Docosyltrimethylammonium methyl sulphate

Endpoint summary

Description of key information

Repeated oral toxicity:
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. 
The 28-day study results are supported by an OECD 421 study with the same substance that reported a parental NOAEL of 30 mg/kg/day.
Additional data on similar supporting substances was also considered.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

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.

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.

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.

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.

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Sex:

male/female

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Sufficient.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

Sufficient.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

Sufficient. Only skin irritation was observed in these studies. Lacking dose-response data (only body-weight related dose information given) do not allow derivation of a DNEL

Additional information

Several studies on similar supporting substances are available:

The key study is a guideline repeated dose 28-day oral toxicity study (OECD TG 407) with a structurally closely related substance. It 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.

The purpose of a supporting dose-range-finder study was to select suitable dosages and appropriate administration method to be used in the subsequent reproduction/developmental toxicity screening test in the Han Wistar rat (key study). The test item was therefore administered by gavage and by feeding. Additional biological assays and clinical pathology were also performed in order to evaluate further findings from previous studies. The test item was administered orally by gavage once daily at dose levels of 50 and 150 mg/kg body weight/day and by feeding at dose levels of 600, 1800 and 6000 ppm. Severe adverse effects occurred at dietary levels of 1800 and 6000 ppm and at the gavage dose level of 150 mg/kg/day. Consequently, these groups were terminated due to humane reasons. At the dietary level of 600 ppm and the gavage dose level of 50 mg/kg/day, similar findings were noted. There were no striking differences in findings when the test item was administered in the diet or by gavege. Based on these results dose levels of 10, 30 and 75 mg/kg bw/day were considered apriopriate for the subsequent Reproduction/Developmental Toxicity Screening Test in the Han Wistar Rat.

A supporting guideline study (OECD TG 421) is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of a similar supporting substance to rats. The test item was administered in Milli-Q-Water as vehicle at dosages of 10, 30, and 75 mg/kg body weight/day, and controls received the vehicle only. The test item was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. Based on the decreased food consumption and lower body weights in males and females and gains in females at 75 mg/kg/day and the lower food consumption in males at 30 mg/kg/day which did not affect body weight, the parental NOAEL (No Observed Adverse Effect Level) was established at 30 mg/kg/day.

In another 28-day oral gavage study, male and female Sprague-Dawley rats received cetrimonium chloride at 30, 100 and 300 mg/kg bw/day for 5 days/week for 28 days, with additional 28-day recovery groups for the control and high dose. No deaths occurred, no effects were found on food consumption and body weight. The mean water intake of the males of the high dose group was higher than that of controls. Ophthalmologic and hematological results revealed no treatment-related changes in any group. At the high dose, a minor increase in serum ALT activity in males and females (within the range of the historical controls) was found. Males showed a slight increase in absolute and relative adrenal weights and slight decrease in absolute and relative spleen weight, however no histopathological alterations were found in these organs. Upon macroscopic examination thickening of the forestomach mucosa, associated with edema and sporadic ulceration in male and female rats was observed. Microscopic examination revealed inflammatory edema of the forestomach mucosa, sporadic ulceration and acanthosis up to papillomatous hyperplasia in both sexes. No other histopathological abnormalities were found. All treatment-related changes were shown to be reversible following the recovery period. The forestomach and stomach changes observed at 300 mg/kg/day were considered to be a result of local irritation and therefore not indicative of systemic toxicity. The slight weight changes of spleen and adrenals and the increase in serum ALT activity were regarded as a possible sign of some systemic toxicity. Therefore, the dose of 100 mg/kg/day was the NOEL.

In a 1-year chronic study male and female Sprague-Dawley rats received cetrimonium bromide in the drinking water at concentrations of 0.007, 0.014 and 0.032 %. These concentrations were calculated to deliver doses of approximately 10, 20 and 45 mg/kg b.w./day. Male and female rats of the high dose groups showed a significantly reduced body weight development, decreased efficiency of food conversion, decreased skeletal growth. In both males and females, an increased relative caecum weight was found in the high (males and females) and mid (males only) dose groups. No compound related changes were observed in haematological and clinical-chemical analyses of blood and urine. No gross necropsy changes were seen, and no microscopic alterations were found in the wall of stomach and small intestine of treated rats. No other tissues were histopathologically examined. The effects on growth and body weight development observed in the study are considered to be an indirect effect of the administration of cetrimonium bromide in the drinking water. The detergent caused diarrhoea which resulted in a faster passage of food through the gastrointestinal tract and reduced absorption of nutrients. The increased relative caecum weight may be due to proliferation of the endothelial cell layer caused by an increased replacement rate due to a faster flux of the intestinal contents. This explanation is supported by the lack of histopathologic alterations of the small intestine. In summary, the effects on growth and body weight were most likely caused by a lower nutrients supply, secondary to diarrhoea. This effect is attributed to the oral administration route and has no relevance for the use of cetrimonium bromide or alkyl (C16, C18, C22) trimethylammonium chlorides in topically applied cosmetic products. The highest dose administered in the study (45 mg/kg bw/day) constitutes a no-observed-adverse-effect-level (NOAEL). The no-observed-effect-level (NOEL) was 10 mg/kg bw/day.

In the 90-day dietary study, male and female Beagle dogs received quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides at 14, 140 and 2800 ppm (approximately 0.5, 5, 100 mg/kg/d) for 7 days/week for 90 days. No treatment-related effects were observed. Therefore, the NOAEL was >100 mg/kg/day.

In a 13-week (extended to 21 weeks) dietary study, male and female Sprague-Dawley rats received quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, methyl sulfates at 0, 0.25, 0.5 and 10% in the diet (0 and approximately 170, 365, and 750 mg/kg/day ingested dose) continuously for 7 days/week for 13 (and 21) weeks. No mortality or significant body weight effects occurred. Increased organ weights of adrenals and liver were observed at all treatment levels. Microscopic evaluation revealed treatment related changes in mesenteric and pulmonary lymph nodes, adrenal glands and liver. Therefore, the LOEL was 170 mg/kg/day.

In a 90-day dietary study, male and female rats received didecyldimethyl ammonium chloride at 0, 100, 300, 600, 1000, or 3000 ppm [0, 6.2, 18.5, 36.8, 60.7, or 175.4 mg/kg (males), 0, 7.5, 22.3, 44.4, 74.3, or 225.5 mg/kg/day (females)] continuously for 7 days/week for 90 days. The high dose group showed increased mortality, decreased mean body weight, body weight gain and food consumption. Gross pathology and microscopic examination revealed higher incidence of glycogen depletion in the liver, contracted spleens, sinus erythrocytosis and lymphoid hyperplasia of mesenteric lymph nodes. Therefore, the NOAEL was 1000 ppm, corresponding to 60.7 mg/kg/day in males and 74.3 mg/kg/day in females.

In a 90-day dietary study, male and female dogs received didecyldimethyl ammonium chloride at 0, 5, 15, or 50 mg/kg/day daily for 90 days. High-dose animals experienced marked decrease in body weight gain, food consumption, and food efficiency, for both males and females. Therefore, the NOAEL was 15 mg/kg/day.

In a 28-day dermal toxicity study in male and female rabbits with ammonium, hexadecyltrimethyl-, chloride applied once daily, 5 d/wk for 28 days at doses of 0 and 2.0 ml/kg of 0.5%(w/w) equalling 10 mg/kg/day only skin irritation, but no systemic effects were observed and the NOAEL was >10 mg/kg/day.

In a 90-day dermal toxicity study in male and female rabbits with quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chloride applied once daily, 5 d/wk for 91 days at doses of 0, 20, 60, 100 and 140 mg/kg/day (0, 1, 3, 5 and 7% aqueous solutions, respectively) only skin irritation, but no systemic effects were observed and the NOAEL was >140 mg/kg/day.

In a 90-day dermal toxicity study in male and female rats with didecyldimethyl ammonium chloride applied once daily for 6 hours/day, 5 days/week for 13 weeks at doses of 0, 2, 6, or 12 mg/kg/day no systemic effects were observed, but at the mid and high dose irritative skin effects were observed. Therefore, the systemic NOAEL was >12 mg/kg/day and the NOAEL for irritative skin effects was 2 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliable study for surrogate substance.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Several supporting studies.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Several supporting studies.

Justification for classification or non-classification

The results of the 28-day key study performed with a structurally closely related substance are discussed to reach a conclusion on the classification of the submission substance according to the criteria of the EU Dangerous Substances Directive (67/548/EEC) (DSD) and according to the criteria of the EU Classification Labeling and Packaging Regulation (1272/2008/EC) (CLP).

Upon start of the treatment period, the animals receiving 50 or 150 mg/kg/d quickly started to show a dose-dependent reduction in absolute and relative food consumption and a lower body weight gain compared to controls. Both effects showed a tendency for a maximum effect after about two weeks of treatment. This was also the time at which the deaths of the three animals occurred. After about two weeks, food consumption and relative body weights stabilised and no further deaths or moribund animals were observed in the remainder of the observation period. The mode of action of these effects is not clear. From the skin and eye irritant properties of the test substance, it seems likely that oral bolus administration of the test substance had led to local gastrointestinal tract irritation and that the surfactant properties of test substance could have affected the intestinal absorption of nutrients. However, the histological examination did not indicate local inflammatory infiltration of the gastrointestinal tract wall. The deaths of three animals in the high dose groups have to be considered substance-related.

Since the guidance values for classification as R48 (DSD) and STOT-RE Cat. 2 (CLP) refer to effects observed in a 90-day study and are increased for a 28-day study by a factor of 3, the mortality observed at 150 mg/kg bw/d would fulfil the classification criteria for a 28-day study of below or equal to150 mg/kg bw/d (DSD) and below or equal to 300 mg/kg bw/d (CLP).

The occurrence of syncytial macrophages and medullary sinus histiocytes in mesenteric lymph nodes can be interpreted as a sign of uptake of the test material and responsive activation of macrophages as part of the immune system. These effects were observable already at 10 mg/kg/d and reached a marked/moderate level at 50 and 150 mg/kg/d.

Taken together, the effects observed suggest that the gastrointestinal tract is the primary target organ for the substance-induced effects.

Further changes in some immune system-associated parameters were observed, such as lower white blood cell, higher neutrophil and lower relative lymphocyte counts, and slightly reduced thymus weight with thymic atrophy. These effects increased with dose and tended to be statistically significant only at 150 mg/kg/d and were minimal to mild/moderate in degree. They are considered to have been caused in a secondary fashion in response to local activation of immune cells in the gastrointestinal tract, and to reflect activation, mobilisation and re-localisation of cells of the immune system in other parts of the body. In this sense, the effects can be considered as part of a responsive reaction and, thus, do not fulfil the classification criteria.

At the high dose of 150 mg/kg/d, signs of impaired bone marrow function were observed includinglower reticulocyte counts and lower mean corpuscular volume and mean corpuscular haemoglobin (and secondary lower bilirubin level), lower white blood cell counts, lower platelet counts, and increased adipocytes in sternum bone marrow. These effects fulfill the classification criteria, but may, in part,have been caused secondary to reduced body weight development and immune system activation.

Increased ALAT (alanine aminotransferase) and ASAT (aspartate aminotransferase) in both sexes were observed. Absolute liver weights were increased only in females, but not in males, while relative liver weights were moderately increased in both sexes. These effects achieved statistical significance mostly at 150 mg/kg/d and are most likely secondary findings to altered liver physiology during the period of decreased food consumption and body weight loss. The lower total protein and lower albumin andlonger prothrombin timealso fit into this picture. The higher urea levels may indicate higher protein degradation. In the absence of any histopathological findings in the liver, these effects cannot be considered so severe that they fulfil the classification criteria.

Some effects were reported in reproductive organs:lower absolute epididymides weight and higher epidydimides/bw ratio, higher testes/bw ratioand minimal to mild/moderate histological findings (diminuition inmyometrial volumeand uterine glands, cervix atrophy and diminished size of seminal vesicles). These findings could not be reproduced in a reproductive toxicity screening assay (OECD 421) and are thus not considered relevant for classification and target organ identification. Some other slightly altered relative and/or absolute organ weights (brain, heart, kidney) did not show any histopathological or functional correlate and are thus not considered relevant for classification. Some changes in electrolytes (higher sodium, potassium and inorganic phosphate levels, lower chloride levels) are considered secondary to body weight and water loss.

 

The data indicate that the gastrointestinal tract is the primary target organ for the test substance-induced effects. The main clinically relevant effects are reduced food consumption resulting in reduced body weight development and, at 150 mg/kg/d in general ill health and delayed mortality. At 150 mg/kg/dsigns of impaired bone marrow function were observed. Signs ofuptake of the test material into the gastrointestinal tract associated lymph nodes and activation of macrophages were found in a dose-dependent manner beginning at the low dose, leading at150 mg/kg/d toresponsivechanges in some immune system-associated parameters. At 150 mg/kg/d, effects on some liver parameterswere observed, which were likely caused in a secondary fashion following reduced food consumption and reduced body weight development. The effects at 150 mg/kg/d support the classification based on delayed mortality.

Inclusion of the dermal route of exposure in the hazard statement according to CLP is not considered necessary, because experiments with analogous quaternary alkylammonium compounds indicate that systemic bioavailability from the dermal route is about three- to tenfold lower than from the oral route and because the gastrointestinal tract is the primary target organ.

Inclusion of the inhalation route of exposure in the hazard statement according to CLP is not considered necessary, because of the very low vapour pressure of the similar substance Fatty alkyl (C20-C22) trimethylammonium chloride, because in the unlikely case that the substance becomes airborne as a inhalable aerosol, an acute irritation of the respiratory tract would, in all likelihood by more relevant,and because the gastrointestinal tract is the primary target organ.

 

In conclusion, the submission substance should be classified like the similar supporting substance as

Xn; R48/22 Harmful: danger of serious damage to health by prolonged exposure if swallowed
according to the criteria of the EU Dangerous Substances Directive (67/548/EEC) and as

Warning; STOT-RE Category 2 - H373: May cause damage to the gastrointestinal tract through prolonged or repeated exposure via the oral route

according to the criteria of the EU Classification Labeling and Packaging Regulation (1272/2008/EC).