000 mg/kg/day males and one 500 mg/kg/day male showed staining around the mouth, whilst incidental episodes of staining were seen throughout the female treatment animals. Furthermore, one 1000 mg/kg/day female also showed episodes of chromodacryorrhea. Increased salivation is often recorded following the oral administration of an unpleasant tasting test material formulation and is considered not to be indicative of systemic toxicity. The remaining observations are considered incidental and not to represent an adverse effect of treatment.
No such effects were detected in males treated with 50 mg/kg/day.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No treatment-related effect on bodyweight development was detected for treated animals, in comparison to controls.
A slight but statistically significant reduction in bodyweight gain (P<0.05) was evident in the 1000 mg/kg/day males during Week 1. Furthermore, a slight but statistically significant increase in bodyweight gain (P<0.05) was detected throughout the male treatment groups during Week 6. In the absence of an overall effect on bodyweight gain or histopathological correlates these findings are considered to be of no toxicological importance.
No such effects were detected in the female treatment groups, in comparison to controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating: No adverse effect on mating performance was detected. All animals from the control and treated groups mated with four days of pairing.

Fertility: No adverse effect on fertility was evident in the treated animals, in comparison to controls. All treated and control females achieved pregnancy.

Gestation Length: There was no adverse effect on gestation lengths between treated and control females. Females from all treated groups went on to give birth following 22 to 23.5 days of gestation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No adverse effect on organ weights was detected for treated animals, in comparison to controls.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Necropsy (Adults): The incidental signs detected in the adults were considered to be incidental and of no toxicological importance.
One control male had small and flaccid testes and one 1000 mg/kg/day male had testes which were both flaccid.
No macroscopic abnormalities were detected in the remaining animals at terminal kill.
The 1000 mg/kg/day female which was found dead was observed with reddened lungs, reddened glandular region of the stomach and fluid filled large intestines.


HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related changes were observed.

All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treated groups, all were considered to be without toxicological significance.

PITUITARY: Isolated instances of vacuolation of the pars anterior and developmental cyst formation were seen among the 1000 mg/kg/day dose group animals and were without toxicological significance.

TESTIS/EPIDIDYMIS: Bilateral atrophy, with associated reduced spermatozoal content and/or cellular debris in the epididymis, was seen for two control and two 1000 mg/kg/day males. Testicular atrophy is seen occasionally as a spontaneous change among laboratory maintained rats of this age and strain.

SEMINAL VESICLES/COAGULATING GLANDS: No histopathological changes were observed.

PROSTATE: No histopathological changes were observed.

MAMMARY GLAND: Glandular hyperplasia was observed in the mammary tissue of all females examined. The appearance of the mammary tissue is consistent with pregnancy and lactation.

OVARY: No histopathological changes were seen.

UTERUS: Focal haemorrhage (with haematoma formation on one occasion), foam cell accumulations and haemosiderin pigment deposition were all observed in the peripheral uterine tissues of all females examined. These changes are commonly encountered consequences of pregnancy and parturition in the female rat.

VAGINA: No histopathological changes were observed.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The oral administration of Diallyl Dimethyl Ammonium chloride to rats for a period of up to forty-five consecutive days at dose levels of up to 1000 mg/kg/day did not result in any treatment-related effects.

Remarks on result:

other: Generation: Parental and offspring (migrated information)

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)

VIABILITY (OFFSPRING)
There were no obvious differences in the number of corpora lutea or implantation sites from treated females when compared to controls, and pre-implantation losses from treated animals were comparable to controls. Furthermore, no obvious effects on litter size, sex ratio and offspring viability were detected from treated animals, in comparison to controls.

CLINICAL SIGNS (OFFSPRING)
No adverse effect on surface righting reflex was detected in treated animals, in comparison to controls.

BODY WEIGHT (OFFSPRING)
No adverse effect on total litter weights, offspring bodyweight change was detected in treated animals, in comparison to controls.

GROSS PATHOLOGY (OFFSPRING)
The incidental signs detected in the offspring were considered to be incidental and of no toxicological importance. One 1000 mg/kg/day litter was terminated early as the dam was found dead. No abnormalities were detected in the offspring. In addition, one male offspring at 1000 mg/kg/day was found dead, no observations for this animal were recorded. The interim death offspring from the 50 mg/kg/day dose group revealed no milk in the stomach. There were no interim death offspring in the 500 mg/kg/day group. At terminal kill one control male had a dark red left testis and one animal female pup in a 50 mg/kg/day litter was small. There were no macroscopic abnormalities detected in the remaining animals at terminal kill.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of Diallyl Dimethyl Ammonium chloride to rats for a period of up to forty-five consecutive days at dose levels of up to 1000 mg/kg/day did not result in any treatment-related effects. The 'No Observed Effect Level' (NOEL) was therefore considered to be 1000 mg/kg/day.

Executive summary:

Introduction.

The study was performed to screen for potential adverse effects of the test material on reproduction including offspring development and provides an initial hazard assessment for effect on reproduction. The study complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995).

This study was also designed to comply with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods.

The test material was administered by gavage to three groups, each of ten male and ten female Wistar Han™:HsdRccHan™:WIST strain rats, for up to forty-five consecutive days, (including a two week maturation phase, pairing, gestation and early lactation for females) at dose levels of 50, 500 and 1000 mg/kg/day (incorporating a correction factor for 63.52% purity). A control group of ten males and ten females was dosed with vehicle alone (Distilled water).

Clinical signs, bodyweight development, dietary intake and waterconsumption were monitored during the study. 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Adult males were terminated on Day 43, and all surviving females and offspring on Day 5 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was perford.

Results.

Mortality.

One female treated with 1000 mg/kg/day was found dead on Day 3 of lactation and consequently the litter was killed. 

There were no further unscheduled deaths.

Clinical Observations.

No toxicologically significant clinical findings were observed throughout the study.

Bodyweight.

No treatment-related effect on bodyweight development was detected for treated animals, in comparison to controls.

Food Consumptions.

No adverse effect on dietary intake was detected for treated animals, in comparison to controls.

Water Consumption.

There was no adverse effect on water consumption for treated animals, in comparison to controls.

Reproductive Screening:

Mating, Gestation and Fertility.

There were no intergroup differences in mating performance or gestation lengths.

Pregnancy was achieved for all female treated and control animals.

Offspring Litter Size, Sex Ratio and Viability. 

No adverse effects on litter size and viability were evident for treated animals, in comparison to controls. There was no obvious difference in sex ratio for litters from treated females in comparison to controls.

Offspring Growth and Development.

There was no adverse effect on offspring growth and development for animals from treated females, in comparison to controls.

Offspring Observations.

The incidental findings detected in the control and treated groups were considered to be unrelated to treatment.

Necropsy.

The incidental signs detected in the adults and offspring were considered to be of no toxicological importance.

Uterine Examination.

From evaluation of the corpora lutea and implantation data, no adverse effects were detected for treated animals, in comparison to controls. 

Organ Weights.

No adverse effect on organ weight measurement was detected for treated animals, in comparison to controls.

Histopathology.

There were no treatment-related changes in the reproductive organs examined.

Conclusion.

The oral administration of Diallyl Dimethyl Ammonium chlorideto rats for a period of up to forty-five consecutive days at dose levels of up to 1000 mg/kg/day did not result in any treatment-related effects.

The ‘No Observed Effect Level’ (NOEL) was therefore considered to be 1000 mg/kg/day.