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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
data is from authoritative database

Data source

Reference
Reference Type:
other: Authoritative database
Title:
Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test for the test chemical
Author:
J-Check
Year:
2018
Bibliographic source:
J-Check, 2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test was performed for the test chemical to evaluate its oral toxicity potential
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report):Tetrabutylammonium bromide
- Molecular formula : C16H36N.Br
- Molecular weight :322.37 g/mol
- Substance type:Organic
- Physical state:White Solid (Crystals)
- Analytical purity:99.9%
- Lot/batch No.:Lot 1/02
- Storage condition of test material:Stored in cool, dry place. Kept in closed container when not in use.
- Other: Handling and Disposal
Safety precautions: Avoided eye and skin contact. Avoided inhalation and spilling. Aprons, caps, mask, gloves and goggles were used to ensure the health and safety of the personnel.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: IGS, Japan Chiyarusu-Lipa Inc., Atsugi breeding center.
- Age at study initiation: 10 weeks old
- Weight at study initiation: Male 389 to 452 g
Female 219 to 266 g
- Fasting period before study: No data available
- Housing: Animals were housed one per by sex in a metal net cage, during the mating period two per sex per cage while mother were housed individually in plastic cage.
- Diet (e.g. ad libitum): Solid feed (NMF, Oriental Yeast Co., Ltd.), ad libitum.
- Water (e.g. ad libitum): Drinking water (NMF, Oriental Yeast Co., Ltd.), ad libitum.
- Acclimatization period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3 ℃
- Humidity (%):50 ± 20 %
- Air changes (per hr): 10-15 times per hour ventilation.
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: No data available
DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available
- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Purified water
- Concentration in vehicle: 0, 60, 180 or 600 mg/kg body weight/day
- Amount of vehicle (if gavage): 10 ml/ kg body weight dose volume
- Lot/batch no. (if required): OU06G, Tokyo Chemical Industry Co
- Purity: No data available
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
42 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 60, 180 or 600 mg/kg body weight/day
Basis: actual ingested
No. of animals per sex per dose:
Total animals: 101
Control: 12 males, 12 females
60 mg/kg/day: 12 males, 12 females
180 mg/kg/day: 12 males, 12 females
600 mg/kg/day: 12 males, 12 females
With 5 male (out of 12) from each group for recovery and 5 female as satellite group.
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
14-day repeated oral dose toxicity study using rats for the test chemical (dose setting test)" was carried out earlier dose (dose: 100,300,1000 mg / kg). 1 rat died on the 14th administration in male 1000 mg / kg dose group, in a general state of deaths loose stools (reddish brown), body weight and food consumption was incresed in 300 mg/kg, liver weight was observed high, low value of the ALP, high total protein, expansion of the cecum at autopsy in male and female were observed at 1000 mg/kg daoe gruop. Therefore, the dose level of 0, 60, 180 and 600 mg/kg/bw/day was chosen in the current study.
- Rationale for animal assignment (if not random): Animals were randomized by body weight.
- Rationale for selecting satellite groups: 5 female as satellite group.
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available

Examinations

Observations and examinations performed and frequency:
Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
Attitude, fistula attack, abnormal behaviour were observed.

- Time schedule: Once, once a week (but 1,7,14 and 20 days of gestation, mating confirmation female animals nursing the 4th calving animals) were observed.

- Cage side observations checked in table [No.?] were included: On 1,7,14 and 20 days of gestation, mating confirmation female animals nursing the 4th calving animals were observed.

DETAILED CLINICAL OBSERVATIONS: Yes
Pupil diameter, abnormal breathing, the state of the fur-skin, secretions of the eyes, nose, exophthalmos,flair closed state, the visible mucous membranes, autonomic function and urination were observed.

- Time schedule: 3 times daily

BODY WEIGHT: Yes
Throughout the recovery period and the period of administration.

- Time schedule for examinations:
At a frequency of twice a week, check mating female animals were weighed on the 4th and lactation, as well as 0 and 20 days 0, 4, 7, 11, 14, 17 pregnancy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data available

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: No data available

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

- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: Yes
Pupil diameter and secretions of the eyes were observed.

- Time schedule for examinations: Daily

- Dose groups that were examined: 0, 60, 180 and 600 mg/kg body weight/day

HAEMATOLOGY: Yes

- Time schedule for collection of blood:
Before autopsy.

- Anaesthetic used for blood collection: Yes

- Animals fasted: Yes
On day 43, overnight (16-20 hours) fasted.
- How many animals: each 5 per sex per group

- Parameters checked in table [No.?] were examined. : RBC, Hb, Ht, MCV, MCH, MCHC, Reticulocyte, Platelet, PT, APTT, Fibrinogen, WBC and differential leucocytes were examined.

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood:
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
Urinalysis of male rat were observed on administration of Tetrabutylammonium bromide and the last week of recovery, urine collected from 5 animals in each group and observed the color, pH, protein, ketone bodies, glucose, occult blood, bilirubin, urobilinogen and urine volume.

- Time schedule for collection of urine: Daily 20 hour.

- Metabolism cages used for collection of urine: No data available

- Animals fasted: Yes
For 4 hours

- Parameters checked in table [No.?] were examined.:
Color, pH, protein, ketone bodies, glucose, occult blood, bilirubin, urobilinogen and urine volume were checked.

NEUROBEHAVIOURAL EXAMINATION: No data available

- Time schedule for examinations: No data available

- Dose groups that were examined: n No data available

- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Grip strength: Grip strength measurement of hindlimb and forelimb were tested.

Spontaneous momentum: Spontaneous movement sensor was counting the momentum of 60 minutes and 10 minute intervals.

OTHER: Auditory response, approaching response, contact reaction, pain reaction, anti-morphism air righting reflex, landing leg width was observed.
Sacrifice and pathology:
Sacrifice and pathology
GROSS PATHOLOGY: Yes
Change attributed in intestinal tracts of male and female of 180 and 600 mg/kg/bw/day group and in liver of male and female of 600 mg/kg/bw/day group.

HISTOPATHOLOGY: Yes
Diffuse hyperplasis in mucosa in cecum,which thought to be related to the macroscopic dilation in lunina of cecum, in the rectum, the debris in crypt were observed in female of 600 mg/kg/bw/day group.

In recovery group of 600 mg/kg/bw/day cell infertilation in mucosa were observed cecum,colon and rectum. In the liver , hypertropy of preiobular hepatocyte were observed.
Statistics:
Statistical analysis was carried out by using x 2 test with continuity correction of Yeates, expected frequency was observed cell of 5 or less, by the direct probability calculation method of Fisher test (significance level for the 4th). Dunnett type test (mean rank test method) (significance level 0,06 ) performed for homogeneity of variance of each group and Bartlett method firstly test was applied. (significance level 0.01, both sides)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Before death, the male showed fractures of incisors, soft stool and a decreased in the amount of feces produced. The female showed staining of lower abdominal fur and staggering gait before death.
Mortality:
mortality observed, treatment-related
Description (incidence):
Deaths occurred in 1 male and 1 pregnant female when treated with 600 mg/kg/day.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
When treated with 600 mg/kg/day, the males showed suppressed body weight gain while females showed a tendency toward high values in body weight during the administration period. In comparison to control, males in the 600 mg/kg/day group showed a low value in body weight during the recovery period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females showed a tendency toward high values in food consumption during the administration period. A high value in food consumption was also observed in females treated with 180 mg/kg/day.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Only a slight increase in water consumption was observed in males with increased treatment concentrations.
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 600 mg/kg/day showed a slight decrease in platelets. Similar effects were observed during the recovery period. An increased level of fibrinogen was also seen in the same animals, i.e. the 600 mg/kg/day-treated males and males from the recovery period.Males in recovery period also showed decreased levels of lymphocyte and eosinophils while the level of neutrophils and monocytes were increased. Females treated with 600 mg/kg/day showed a slight increase in RBC and a slight decrease in platelets. These effects were not observed during the recovery period. In addition, a decreased level of fibrinogen was also seen in the same animals, i.e. the 600 mg/kg/day-treated females.Females in recovery period showed decreased levels of lymphocytes and increased levels of monocytes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In male rat, Ast and Cl were increased, during recovery total cholestrol, PL, glucos and Tp were decreased and Cl were increased in 600 mg/kg/bw/day group.In female, AST, LDH and Ca wee increased and BUN and creatinine level were decreased in 600 mg/kg/be/day group. creatinine level were also decreased in 60 mg/kg/bw/day group female rat. These changes were not observed after the end of recovery period showed reversibility of the change.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
In male rat treated with 600 mg/kg/day showed red urine sporadically and light brown urine were observed.
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In males treated with 600 mg/kg/day, the absolute organ weight was decreased for thymus, heart, kidneys and epididymis.In the male recovery period, the absolute organ weights for thyroids, heart and liver were decreased.In females treated with 600 mg/kg/day, the absolute organ weight was decreased for brain and thymus, while the organ weight for thyroids, heart, liver, spleen and kidneys increased. In the female recovery period, the absolute organ weights for thyroids, heart and kidneys increased.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Change attributed in intestinal tracts of male and female of 180 or 600 mg/kg/day groups and in liver of male and female of the 600 mg/kg/day group. Dilatation in lumina of the cecum was also observed.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Diffuse hyperplasia in mucosa in the cecum was observed. In rectum, cell debris in crypt was observed in females.In the recovery group, these changes were no longer observed. However in the recovery group, cell infiltration in mucosa was observed in cecum, colon and rectum. In the liver, hypertrophy of perilobular hepatocytes was observed.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Effect levels

Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Thus, from all the above observations, it was concluded that the observed NOAEL for males and female rats was 180 mg/Kg body weight and LOAEL was considered to be 600 mg/Kg/Day for the test chemical male and female rats.
Executive summary:

A Combined Repeated Dose and Reproductive Toxicity Screening Test of the test chemical was performed to evaluate general toxicity and toxicity to reproductive organs and performance and effects on the developing fetus. The study was performed according to OECD 422 Guidelines. A total 96 animals were included in the main group and 20 animals in the recovery groups were used. A total of 12 males and 12 females per group in the main study and 5 males and 5 females per group in the recovery were grouped. A recovery group was included to check the reversibility of the effects that were observed in the main study. The animals that were used in the study were Crl:CD (Sprague Dawley) rats. The animals in all the groups were dosed via the oral route by gavage, using an intubation canula. The doses were prepared in the concentration of 0, 60, 180 and 600 mg/Kg body weight. The vehicle that was used to prepare the doses was purified distilled water. The male animals were dosed for 42 days and the females were dosed from approximately 41 to 53 days (including from 14 days before mating to day 4 lactation period). The recovery group was kept under observation for 28 days to see any reversal of effects that are observed in the main groups. The animals were observed for mortality and clinical signs, Functional Observation Battery, Body weight gain and food consumption, Hematology and Clinical Chemistry parameters. During the observations, at 600 mg/Kg body weight, one male and one female rat were found dead. Before death, the male animals showed fractures of incisors, soft stool and a decrease in the amount of feces. In female rat, staining of lower abdominal fur and a staggering gait was observed. Animals that survived at 600 mg/Kg dose showed presence of red urine sporadically. A light brownish urine was observed during urinanalysis, but the change was transient. Also, at 600 mg/kg male rats showed a tendency of suppressed body weight while the female rats showed signs of increased body weight and food consumption. At 180 mg/Kg body weight, increased food consumption was observed in female rats. In recovery group, at 600 mg/kg, the male animals showed lowering of body weights they were not considered to be of toxicological importance since the same effect was observed in the recovery control group. In clinical chemistry parameters, male rats in 600 mg/Kg showed high values of Aspartate Amino Transfrase (AST) and lower concentration of blood urea nitrogen (BUN), while female rats showed high levels of Aspartate Amino Transferase (AST) and Lactate Dehydrogenase (LDH) and lower levels of blood urea nitrogen (BUN) and creatinine. The females who received dose of 600 mg/Kg showed an increase in the calcium concentration. These changes were found to be inconsistent as the same symptoms were not observed in high dose recovery group. In hematology analysis, no test chemical related adverse effect was observed at any dose levels in main study and the recovery group. The animals were also observed for function observation battery, where all animals were found to be normal and there was no test item related adverse effects in any animals in the main study and the recovery group. The animals were also tested for grip strength analysis wherein there was no change in the animals from all the dose groups when compared to the control group. At 180 mg/Kg and 600 mg/Kg, changes in intestinal tracts in males and female rats. Also, changes in liver morphology were observed at 600 mg/Kg in both males and females. In histopathological examinations, diffused hyperplasia in the mucosa in the cecum was observed, which was assumed to be related to the macroscopic observation in In the rectum, cell debris in crypt was observed in females. In recovery group, all the changes were reverted and thus recovery from these observations was observed. However, cell infiltration in mucosa was observed in cecum, colon and rectum and in liver hypertrophy of perilobular hepatocytes were observed. Thus, from all the above observations, it was concluded that, the observed NOAEL for males and female rats was 180 mg/Kg body weight and LOAEL was considered to be 600 mg/Kg/Day for the test chemical male and female rats.