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Description of key information

The registrations substance was tested in a 28-day oral gavage study in rats that received daily doses of 0, 10, 50 and 150 mg/kg/d. 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/d showed various effects on body weight, hematology, clinical chemistry, organ weights, macroscopic and microscopic examination. In animals at 10 mg/kg/d, mesenteric lymph nodes were affected to a minimal or mild degree only and no further treatment-related changes were noted. NOAEL of 10 mg/kg/d was obtained.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07.04.2008 - 22.05.2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
other: dose range finding
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.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
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 sacrifice 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.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
other: general health affected
Organ:
other: general health affeced
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The registration substance was tested in a 28-day oral gavage study in rats that received daily doses of 0, 10, 50 and 150 mg/kg/d. 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/d 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. NOAEL of 10 mg/kg/d was obtained
Executive summary:

The registration substance was tested  in a 28-day oral gavage study in rats that received daily doses of 0, 10, 50 and 150 mg/kg/d. 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/d, mesenteric lymph nodes were affected to a minimal or mild degree only and no further treatment-related changes were noted in these animals. The NOAEL of 10 mg/kg/d was obtained.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rat

Mode of Action Analysis / Human Relevance Framework

Presumed mode of action in the repeated dose toxicity study:

The doses used in the 28 -day toxicity study were 0, 10, 50 and 150 mg/kg/d.

At 150 mg/kg/d the most remarkable effect was the clear tissue damage of GI tract upon macroscopic investigation and the mesenteric lymph node changes upon microscopic investigation. Simulaneously body weight decrease, thymus atrophy and inflammatory changes in multiple organs were osberved.

At 50 mg/kg/d similar effects were found with dose dependant manner. Down to 10 mg/kg/d the mesenteric lymph node was affected.

Taking account that the registration substance is a cationic surfactant and known to cause cell membrane destruction, it is reasonable to consider the portal of entry effect as the underlying mode or action. Damage on the direct contact tissue led to the local inflammatory response, which in turn caused systemic inflammatory response and bad health conditions.

Additional information

Justification for classification or non-classification

The registration substance should be classified as 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).

The results obtained in the 28 -day toxicity study indicate that the gastrointestinal tract is the primary target organ for the test substance-induced effects, as evidences by the pathologically changed mesenteric lymph nodes. The main clinically relevant effects are mortality at 150 mg/kg/d and general ill health at 50 mg/kg/d. The NOAEL of 10 mg/kg/d was obtained.