Docosyltrimethylammonium methyl sulphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

07.04.2008 - 22.05.2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 407). Justification for read-across see chapter 1 of the chemical safety report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

EC Number:

271-756-9

EC Name:

Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

Cas Number:

68607-24-9

IUPAC Name:

68607-24-9

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Approximately 190 g males, 140 g females
- Fasting period before study: not applicable
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in MIII type; height 15 cm.) with sterilized sawdust as bedding material and paper as cage-enrichment.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 - 21.8
- Humidity (%): 31 - 71
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing, and were homogenized to visually acceptable levels. In order to obtain homogeneity, the test substance formulations were heated in a water bath with a maximum temperature of 39°C for a maximum of 33 minutes. No correction was made for the purity of the test substance.

DIET PREPARATION
- Rate of preparation of diet (frequency): daily within 4 hours prior to dosing
- Storage temperature of food: At ambient temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0, 10, 50, 150 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg b.w.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed on a single occasion after the in-life phase for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). The concentrations analysed in formulations were in agreement with target concentrations (i.e. mean accuracies between 85% and 115 %.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose.

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 50, 150 mg/kg/day
Basis:
nominal in water

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels are based on results of a 5-day dose range finding study withthe test substance.

- Rationale for animal assignment: random, by computer-generated algorithm.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations: Mortality / Viability


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly


FOOD CONSUMPTION: Weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.


FUNCTIONAL OBSERVATIONS: Yes
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all groups
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength, motor activity test

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination at the end of the treatment
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight (with a maximum of 20 hours)
- How many animals: all animals
- Parameters examined: white blood cells, differential leucocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination at the end of the treatment
- Animals fasted: Yes (iso-flurane)
- How many animals: all animals
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate


URINALYSIS: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Adrenal glands, (Aorta), Brain [cerebellum, mid-brain, cortex], Caecum, Cervix, (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve [if detectable] and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Oesophagus), Ovaries, (Pancreas), Peyer's patches [jejunum, ileum] if detectable, (Pituitary gland), (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid [if detectable], (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions

Tissues/organs mentioned in parentheses were not examined by the pathologist since there were no changes in macroscopic appearance indicative of (potential) toxicity.

The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus


HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Main group 1 and 4 animals,
- all tissues from animal nos. 19, 38 and 39 which died spontaneously or were terminated in extremis,
- all gross lesions.

Statistics:

The following statistical methods were used to analyze the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
One male at 150 mg/kg/day (no.19) was found dead on day 16 and two females at 150 mg/kg/day (nos. 38 and 39) were sacrificed in extremis on days 16 and 8, respectively.
No further mortality occurred during the study period.
Lethargy, hunched posture, abdominal swelling, piloerection, maculate erythema of the tail, dehydration, a lean appearance and/or hypothermia were noted in all animals at 150 mg/kg/day during the observation period. In addition, the female that was sacrificed in extremis on day 8 appeared moribund and showed a flat posture.

No clinical signs (of toxicity) were noted in control animals and animals at 10 and 50 mg/kg/day.

Salivation noted among males at 50 mg/kg/day and males and females at 150 mg/kg/day was considered to be a physiological response rather than a sign of systemic toxicity considering the nature of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance.
Rales were noted in one female at 50 mg/kg/day and one male at 150 mg/kg/day and alopecia and scabs were noted in all females at 50 mg/kg/day. These findings are occasionally noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed or in the absence of a treatment-related distribution, these were considered signs of no toxicological significance.


BODY WEIGHT AND WEIGHT GAIN
Lower body weights and body weight gain were noted for animals at 50 and 150 mg/kg/day throughout the treatment period, achieving a level of statistical significance on most occasions. Most animals at 150 mg/kg/day showed body weight loss during the treatment period.
Body weights and body weight gain of animals at 10 mg/kg/day remained in the same range as controls over the study period.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Lower food consumption and relative food consumption were noted for males at 150 mg/kg/day and females at 50 and 150 mg/kg/day. Males at 50 mg/kg/day showed minor lower food consumption than control males, but relative food consumption of these animals remained in the same range as relative food consumption of control males over the study period.
Food consumption and relative food consumption were similar between control animals and animals at 10 mg/kg/day.

FUNCTIONAL OBSERVATIONS
A lower motor activity as recorded by both high and low sensors was observed for males at 150 mg/kg/day, achieving statistical significance for high sensor recordings. Females showed a lower motor activity as recorded by the low sensor (not statistically significant).
Motor activity was similar in control animals and animals at 10 and 50 mg/kg/day.
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all surviving animals.

HAEMATOLOGY
The following (statistically significant) changes in haematology parameters distinguished treated animals from control animals:
- Lower white blood cell (WBC) counts in males at 150 mg/kg/day and in 1 female at 150 mg/kg/day (not statistically significant).
- Higher relative neutrophil counts (not statistically significant) in males and females at 50 and 150 mg/kg/day.
- Lower relative lymphocyte counts in males and females at 50 and 150 mg/kg/day (in females at 50 mg/kg/day not statistically significant).
- Lower relative eosinophil counts in females at 150 mg/kg/day.
- Lower reticulocyte counts in males and females at 150 mg/kg/day.
- Lower mean corpuscular volume (MCV) in males at 150 mg/kg/day.
- Lower mean corpuscular haemoglobin (MCH) in males at 150 mg/kg/day.
- Lower platelet counts in females at 150 mg/kg/day.
- Longer prothrombin time (PT) in males at 150 mg/kg/day.

Statistically significant higher platelet counts in males at 10 mg/kg/day, longer activated partial thromboplastin time (APTT) in females at 10 mg/kg/day and lower relative eosinophil counts in males at 50 mg/kg/day occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. Therefore, these changes were considered to be of no toxicological significance.


CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
- Higher alanine aminotransferase (ALAT) levels in males at 50 mg/kg/day (not statistically significant) and males and females at 150 mg/kg/day.
- Higher aspartate aminotransferase (ASAT) levels in males at 50 mg/kg/day (not statistically significant) and males and females at 150 mg/kg/day.
- Lower alkaline phosphatase (ALP) levels in males at 50 and 150 mg/kg/day.
- Lower total protein levels in females at 50 mg/kg/day and males and females at 150 mg/kg/day.
- Lower albumin levels in females at 50 mg/kg/day and males and females at 150 mg/kg/day.
- Lower total bilirubin levels in females at 50 and 150 mg/kg/day.
- Higher urea levels in males and females at 150 mg/kg/day.
- Higher sodium levels in females at 50 and 150 mg/kg/day.
- Higher potassium levels in females at 150 mg/kg/day.
- Lower chloride levels in females at 150 mg/kg/day.
- Higher inorganic phosphate levels in females at 150 mg/kg/day.

The statistically significant higher creatinine levels in females at 50 mg/kg/day occurred in the absence of a dose related effect and were considered to be of no toxicological significance.


ORGAN WEIGHTS
The following (statistically significant) changes in organ weights and organ to body weight ratios distinguished treated animals from control animals:
- Lower brain weight in males at 150 mg/kg/day and higher brain to body weight ratio in males and females at 150 mg/kg/day.
- Lower heart weight in males at 50 and 150 mg/kg/day.
- Higher liver weight in females at 150 mg/kg/day and higher liver to body weight ratio in males and females at 50 mg/kg/day and 150 mg/kg/day.
- Lower thymus weight in males at 50 mg/kg/day and males and females at 150 mg/kg/day and lower thymus to body weight ratio in males and females (not statistically significant ) at 150 mg/kg/day.
- Lower kidneys weight and higher kidneys to body weight ratio in males and females at 150 mg/kg/day.
- Lower testes weight (not statistically significant) and higher testes to body weight ratio in males at 150 mg/kg/day.
- Lower epididymides weights in males at 150 mg/kg/day and higher epididymides to body weight ratio in males at 50 and 150 mg/kg/day.

The higher adrenal to body weight ratio in males at 50 and 150 mg/kg/day was attributed to lower terminal body weight. Higher adrenal weight in males at 50 mg/kg/day occurred in the absence of a treatment-related distribution. Therefore, these changes were therefore considered of no toxicological significance.

Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.


GROSS PATHOLOGY
Necropsy findings in both surviving and moribund animals included an emaciated appearance in males and females at 150 mg/kg/day and one male at 50 mg/kg/day and reduced size of the thymus in males and females at 150 mg/kg/day.
Additional findings in moribund animals at 150 mg/kg/day included many dark red foci on the skin of the tail, distention of caecum with faeces, dark red discolouration of the glandular mucosa of the stomach, a stage of beginning or advanced autolysis and death before necropsy.
No macroscopic abnormalities were noted in control males, males and females at 10 mg/kg/day and females at 50 mg/kg/day.
Incidental findings among control females, males at 50 mg/kg/day and surviving males and females at 150 mg/kg/day included fluid in the uterus, isolated red foci on the glandular mucosa of the stomach, enlargement of the spleen, reduced size and black/brown discolouration of the kidneys, a disfigured pituitary gland and a red nodule at the base of the skull. These findings were considered changes of no toxicological significance, because they are occasionally seen among rats used in these types of studies and/or occurred in the absence of a treatment-related distribution.

The following treatment related microscopic findings were noted:
- Syncytial macrophages in mesenteric lymph nodes in 3/5 males and 4/5 females at 10 mg/kg/day (minimal or mild degree), 4/5 males and 4/5 females at 50 mg/kg/day (minimal to marked degree) and 5/5 males and 4/5 females at 150 mg/kg/day (minimal to marked degree).
- Medullary sinus histiocytosis in mesenteric lymph nodes in 5/5 males and 3/5 females at 10 mg/kg/day (minimal or mild degree), 5/5 males and 5/5 females at 50 mg/kg/day (mild or moderate degree) and 4/5 males and 5/5 females at 150 mg/kg/day (minimal to marked degree).
- Thymus atrophy in 3/5 males and 3/5 females at 150 mg/kg/day (minimal to marked degree).
- Increased adipocytes in bone marrow of the sternum in 3/5 males and 3/5 females at 150 mg/kg/day (minimal or mild degree).
- Foamy alveolar macrophages in lungs of 2/5 males and 2/5 females at 50 mg/kg/day and 3/5 males and 3/5 females at 150 mg/kg/day (minimal or mild degree).
- Mandibular lymph node atrophy in 3/5 males and 3/5 females at 150 mg/kg/day (minimal to moderate degree).
- Diminution in myometrial volume of the uterus of 5/5 females at 50 mg/kg/day (minimal or mild degree) and 4/5 females at 150 mg/kg/day (minimal to moderate degree).
- Diminution in uterine glands of the uterus of 2/5 females at 50 mg/kg/day (minimal degree) and 5/5 females at 150 mg/kg/day (minimal to moderate degree).
- Cervix atrophy in 3/5 females at 150 mg/kg/day (minimal or mild degree).
- Diminished size of seminal vesicles in 2/5 males at 150 mg/kg/day (minimal or mild degree).

All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain under the conditions in this study.

Effect levels

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

A similar supporting substance was tested in a 28-day oral gavage study in rats that received daily doses of 0, 10, 50 and 150 mg/kg/day. One male of the high dose group died and two females of the high dose group had to be humanely killed during the treatment period. In a dose dependent fashion, animals at 50 and 150 mg/kg/day showed various effects on body weight, hematology, clinical chemistry, organ weights, macroscopic and microscopic examination. In animals at 10 mg/kg/day, mesenteric lymph nodes were affected to a minimal or mild degree only and no further treatment-related changes were noted in these animals. Therefore, a No Observed Adverse Effect Level (NOAEL) for the test substance of 10 mg/kg/day was established.

Executive summary:

Repeated dose 28-day oral toxicity study with a similar supporting substance by daily gavage in the rat.

Guidelines

The study was based on the following guidelines.

-     EC Directive 96/54/EEC, B.7 Repeated Dose (28 days) Toxicity (oral), 1996

-     OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 1995

-     OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000

Rationale for dose levels

Based on the results of a range finding study (NOTOX Project 487887), the dose levels for this 28-day oral gavage study were selected to be 0, 10, 50 and 150 mg/kg/day.

Study outline

The test substance, formulated in water (Elix), was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females.

Evaluated parameters

Chemical analyses of formulations were conducted once after the in-life phase of the study to assess accuracy and homogeneity.

The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Results

Accuracy and homogeneity of formulations of test substance in water (Elix) were demonstrated by analyses.

One male and two females at 150 mg/kg/day did not survive until their scheduled termination.

Except for the moribund appearance and macroscopic abnormalities, such as beginning or advanced autolysis, the changes in these animals were essential similar to those observed in surviving animals at 150 mg/kg/day.

In a dose dependent fashion, animals at 50 and 150 mg/kg/day showed various changes which were indicative of general ill health. During treatment, these changes included (but were not restricted to) body weight loss, lower body weight gain, lower food intake, lower motor activity, lethargy, hunched posture, piloerection, dehydration, a lean appearance and/or hypothermia. In addition, several changes in clinical biochemistry and haematology parameters, such as lower relative lymphocyte counts and higher relative neutrophil counts, higher urea levels and an imbalance in plasma electrolytes, were observed. In addition, effects in kidneys, thymus, brain, heart, bone marrow of the sternum, testes, epididymides, seminal vesicles and cervix were predominately observed in animals at 150 mg/kg/day.

Moderate diminution of myometrial volume and uterine glands of the uterus was found only among females at 150 mg/kg/day. Minimal or mild diminution of myometrial volume was found among females of all dose groups and control females. Minimal or mild diminution of uterine glands was found among females of all dose groups.

A higher liver weight and/or higher liver to body weight ratio were noted in males and females at 50 and 150 mg/kg/day. Although morphological lesions indicative of hepatocytotoxicity were absent, the higher liver to body weight ratio was considered treatment-related since a number of effects on clinical biochemistry parameters were observed that suggest an effect of the test substance on liver function. These parameters included higheralanine aminotransferase and aspartate aminotransferase levels, lower alkaline phosphatase levels and/or lower total protein and albumin levels in males and females at 50 and 150 mg/kg/day.

Foamy alveolar macrophages were observed in lungs of males and females at 50 and 150 mg/kg/day in a minimal or mild degree. In addition, syncytial macrophages and medullary sinus histiocytosis was observed in mesenteric lymph nodes of animals at 10, 50 and 150 mg/kg/day, in an increasing incidence and severity.

Conclusion

Several signs of ill health and effects on liver, lungs and mesenteric lymph nodes were noted in animals at 50 and 150 mg/kg/day. In animals at 10 mg/kg/day, mesenteric lymph nodes were affected to a minimal or mild degree only and no further treatment-related changes were noted in these animals. Therefore, a No Observed Adverse Effect Level (NOAEL) of

10 mg/kg/day was established.