Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-03-29 until 2013-11-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
other: GB/T21763-200/ Chemicals-Test method of repeated dose oral toxicity study in rodents
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: bulk
Details on test material:
Batch ST02697671
Specific details on test material used for the study:
purity: 86.8%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4-6 weeks old
- Weight at study initiation: males 84-115 g and females 86-110 g
- Fasting period before study: no data
- Housing: 3 rats/per cage of the same sex in stainless steel screen bottom cages
- Diet (e.g. ad libitum): basal diet was ground Purina Certified Rodent Chow 95002
- Water (e.g. ad libitum): ad libitum - pure water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25 °
- Humidity (%): 50 -68 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test substance for the three dose groups and high dose satellite group were administrated to the animals daily for a period of 90 days by oral gavage at the same time each day. The control group and satellite control group were received distilled water. The volume was 5 mg/kg bw/d, and was be adjusted weekly according to the changes of body weight.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in distilled water into the concentration of 8, 40, 200 mg/ml for low, medium, high dose groupds and high dose satellite groups respecitvely. The test substances were prepared daily throughout the treatment-perio of the study.



Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily at the same time.
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 animals per sex per dose for control group and three dose groups
6 animals per sex for control satellite group and high dose satellite group
Control animals:
yes
other: no positive control, but not necessarily needed according to OECD 408
Details on study design:
- Dose selection rationale: according to preliminary test with 1000 mg/kg bw/d and five SD rats by oral gavage for total 5 days. No significant toxicity was observed. (acute toxicity data showed LD50>5000 mg/kg.)
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: one control satellite group and one high dose satellite group were kept for 14 days without treatment for follow-up observations.
- Section schedule rationale (if not random): random
Positive control:
no positive control

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- All animals were observed at least daily for moribundity, death and obvious indications of a toxic effect. At least once each week each animal was removed from its cage and carefully examined for abnormalities and clinical signs of toxic or pharmacologic effects.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day for 90 day.
include changes in skin, fur, eyes, mucous membranes, respiratory, circulatory system, ANS and CNS, autonomic activity, food intake, the properties of diachorema and mortality in all animals. Recorded all toxicity symptom, including the occurrence time, degree and persistence of toxic effect. The dying or dead animals during the study should be subjected to a full, detailed gross necropsy at once.

BODY WEIGHT: Yes
- Time schedule for examinations: at initiation and weekly through termination of study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- measurements of food consumption were made weekly.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

Haematology: Yes
- Time schedule for collection of blood: at the end of the test period (fasted for 16 h) blood samples were obtained from the cervical vena.
- Anaesthetic used for blood collection: no
- Animals fasted: no data
- How many animals: all
- Parameters examined: total and differential leukocyte count, erythrocyte count (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), erythrocyte haemoglobin distribution width (RDW), platelet count (PLT) and mean platelet volume (MPV), platelet distribution width (PDW) and mean platelet volume (MPV), platelet distribution width (PDW), thrombocytocrit (PCT), pro-time prothrombin time (PT), partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Parameters examined in serum: blood serum aspartate aminotransferase (AST), blood serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (CREA), total protein (TP), albumin (ALB), globulin (GLB), glucose (GLU), total bilirubin (TBIL), triglyceride (TG), serum total cholesterol (CHOL), ɤ-glutamyl transpeptidase (GGT), serum potassium (K), serum sodium (Na).

URINALYSIS: before the end of the test period, urine samples of 6 female and 6 mals animals of each group were dollected.
urine pH, nitrite, protein, glucose and blood/blood cells, specific gravity, bilirubin, urobilinogen, ketones, WBC and the settling in the urine were performed.

NEUROBEHAVIOURAL EXAMINATION: No

GROSS NECROPSY:
detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
The following tissues would be preserved: the brain, pituitary, heart/thoracic aorta, thyroid and parathyroid, oesophagus, trachea and lungs, thymus, stomach, dodecadactylon, small and large intestines, liver, kidneys, spleen, pancreas, adrenals, ovary, tisticle, epididymis, representative lymph node, spinal cord, uterus, prostate, gladder, peripheral nerve, sternum.
If changes were observed during gross examinations, female mammary gland, skeletal muscle, femur, tear gland, salivary glands and eyes would be preserved.

ORGAN WEIGHT
liver, kidneys, adrenals, testes, epididymis, uterus, ovaries, thymus, spleen, brain and heart
Sacrifice and pathology:
All animals were killed within 3 days after completion of the 91-day dosing period. The animals were weighed on the day of necropsy, anesthetized with ether and killed by exsanguination.

GROSS PATHOLOGY: Yes
All animals were examined macroscopically. Relative and absolute organ weights of liver and kidneys were recorded.

HISTOPATHOLOGY: Yes
All tissues were identified only by a random number. The following tissues and organs were examined and preserved in enough 10 % phosphate-buffered formalin to ensure adequate fixation.

Bone marrow, lung, heart, aorta (thoracic), tongue, trachea, esophagus, thyroid, submandibular lymph nodes, lymph nodes (ileocecocolic and
anterior mesenteric), stomach (fundic, cardiac pyloric regions), liver, duodenum, jejunum, ileum, cecum, colon urinary bladder, kidneys (left kidney
longitudinally, right kidney transversely to show cortex medulla and pelvis)
Reproductive organs males: prostate and seminal vesicle, testis with tunic nicked and epididymis attached.
Reproductive organs females: ovaries cross section of both uterine horns cross section of vagina.
Adrenals, thymus, gluteal plus biceps muscle, spleen, pancreas, distal femur (with marrow), brain (incl. cerebrum, mid-brain, cerebellum and stem),
lumbar spinal cord, sciatic nerve, submandibular salivary glands, pituitary gland, eyes (fixed in Zenker´s solution)

Lesions were collected.

Statistics:
The parameters and datas were analysed statisticaly by SPSS 11.5 software. One-way ANOVA or rank sum test was chosen. The significance level was 0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
one female of the high-dose group, one female of the mid-dose group and one male of the vehicle-control group were dead in the day 30th, 33th, 52th of administration respectively because of laboratory personnel operating errors.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
TBIL of females and TG of males in the high dose group increased compared with the control group (p<0.05)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no treatment related clinical signs observed. Three animals died, the observed mortalities were not treatment related. One female of the high-dose group, one female of the mid-dose group and one male of the vehicle-control group were dead in the day 30th, 33th, 52th of administration respectively because of laboratory personnel operating errors.

BODY WEIGHT AND WEIGHT GAIN
No significant differences in mean body weights or body weight gains were detected.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No significant differences were detected for any treated animals.

FOOD EFFICIENCY
No significant differences for food efficiencies were detected in female dose groups and the male 10 mg/kg bw/day dose group. Significantly lower mean values were detected during week 1 for male rats treated with 100 mg/kg bw/day and during weeks 1 and 2 for males treated with 1000 mg/kg bw/day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
No data

OPHTHALMOSCOPIC EXAMINATION
No data

HAEMATOLOGY
In general there were no significant differences between treated groups and controls. Minor findings were in the scope of the historical control values and without clinical significance.

CLINICAL CHEMISTRY
Treatment related effects were observed.
TBIL of females and TG of males in the high dose group increased compared with the control group (p<0.05)
There were no significant differences detected for males treated with 100 mg/kg bw/day or females of all dose groups. Significantly higher mean values for total protein and SGOT were detected in males treated with 10 mg/kg bw/day. A significantly lower value for total protein and significantly higher values for SGOT and SGPT levels were detected in the male 1000 mg/kg bw/day group.

URINALYSIS
No significant differences were detected for any treated animals.

NEUROBEHAVIOUR
No data

ORGAN WEIGHTS
No significant differences were detected in absolute or relative organ weights for female and male rats in the three dose groups.


GROSS PATHOLOGY:
All findings were considered incidential and not treatment related.

HISTOPATHOLOGY: NON-NEOPLASTIC
No obvious pathology changes occured.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No neoplastic lesions are described in the study report.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
clinical biochemistry

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Treatment related effects were limited to lower absolute and relative liver weight in the male high dose group with concomitant lower total plasma protein and higher SGPT and SGOT values for this treatment group.
Executive summary:

In a subchronic toxicity study comparable to OECD guideline 408 (1981), the partially unsaturated IQAC, DMS quaternised (tallow fatty acid based; CAS-no. 68122-86-1) was administered to 20 Sprague-Dawley rats/sex/dose by oral feed at dose levels of 0, 10, 100 and 1000 mg/kg bw/day (active ingredient). No satellite group was included to assess the reversibility of any potentially adverse effects.

The active ingredient in diet was analysed in all preparations and actual test material consumption closely resembled theoretical values (males 94 to 95 % and females 86 to 93 %).

There were no treatment related clinical signs observed, no animal died or was sacrificed during the study. Body weights and weight gains were in a normal range. No significant differences in food consumption were detected for any treated male group. Food consumption in the 10 and 100 mg/kg bw/day female groups were significantly increased during some weeks during the study. There were no consistent differences in food efficiencies.

Decreased values of absolute and relative liver weights in the male high dose group were probably treatment related and were accompanied by lower total serum protein and higher SGPT and SGOT concentrations for this treatment level. Correlating microscopic lesions were not detected. A dose dependency for these effects was not found.

No other treatment related effects on organ weights, hematological or clinical chemistry parameters were observed. All macroscopic observations and microscopic lesions were considered incidental and not treatment related. Microscopic examinations revealed no treatment related changes.

The histological examination of the reproductive organs (testes, epididymis, prostate, seminal vesicle, ovary, uterus and vagina) did not reveal any treatment related abnormalities.

A pretest health examination, conducted on 10 animals/sex, showed five animals with positive background endoparasites or viral titers.

The NOEL was 100 mg/kg bw/day.

The study suffers, however, from the excessively wide dose spacing, which would not be the standard for present day testing protocols. Considering the overall toxicological profile, there is no evidence for a potential serious health risk for humans upon ingestion of members of this chemical structure family. As a consequence, the reported NOEL has been recalculated for the derivation of an acceptable DNEL longterm oral, departing from the highest dose as the LOAEL to arrive at a realistic basis for DNEL derivations, resulting in the recalculated NOAEL of 300 mg/kg bw/day.