5-amino-2-anilinobenzenesulphonic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from J check

Data source

Reference

Reference Type:

other: J check

Title:

28 day repeated oral dose toxicity study for the test compound 4-amino-1-sodium naphthalene sulfonic acid

Author:

Japan Chemicals Collaborative Knowledge Database

Year:

2016

Bibliographic source:

National institute of technology and evaluation, 2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Mammalian-induced repeated dose toxicity test using 28 days

Principles of method if other than guideline:

Repeated dose oral toxicity study was performed to evaluate the toxic nature of the test compound upon repeated application by oral route of administration

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium naphthionate

IUPAC Name:

Sodium naphthionate

Constituent 2

Reference substance name:

Sodium 4-aminonaphthalene-1-sulphonate

EC Number:

204-975-5

EC Name:

Sodium 4-aminonaphthalene-1-sulphonate

Cas Number:

130-13-2

IUPAC Name:

sodium 4-aminonaphthalene-1-sulfonate

Details on test material:

- Name of test material: 4-amino-1-sodium naphthalene sulfonic acid
- Molecular formula: C10H9NO3S.Na
- Molecular weight: 245.233 g/mol
- Substance type: Organic
- Physical state: No data available
- Impurities (identity and concentrations): 76.1% pure

Test animals

Species:

rat

Strain:

other: Sprague Dawley strain (Crj: CD) SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Japan Charles River
- Age at study initiation: 5 weeks
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: The animals were kept in a breeding room and were housed independently in each cage
- Diet (e.g. ad libitum): solid feed (CE2, Japan Clea) ad libitum
- Water (e.g. ad libitum): tap water (Hadano Waterworks water supply) ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24.0 to 25.5 ° C
- Humidity (%): 44 to 64%
- Air changes (per hr): 15 times / hour
- Photoperiod (hrs dark / hrs light): 12 hours (lights of 7 to 19 times)

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test compound 20% w/v was dissolved in water. It was serially diluted to 6% and 2% dispensed into a brown bottle and kept in a cool dark place. Dosing specimens were removed from the cool dark place on the previous day and returned to room temperature before use. Dosing specimens were prepared at a frequency of once every 4 or 6 days. As a result of conducting the stability test and the content test of the prepared specimens, the test substances in the 0.2 and 20.0% (w / v) solutions were stable for 6 days in a cold dark place followed by 24 hours at room temperature, , It was confirmed that the content of the test substance in the administered specimen was 102 to 105% of the prescribed concentration

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water
- Concentration in vehicle: 0, 100, 300 and 1000 mg/kg
- Amount of vehicle (if gavage): 5 mL/Kg
- Lot/batch no. (if required): No data
- Purity: No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

28 days + 14 days recovery period

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
Test group: 0, 100, 300 or 1000 mg/kg Recovery group: 0 or 100 mg/Kg Dose volume: 5mL/Kg
Basis:

No. of animals per sex per dose:

Total: 50
0 mg/Kg bw: 10
100 mg/Kg bw: 5
300 mg/Kg bw: 5
1000 mg/Kg bw: 10

Recovery period:
0 mg/Kg bw: 10
1000 mg/Kg bw: 10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose for the main study was determined by referring to the results of preliminary 7-day repeated oral administration toxicity test in rats of sodium 4-amino-1-naphthalenesulfonate conducted at Hatano Laboratory. Male Sprague-Dawley rats were repeatedly administered sodium 4-amino-1-naphthalenesulfonate at doses of 100, 300 and 1000 mg / kg for 7 days. There was no death and a change was observed in the general state. Also, at autopsy, no abnormal findings which were thought to be caused by administration of the test substance were observed.
- Rationale for animal assignment (if not random): The grouping was carried out by random weighted stratified extraction method based on body weight on the day before the start of administration from animals without abnormality during the quarantine period
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: Yes, 14 days
- Section schedule rationale (if not random): No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily for mortality; For all cases, the general condition was observed daily during the administration period, twice a day before administration and once a day (once during the recovery test period).
- Cage side observations checked in table [No.?] were included. Mortality, General condition

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were measured at the start of administration week, a frequency of twice a week for all cases immediately before the administration, on the fourth day of administration, on the administration period after the second week and on and during the recovery test period, and the administration period or the recovery test period. The body weight was also measured on the end date and on the autopsy date.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, food intake was measured at the start of administration, at a frequency of once a week for all the cases during the administration period, after the second week and the recovery test period on the administration start day.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of administration period and recovery test period
- Anaesthetic used for blood collection: Yes, Pentobarbital
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table [No.?] were examined. the number of red blood cells by the , the number of white blood cells, the amount of hemoglobin, mean corpuscular volume and the number of platelets. The average red blood cell hemoglobin amount, average red blood cell hemoglobin concentration, and hematocrit value were calculated based on these values. In addition, a part of the blood was used as a smear, and white blood cell classification and reticulocyte ratio were obtained. The measurement of prothrombin time and activated partial thromboplastin time was also measured

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of administration period and recovery test period
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table [No.?] were examined. Total protein concentration, albumin concentration, total cholesterol concentration, glucose concentration, urea nitrogen concentration, creatinine concentration, alkaline phosphatase activity, GOT activity, GPT activity, LDH activity, calcium concentration, inorganic phosphorus concentration, Triglyceride concentration, γ-GTP activity, A / G ratio were measured. In addition, sodium concentration, potassium concentration and chlorine concentration were measured.

URINALYSIS: Yes
- Time schedule for collection of urine: Urinalysis was performed in 5 animals at the end of the administration period (administration day 23), and all of the recovery test cases were selected at the end of the recovery test period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. Color tone and turbidity (vision), specific gravity in urinal volume, Inspection of pH, occult blood, proteins, sugar, ketone bodies, bilirubin, urobilinogen and sediments was conducted using urine collected within about 4 hours in a metabolism cage using the test paper method and microscopic inspection

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations: No data
- Dose groups that were examined: No data
- Battery of functions tested: sensory activity / grip strength / motor activity / other: No data

OTHER: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, the brain, liver, kidney, adrenal gland, testis or ovary of each animal was weighed, and the weight of each organ was divided by the body weight on the day of necropsy, and the relative weight of each was calculated.

HISTOPATHOLOGY: Yes, Histopathological examination of formalin stained tissues of the duodenum, jejunum, ileum, colon, rectum, testis or ovary, seminal vesicle, bladder, prostate, bone marrow (femur), sciatic nerve, skeletal muscle (lower thigh) and lesions was performed by embedding paraffin for the brain, spinal cord, heart, liver, kidney, spleen, adrenal gland, and sciatic nerve of 1000 mg / kg administration group and solvent control group of autopsy animals at the end of the administration period, - Eosin stained specimens were prepared and carried out. In addition, lesions of animals in which macroscopic abnormality was observed at the end of the administration period and at the end of the recovery test period were also performed.

Other examinations:

No data

Statistics:

Averages and standard deviations were determined for each group on body weight, food intake, urine test (urine volume, specific gravity) and hematology test, blood biochemical examination and organ weight values of regular dissection cases. In addition, when there are three or more groups in the test group including the solvent control group, a test of uniformity of dispersion by the Bartlett method (significance level: 5%) is carried out, then if the variance is uniform, It performs a one-way ANOVA type, significant (significance level: 5%) at the time of the Dunnett or Scheff was multiple comparison in the method method. On the other hand, if the variance is not uniform performs a rank test of Kruskal-Wallis, significant (significance level: 5%), then Dunnett type or Scheff was multiple comparison in of the method. In addition, when the test group consists of two groups including the solvent control group, the test of the difference between the mean value of the solvent control group and the average value of the test substance administration group is Student's t test, unequal variance if equal variance If so, Aspin-Welch's t test was done. Regarding the histopathological findings, the grade-separated data was obtained by Mann-Whitney U test and the total value of positive grade was determined by Fisher's direct probability one-sided test between the solvent control group and each test substance administered group A significant difference test was conducted (significance level: 5%).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not specified

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

Histopathological findings: neoplastic:

not specified

Details on results:

Clinical signs and mortality
Clinical signs: As a change in general condition, crust, ulcer and hair loss were observed in the skin of the neck in one males, crusts and hair loss were observed even during the recovery test period in the solvent control group during the administration. In the test animals, depilation was observed in both forelimbs during the administration period and recovery test period. In addition, in one female, there were crusts in the skin of the neck during the administration period, and crust, ulcer, hair loss during the recovery test period. Also, during the administration period, in the 1000 mg / kg administration group, two cases of male had crusts in the skin of the neck, and in one of them, ulcer was also observed. In addition, in one female of the same group, transient salivation after administration on day 19 of administration was observed.

Mortality: No deaths were noted during the study

Body weight and weight gain No significant difference in mean body weight between the solvent control group and the test substance administration group in both males and females during the administration period and the recovery test period were noted.

Food consumption and compound intake: The average feed intake of male significantly increased on day 8 of administration of 1000 mg/kg administration group compared with the solvent control group, but in females there was no significant difference in the any of the group throughout the administration period and the recovery test period.

Food efficiency: No data

Water consumption and compound intake No data

Opthalmoscopic examination No data

Haematology No significant difference was found between the solvent control group and the test substance administered group in any test items in males and females.

The hemoglobin content and hematocrit value were significantly decreased in males in the 1000 mg/kg administration group in the test at the end of recovery period, and the active partial thromboplastin time was significantly shortened. In females, white blood cell count decreased significantly.

Clinical chemistry: At the end of the administration period, the calcium concentration in the 100 mg/kg administration group and the total protein concentration in the 300 mg/kg administration group in the male decreased significantly between the solvent control group and the test substance administration group In females, the inorganic phosphorus concentration in the 300 mg / kg administration group significantly increased, but none of them were dose - dependent changes. There was also a significant increase in the GPT activity of the male 1000 mg / kg administration group, and in the female 1000 mg / kg administration group, a significant rise in creatinine concentration and calcium concentration and an increase in GPT activity were observed.

At the end of the recovery test period, the sodium concentration and GPT activity increased significantly in the male and the potassium concentration decreased significantly in the male group at 1000 mg/kg. In females, sodium concentration increased significantly.

Urinanalysis: 3 cases in the male 1000 mg / kg administration group and 2 cases in the female 300 mg / kg administration group were urinary bilirubin positive.

No dose-dependent change was observed in the number or degree of appearance in any examination items. There was no clear difference that could be attributed to administration of the test substance between the solvent control group and the test substance administration group.

Neurobehaviour: No data

Organ weights There was no significant difference in absolute weight and relative weight between the solvent control group and the test substance administration group in males and females in the organ weights of autopsy cases at the end of administration period.

However, at the end of the recovery test period, the absolute weight of each organ in the 1000 mg/kg administration group of the autopsy case showed a significant decrease in female liver. Also, the relative weight was significantly decreased in male testis and female liver and significantly increased in female kidney.

Gross pathology: Autopsy cases showed changes in lung, kidney, liver, adrenal glands, mandibular lymph nodes and skin at the end of administration and recovery period

Histopathology: Periportal lipidation was observed in the male and female solvent control group and 1000 mg/kg administration group, but there was no clear difference in frequency and extent between the two groups. In one male in the 300 mg/kg administration group where grossly depressed parts were observed, focal necrosis was observed histologically, and in one female of the same group in which a light color region was observed, the portal vein. There was no abnormality other than sexual fat. In the spleen, extramedullary hematopoiesis was observed in both the male and female solvent control group and in the 1000 mg/kg administration group, brown pigmentation was found in all of the female and female groups.

In the kidney, basophilic tubules were found in the cortex of male solvent control group, 4 cases of each of 1000 mg/kg administration group and 3 cases of each group of females, and basal tubules in 2 cases of both female groups Mineral deposition was found at the boundary, but there was no apparent difference in frequency and degree between the two groups. Cysts were found in one male in the solvent control group, and renal pelvic dilation was observed in one male in the 1000 mg / kg administration group. In addition, cysts were confirmed histologically in males of 100 mg / kg group in which cysts were found macroscopically in the cortex, and basophilic renal tubules were found in the cortex.

Besides, there were no abnormalities in the brain, spinal cord, heart, adrenal gland and sciatic nerve, and there were no histopathologic changes thought to be attributable to the administration of the test substance to other macroscopically abnormal organs. No statistically significant differences were found in any histopathological findings.

At necropsy at the end of the recovery test period, there were no changes that could be attributed to the administration of the test substance to the organ in which macroscopically abnormalities appeared.

Effect levels

Dose descriptor:

NOAEL

Effect level:

300 other: mg/Kg bw

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant alteration were noted at the mentioned dose level

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The No observed adverse effect level (NOAEL) for 4-amino-1-sodium naphthalene sulfonic acid in male and female SPF strain Sprague Dawley rats is found to be 300 mg/Kg bw/day.

Executive summary:

Repeated dose oral toxicity study was performed to evaluate the toxic nature of 4-amino-1-sodium naphthalene sulfonic acid upon repeated application by oral route of administration. The test chemical was dosed at levels of 0, 100, 300 or 1000 mg/Kg once daily for 28 days. Recovery test was set up in the study designed.

 

During the administration period and during the recovery test period, no deaths were observed in any of the treatment groups, and the results of general condition, body weight, food intake and urinalysis, hematology examination, pathological examination were also used for administration of the test substance There was no change thought to be caused.

 

On the other hand, in the blood biochemical test at the end of the administration period, high GPT activity was observed in the group administered 1000 mg / kg of male and female, and it was judged that it is highly likely that it is a finding attributable to administration of the test substance.

 

Hence, The No observed adverse effect level (NOAEL) for 4-amino-1-sodium naphthalene sulfonic acid in male and female SPF strain Sprague Dawley rats is found to be 300 mg/Kg bw/day.