1-Propanaminium, N,N,N-trimethyl-3-(octadecyloxy)-, chloride (1:1)

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:

28 August 2007 to 20 May 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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)

Remarks:

Date of inspection: 21 August 2007 Date of Signature: 15 October 2007

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

EC Number:

700-414-8

Cas Number:

23328-71-4

Molecular formula:

C24H52ClNO

IUPAC Name:

trimethyl[3-(octadecyloxy)propyl]azanium chloride

Details on test material:

- Name of test material (as cited in study report): 3-Octadecyloxypropyl-N,N,N-trimethylammonium chloride
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other than submission substance): Not applicable
- Substance type: white solid
- Physical state: solid
- Analytical purity: Not reported
- Impurities (identity and concentrations): Not reported
- Composition of test material, percentage of components: Not reported
- Isomers composition: Not reported
- Purity test date: Not reported
- Lot/batch No.: Exp. I-070518
- Expiration date of the lot/batch: Not reported
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Not reported.
- Storage condition of test material: room temperature in the dark
- Other: Not reported.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, UK.
- Age at study initiation: 8 weeks of age.
- Weight at study initiation: The males weighed 306 to 360g; the females weighed 209 to 274 g
- Fasting period before study: Not reported.
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel grid lids and softwood flake bedding (Datesand Ltd. Cheshire, UK). During the mating phase, animals were transferred to similar cages on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females remained in the mating cage, housed individually during gestation and lactation, with softwood flakes.
- Diet (e.g. ad libitum): A pelleted diet Rodent PMI 5002 (Certified) diet, BCM IPS Limited, London, UK ad libitum. The diet was routinely analysed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
- Water (e.g. ad libitum): Mains drinking water ad libitum. The water was routinely analysed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC.
- Humidity (%): 55 ± 15%.
- Air changes (per hr): At least 15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.

IN-LIFE DATES: From: Day 0 To: Day 43 (males); Day 0 To: Day 5 post partum (females).
Administration / exposure

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was prepared at 1, 2, 5 and 25 mg/kg/day as a suspension in Arachis oil BP. The stability and homogeneity of the test material formulations were determined at the test laboratory. The formulations to be stable for at least 14 days. Formulations were therefore prepared weekly and stored at approximately 4ºC in the dark.

DIET PREPARATION
Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Analysis of the methanol and a blank Arachis Oil BP produced no signal that interfered with the signal due to the test material.
- Amount of vehicle (if gavage): 4 ml/kg.
- Lot/batch no. (if required): Not reported.
- Purity: Not reported.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Summary; The concentration of 3-Octadecyloxypropyl-N, N, N-Trimethylammonium chloride in the test material formulations was determined by high performance liquid chromatography mass selective (HPLC/MS) using an external standard technique.

Materials and methods; The test material formulations were extracted with methanol to give a final, theoretical test material concentration of approximately 0.0002 mg/ml.
Standard solutions of test material were prepared in methanol at a nominal concentration of 0.0002 mg/ml.
The standards and samples were analysed by HPLC.
The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
The test material formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for fourteen days.
The test material formulations were sampled and analysed within three days of preparation.

Results; A range of standard solutions covering the concentration range 0 to 0.000297 mg/ml, were prepared and analysed.
The detector response was shown to be linear up to 0.000297 mg/ml.
Analysis of the solvent and a blank Arachis Oil BP (control) produced no signal that interfered with the signal due to the test material.
The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.
Conclusion; The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

Duration of treatment / exposure:

From: Day 0 To: Day 42 (males); Day 0 To: Day 4 post partum (females).

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1 mg/kg/day
Basis:
other: nominal in vehicle

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2 mg/kg/day
Basis:
other: nominal in vehicle

Doses / concentrations 3

Remarks:

Doses / Concentrations:
5 mg/kg/day
Basis:
other: nominal in vehicle

Doses / concentrations 4

Remarks:

Doses / Concentrations:
25 mg/kg/day
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

10 animals per sex per dose and the control group.

Control animals:

yes, concurrent vehicle

Details on study design:

i) Groups of 10 male and 10 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) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iii) One day prior to pairing (Day 14), blood samples were taken from five selected males and females, from each dose group and analysed for haematological and blood chemical assessment.
iv) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of 14 days.
v) 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.
vi) On completion of mating (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vii) 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.
viii) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
ix) Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically.
x) At Day 5 post partum, all surviving offspring were killed and examined macroscopically.

- Dose selection rationale: The dose levels were chosen based on the results of a preliminary range-finder performed as part of the study.
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not applicable

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to 30 minutes after dosing, and 1 and 5 hours after dosing, during the working week. Animals were observed immediately before dosing,
after dosing, and 1 hour after dosing at weekends and public holidays (except for females during parturition where applicable).
- Cage side observations checked: Overt signs of toxicity, ill-health and behavioural change.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately before dosing, up to 30 minutes after dosing, and 1 and 5 hours after dosing, during the working week. Animals were observed immediately before dosing,
after dosing, and 1 hour after dosing at weekends and public holidays (except for
females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident (or at death).
Bodyweights were then recorded for females 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. This was continued for males after the mating phase. For females showing
evidence of mating, food consumption was recorded for the periods covering Days 0-7,
7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1
and 4 post partum.

FOOD EFFICIENCY: Yes
- Food efficiency (the ratio of bodyweight change/dietary intake) was calculated
retrospectively for males throughout the study period and for females during maturation and the first two weeks of gestation. Due to offspring growth and milk production, food efficiency could not be accurately calculated during the final week of gestation and during lactation.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Haematological and blood chemical investigations were performed on Day 14 (day prior to pairing). Blood samples were obtained from the lateral tail vein at Day 14.
- Anaesthetic used for blood collection: Not reported.
- Animals fasted: No
- How many animals: 5 males and 5 females selected from each test and control group.
- Parameters checked in tables 16 -19 which are attached were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Behavioural assessment: Prior to the start of treatment and at weekly intervals thereafter.
Functional Performance Tests: Prior to termination.
Sensory Reactivity: Prior to termination.

- Dose groups that were examined:
Behavioural assessment: All animals.
Functional Performance Tests: Five selected males and females per dose level.
Sensory Reactivity: Five selected males and females per dose level.

- Parameters examined:
Behavioural 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 colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation and lachrymation.
Functional Performance Tests: Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall time period and also during the final 20% of the period (considered to be the asymptotic period).
Forelimb/Hindlimb Grip Strength. An automated 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. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.
Sensory Reactivity: Animals were assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The following parameters were observed:
Grasp response, touch escape, vocalisation, pupil reflex, toe pinch, startle reflex, tail pinch, blink reflex and finger approach.

OTHER:
MATING
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of
up to 14 days. 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) and the males were subsequently returned to their original holding
cages (unless required for additional pairing). Mated females were housed individually
during the period of gestation and lactation.

PREGNANCY AND PARTURITION
Each pregnant female was observed at approximately 08:30, 12:30 and 16:30 hours and
around the period of expected parturition. Observations were carried out at
approximately 08:30 and 12:30 hours at weekends. The following was recorded for each female:
i) Date of mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

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

PHYSICAL DEVELOPMENT
All live offspring were assessed for surface righting reflex on Day 1 post partum.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by
intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.
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 1% ammonium polysulphide solution. In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ weights:
Parental animals: The following organs, removed from the 5 selected males and parental females from each group that were killed at the end of the study, were dissected free from fat and weighed before fixation.
Adrenals, ovaries, brain, spleen, epididymides, testes, heart, thymus, kidneys, thyroid and liver.
The following reproductive organs were weighed from all animals that were killed at the end of the study: ovaries, epididymides and testes.
Offsprings: Necropsy findings checked in table 26 were included.

HISTOPATHOLOGY: Yes
Samples of the following tissues were preserved from 5 adult males and 5 females from each dose group, in buffered 10% formalin except where indicated.
Adrenals, aorta (thoracic), bone & bone marrow (femur including stifle joint), bone & bone marrow (sternum), brain (including cerebrum, cerebellum and pons), caecum, coagulating gland, colon, duodenum, epididymides (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), eyes (fixed in Davidson’s fluid), gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi. Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), lymph nodes (cervical and mesenteric), mammary gland, muscle (skeletal), ovaries, pancreas, pituitary, prostate, oesophagus, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), spleen, stomach, thyroid/parathyroid, trachea, testes (preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) up to 48 hours later), thymus, urinary bladder, uterus/cervix and vagina.
The following tissues were also removed from the remaining animals:coagulating gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes and uterus/cervix.

All tissues were despatched to Propath UK Ltd (Principal Investigator: T Hilling). The tissues from 5 selected control and 25 mg/kg/day dose group animals and those animals dying during the study, were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues from oagulating gland, epididymides, ovaries, pituitary, prostate, seminal vesicles, testes and uterus/cervix from the remaining control and 25 mg/kg/day were also processed.
Since there were indications of treatment-related histopathological changes in the trachea, oesophagus, stomach and thymus, examination was subsequently extended to include sections of sections of these tissues from 5 male and 5 female animals in the remaining groups.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Other examinations:

Not reported.

Statistics:

The following parameters were subjected to statistical analysis:
Quantitative functional performance data
Bodyweight and bodyweight change
Food consumption during maturation, gestation and lactation
Haematology, blood chemistry and absolute and bodyweight relative organ weights
Litter data
Sex ratio
Implantation losses and viability indices
Offspring bodyweight and bodyweight changes
Offspring surface righting reflex
The following statistical procedures were used:
Data were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
Non-parametric methods were used to analyse offspring sex ratio.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Probability values (p) were calculated as follows:
p<0.001 +++ --- ***
p<0.01 ++ -- **
p<0.05 + - *
p<0.1 (+) (-) (*)
p≥0.01 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs indicate negative differences. Asterisks refer to overall differences between group variation which is non-directional.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional 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:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Systematic toxicity in parental animals: Clinical signs and mortality; Summary incidences of daily clinical observations are given in Tables 1 and 2. A death occurred at a high dose (25 mg/kg/day) and a male were killed on welfare grounds after three weeks of treatment. No toxicologically significant clinical observation was observed. However, instances of respiratory abnormalities with or without excessive transient salivation developed during the first two weeks of dosing in animals of either sex dosed at 25 mg/kg/day. During the third week of treatment, one 25 mg/kg/day male, following a marked deterioration in condition in which pronounced findings of respiratory distress, diarrhoea, distended abdomen, was terminated on humane grounds. Further animals showed sporadic signs of pallor of the extremities, hunched posture and staining of the fur around the snout and mouth (the latter, probably resulting from the excessive salivation). Isolated instances of transient salivation around the time of dosing and noisy respiration were observed in males dosed at 5 mg/kg/day and one instance of hunched posture was detected in one female dosed at 2 mg/kg/day. The isolated nature of these changes would suggest they were associated with the treatment procedure (particularly at 2 mg/kg/day) rather than toxicity.

Body weight and and weight gain: Treatment had no toxicological impact on bodyweight of either sex, including females during gestation and lactation.

Food consumption: Food consumption of males throughout the study and of females during maturation showed no significant divergence from controls.

Water consumption: Daily visual inspection of water bottles revealed no intergroup differences.

Haematology: No treatment-related changes were detected in the haematological parameters examined. Statistical analysis of the data revealed no significant intergroup differences.

Clinical chemistry: No treatment-related changes were detected in the blood chemical parameters examined. Statistical analysis of the data revealed no significant intergroup differences.

Neurobehaviour: No toxicological significant functional observations.

Gross pathology: No treatment-related macroscopic abnormalities were detected for adult animals at terminal kill.

Organ weights: No toxicologically significant effects of treatment were detected.

Histopathology: Summary incidences of histopathological findings are given in Tables 3 and 4 attached. Histopathology findings further confirmed that the only clear treatment-related change identified ie. deciliation and flattening of the tracheal epithelial lining and changes to the oesophagus and stomach primarily present among animals dosed at 25 mg/kg/day was considered to be associated with the irritant properties of the test material and not systemic toxicity. Also detected was lymphoid atrophy in three 25 mg/kg/day females and one 5 mg/kg/day female. While the aetiology is uncertain, such changes are sometimes associated with stress responses to treatment.

Reproductive and developmental toxicity in parental animals
Mating: One high dose (25 mg/kg/day) female showed positive evidence of mating after three days of co-habitation with its male partner but subsequently was not observed to litter. The female was found not to have any evidence of implantation sites at necropsy and therefore had not achieved pregnancy. This isolated finding was considered more likely attributable to biological variability rather than toxicity.

Fertility: No adverse effects were detected.

Gestation length: No adverse effects were detected.

Reproductive and developmental toxicity in offspring
In total 9 females from the control and 25 mg/kg/day dose groups and all from 1, 2 and 5 mg/kg/day dose groups gave birth to a live litter and successfully reared young to Day 5 of age. The assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.
One high dose female was found not to be pregnant. Data for this single animal was considered to be atypical for the group, the assessment of litter responses is therefore based on animals which successfully reared young to termination (Day 5 of lactation).

Litter size and viability: Pre and post-implantation and post natal losses for all treatment groups were similar to the control. Sex ratio at birth, on Day 1 and on Day 4 were similar in all groups and did not indicate any obvious selective effect on survival for either sex.

Litter observation: No toxicologically significant clinical findings were observed.

Offspring growth and development: Offspring bodyweight for all dose groups on Day 1 of age and subsequent bodyweight gain to Day 4 of age was unaffected by treatment. Assessment of surface righting reflex did not indicate any adverse effect on offspring development at dosages up to 25 mg/kg/day.

Necropsy: No treatment related macroscopic abnormalities were detected for offspring.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

5 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Remarks:

systemic toxicity

Effect level:

2 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: No treatment-related findings were detected in rats dosed at 2 mg/kg/day.

3

Dose descriptor:

NOEL

Remarks:

reproductive and development toxicity

Effect level:

25 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Due to the absence of systemic toxicity at any doses tested.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1 Clinical Observations for Males - Summary Incidences

Dose Level (mg/kg/day)	Number of Animals	Clinical Observations	Number Showing Effects between Days:
			1-7	8-14	15P-21	22 - 28	29 - 35	36 - 42
0 (Control)	10	No abnormalities detected	10	10	10	10	10	10
1	10	Staining around the snout and mouth	0	0	0	0	1	0
		No abnormalities detected	10	10	10	10	9	10
2	10	Staining of fur	0	0	0	1	0	0
		No abnormalities detected	10	10	10	9	10	10
5	10	Increased salivation	0	0	1	0	0	1
		Noisy respiration	1	1	0	0	0	1
		No abnormalities detected	9	9	9	10	10	8
25	10-Sep	Decreased respiration	0	0	1	0	0	0
		Diarrhoea	0	0	1	0	0	0
		Distended abdomen	0	0	1	0	0	0
		Gasping respiration	0	0	1	0	0	0
		Increased salivation	0	4	9	7	5	7
		Irregular breathing (post dose)	0	0	0	1	0	0
		Increased respiration	0	0	0	1	0	0
		Killed in extremis	0	0	1	0	0	0
		Laboured respiration	0	0	1	0	0	1
		Lethargy	0	0	1	0	0	0
		Noisy respiration	2	6	8	7	7	5
		Pallor of the extremities	0	0	1	0	0	0
		Staining around the mouth	0	0	3	1	1	0
		Staining around the snout	0	0	0	0	1	0
		No abnormalities detected	8	3	0	0	0	1

P= animals paired for mating on Day 15

Table 2 Clinical Observations for Females - Summary Incidences

Dose Level (mg/kg/day)	Number of Animals	Clinical Observations	Number Showing Effects between Days:
			1-7	8-14	15P-21	22 - 28	29 - 35	36 - 42	43 - 49
0 (Control)	10-Sep	Generalised fur loss	0	1	1	0	2	3	3
		Scab formation	0	0	0	0	0	1	1
		Swelling (mass) behind left forelimb	0	0	0	0	1	1	1
		Died on Day 14 (during blood sampling)	0	1	0	0	0	0	0
		Removed for necropsy - Day 5 post partum	0	0	0	0	0	3	6
		No abnormalities detected	10	8	8	9	7	6	6
1	10	Diarrhoea	0	0	1	0	0	0	0
		Generalised fur loss	0	0	0	0	0	2	3
		Staining - ano-genital region	0	0	1	0	0	0	0
		Removed for necropsy - Day 5 post partum	0	0	0	0	0	3	7
		No abnormalities detected	10	10	9	10	10	8	7
2	10	Generalised fur loss	0	0	0	0	2	2	1
		Hunched posture	0	0	0	0	0	0	1
		Removed for necropsy - Day 5 post partum	0	0	0	0	0	2	8
		No abnormalities detected	10	10	10	10	8	8	6
5	10	Generalised fur loss	0	0	0	0	2	2	2
		Removed for necropsy - Day 5 post partum	0	0	0	0	0	1	9
		No abnormalities detected	10	10	10	10	8	8	7
25	10	Generalised fur loss	0	0	0	0	1	1	1
		Hunched posture	0	0	0	0	0	0	1
		Increased salivation	0	1	0	2	0	2	0
		Noisy respiration	0	2	3	1	1	2	0
		Staining around the mouth	0	0	0	0	2	0	0
		Removed for necropsy - Day 5 post partum	0	0	0	0	0	0	10
		No abnormalities detected	10	8	7	8	7	6	8

P= animals paired for mating on Day 15

Discussion

The oral administration of 3-Octadecyloxypropyl-N, N, N-trimethylammonium chloride to rats for a period of up to forty-five consecutive days at dose levels of 1, 2, 5 and 25 mg/kg/day was not associated with any toxicological significant clinical or functional observations. Treatment had no toxicological impact on bodyweight of either sex, including females during gestation and lactation. Similarly food consumption of males throughout the study and of females during maturation showed no significant divergence from controls. Assessment of haematological and blood chemistry parameters did not reveal any adverse effects of treatment. Mating performance was unaffected by treatment, with all but one high dose female successfully giving birth and rearing offspring to termination (Day 5 of age). Organ weight analysis and macroscopic necropsy of adults killed at study termination did not reveal any adverse effects of treatment.

The most remarkable treatment-related changes associated with the test material were related to its local irritancy. Preliminary investigations had already demonstrated that dosages of 50 mg/kg bodyweight and above resulted in premature deaths resulting from respiratory distress.

The clinical findings identified in the main phase of this study of respiratory abnormalities, increased salivation around the time of dosing and associated soiled/stained fur together with slightly lower dietary intake all most prevalent in animals receiving 25 mg/kg/day are all common observations in animals administrated unpalatable or slightly irritant test formulations. Gastric irritation was identified in the preliminary investigations and in the main phase of this study there was one treatment-related death at 25 mg/kg/day. Similar changes of gastric irritation and respiratory distress seen in the one treatment-related decedent were also identified in premature death preliminary animals.

Histopathology findings further confirmed that the only clear treatment-related change identified ie. deciliation and flattening of the tracheal epithelial lining and changes to the oesophagus and stomach primarily present among animals dosed at 25 mg/kg/day was considered to be associated with the irritant properties of the test material and not systemic toxicity.

Also detected was lymphoid atrophy in three 25 mg/kg/day females and one 5 mg/kg/day female. While the aetiology is uncertain, such changes are sometimes associated with stress responses to treatment.

Responses to treatment among animals dosed at 5 mg/kg/day were limited to isolated instances of increased salivation and noisy respiration and one instance of deciliation and flattening of the tracheal epithelial lining. None of these were considered to be toxicologically important and on this basis 5 mg/kg/day was considered to be the No Observed Adverse Effect Level.

Applicant's summary and conclusion

Conclusions:

The oral administration of the test material to rats by gavage at a maximum dose level of 25 mg/kg/day resulted in no toxicologically significant findings. However, secondary changes related to the irritancy of the test material including the death of one 25 mg/kg/day male were identified. There were also sporadic changes associated with the irritancy of the test material seen in rats dosed at 5 mg/kg/day, however, given their isolated nature 5 mg/kg/day can be considered the No Observed Adverse Effect Level (NOAEL).
No treatment-related findings were detected in rats dosed at 2 mg/kg/day, the No Observed Effect Level (NOEL) can therefore be cited as 2 mg/kg/day.
In the absence of systemic toxicity the NOEL for reproductive toxicity was considered to be 25 mg/kg/day. The test material does not meet the criteria for classification according to EU classification system.

Executive summary:

Introduction.The study was designed to investigate the systemic toxicity and potential adverse effects of the test material on reproduction (including offspring development) and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996).

Methods. The test material was administered by gavage to four groups each of ten male and ten female Sprague-Dawley Crl:CD^®(SD) IGS BR strain rats, for up to forty-five consecutive days, at dose levels of 1, 2, 5 and 25 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated prior to mating.

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.

Extensive functional observations were performed on five selected males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group on Day 4 post partum.

Males were terminated on Day 43, followed by the termination of all surviving females and offspring on Day 5post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Adult Responses:

Mortality.There were two deaths during the study, however only one (25 mg/kg/day) was related to treatment. There were no other unscheduled deaths during this study.

Clinical Signs.Instances of respiratory abnormalities developed in 25 mg/kg/day animals of either sex from the first week of dosing through to termination. No other treatment-related findings were identified.

Behavioural Assessment.Detailed behavioural assessments revealed no abnormalities.

Functional Performance Tests.No abnormalities were detected. Statistical analysis of the quantitative data revealed no significant intergroup differences.

Sensory Reactivity Assessments.There were no treatment-related changes in sensory reactivity. Statistical analysis of the quantitative data revealed no significant intergroup differences.

Bodyweights.Bodyweight development in males treated with 25 mg/kg/day was generally slightly lower than controls throughout the study. Females that received 2, 5 and 25 mg/kg/day showed slightly lower weight gains than controls during maturation and lactation but not during gestation.

Food Consumption and Food Efficiency.There was no toxicologically important adverse effect on food consumption identified throughout the study. Similarly food efficiency remained similar for all groups throughout the observation period.

Water Consumptions.No intergroup differences were noted.

Haematology.No treatment-related effects were detected in the haematological parameters measured.

Blood Chemistry.No treatment-related effects were detected in the blood plasma parameters measured.

Reproductive Performance:

Mating.No adverse effects were detected on mating performance.

Fertility.No adverse effects were detected on fertility performance.

Gestation Length.No adverse effects were detected on gestation length.

Litter Responses.No adverse effects in litter response were detected.

Offspring Litter Size and Viability.No adverse effects were detected.

Offspring Growth and Development.No adverse effects were detected.

Pathology.

Necropsy.No treatment-related findings were identified in animals sacrificed at study termination.

Organ Weights.There were no toxicologically significant differences between control and test treatment groups.

Histopathology.

Trachea:Deciliation and flattening of the epithelial lining were seen for four male rats, including the premature death animal, and for one female rat dosed at 25 mg/kg/day. One female rat dosed at 5 mg/kg/day was similarly affected. These findings likely represents the consequence of accidental instillation of the test material into the lower respiratory tract during dosing and is thus not a systemic effect of treatment.

Oesophagus: Mononuclear cell infiltration of the peripheral musculature is a common finding in gavage dosed animals and likely results from the physical trauma of dosing. In this investigation all surviving male rats and one female dosed at 25 mg/kg/day were affected against two female control rats indicating the possibility that the condition was exacerbated by the test material. Instances of the condition were also seen among animals from the remaining treatment levels but there was no convincing evidence of an influence of exposure to the test material other than among high dose males.

Stomach:  Hyperkeratosis and or acanthosis were seen in the forestomach of two male and of two female rats dosed at 25 mg/kg/day, but not at any other treatment level. Focal ulceration was also seen for one male rat dosed at 25 mg/kg/day.

Thymus Lymphoid atrophy was observed for three female rats dosed at 25 mg/kg/day and for one female rat dosed at 5 mg/kg/day. Although lymphoid atrophy is occasionally seen among control animals and the severity in each case in this investigation was minimal a true effect of treatment cannot be excluded at the high dose level.

Conclusion.The oral administration of 3-Octadecyloxypropyl-N, N, N-trimethylammonium chloride to rats by gavage at a maximum dose level of 25 mg/kg/day resulted in no toxicologically significant findings. However, secondary changes related to the irritancy of the test material including the death of one 25 mg/kg/day male were identified. There were also sporadic changes associated with the irritancy of the test material seen in rats dosed at 5 mg/kg/day, however, given their isolated nature 5 mg/kg/day can be considered the No Observed Adverse Effect Level (NOAEL).

No treatment-related findings were detected in rats dosed at 2 mg/kg/day, the No Observed Effect Level (NOEL) can therefore be cited as 2 mg/kg/day.

In the absence of systemic toxicity the NOEL for reproductive toxicity was considered to be 25 mg/kg/day. The test material does not meet the criteria for classification according to EU classification system.