2-chloro-6,7-dimethoxyquinazolin-4-amine

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 J check

Data source

Reference

Reference Type:

other: J check

Title:

Combined Repeat Dose and Reproductive / Developmental Toxicity Screening Test of 2-Amino-1-naphthalenesulfonic acid by Oral Administration in Rats

Author:

National Institute of Technology and Evaluation

Year:

2016

Bibliographic source:

J check, 2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Combined repeated dose repro-devp. Screen was performed for the test chemical 2-Amino-1-naphthalenesulfonic acid to evaluate its toxic nature upon repeated application by the oral route of exposure.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-aminonaphthalene-1-sulphonic acid

EC Number:

201-331-5

EC Name:

2-aminonaphthalene-1-sulphonic acid

Cas Number:

81-16-3

Molecular formula:

C10H9NO3S

IUPAC Name:

2-aminonaphthalene-1-sulfonic acid

Constituent 2

Reference substance name:

2-Amino-1-naphthalenesulfonic acid

IUPAC Name:

2-Amino-1-naphthalenesulfonic acid

Test material form:

other: Solid

Details on test material:

- Name of test material: 2-Amino-1-naphthalenesulfonic acid
- Molecular formula: C10H9NO3S
- Molecular weight: 223.2511 g/mol
- Substance type: Organic
- Physical state: Yellowish white to very light red solid
- Impurities: Purity: 98.9%

Specific details on test material used for the study:

- Name of test material: 2-Amino-1-naphthalenesulfonic acid
- IUPAC name: 2-Amino-1-naphthalenesulfonic acid; 2-aminonaphthalene-1-sulfonic acid
- Molecular formula: C10H9NO3S
- Molecular weight: 223.2511 g/mol
- Substance type: Organic

Test animals

Species:

rat

Strain:

other: Crj: CD (SD), (SPF)

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Source: Japan Hokkaido Center, Charles River Japan Co., Ltd.
- Age at study initiation: 9 week and 5 days
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: During the quarantine / acclimation period, stainless steel suspended cages were used to keep up to 5 groups per cage, and after grouping they were individually raised using a stainless steel 5 cage
- Diet (e.g. ad libitum): CRF-1,
Oriental Yeast Industry Co., Ltd. Ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period:
Acclimation period: 7 days
Quarantine period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24 ° C
- Humidity (%): 40 to 70%
- Air changes (per hr): 12 times / hour
- Photoperiod (hrs dark / hrs light): bright and dark each for 12 hours

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

No data

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: Test sample solution was prepared by suspending the test substance in 0.5% CMC aqueous solution.

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): 0.5% CMC aqueous solution
- Concentration in vehicle: 0, 8, 40, 200 or 1000 mg/Kg bw
- Amount of vehicle (if gavage): 5 mL/Kg
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance concentration in each administration sample solution was measured by HPLC method in the test facility twice before administration and before the administration period.

Duration of treatment / exposure:

The administration period was 49 days in males for the total of 49 days before mating and for 35 days thereafter in the female, 14 days before mating in the female, during the mating period (maximum 8 days), the day before the pregnancy period and 4th day of autopsy on the 4th day of nursing (41 to 48 Days).

Frequency of treatment:

Daily, Once a day

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 8, 40, 200 or 1000 mg/Kg bw
Basis:

No. of animals per sex per dose:

Total: 120
0 mg/Kg bw: 12 male and 12 female
8 mg/Kg bw: 12 male and 12 female
40 mg/Kg bw: 12 male and 12 female
200 mg/Kg bw: 12 male and 12 female
1000 mg/Kg bw: 12 male and 12 female

Control animals:

yes, concurrent vehicle

Details on study design:

Details on study design
- Dose selection rationale: As a result of preliminary test (oral administration: 0, 30, 100, 300 and 1000 mg / kg, 5 groups per group) for oral administration for 2 weeks for setting dose using male rats, 1000 There was no mortality in the mg / kg group, and no abnormalities were observed in general condition, weight shift and necropsy.
- Rationale for animal assignment (if not random): The grouping was performed on the day before the administration start date so that the average body weight and dispersion of each group were almost equal by random sampling method after separating body weight by stratum.
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: No data available
- Section schedule rationale (if not random): No data available

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the administration period, observation was made twice before and after administration daily
- Cage side observations checked in table [No.?] were included. Clinical signs and mortality

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

BODY WEIGHT: Yes
- Time schedule for examinations:
Males:Twice per week and measured on autopsy day

Females:Twenty four days before the mating and twice weekly during the mating period, gestation on 0, 7, 14 and 21 gestation during pregnancy, and during nursing at 0 and 4 days of feeding, respectively

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes,
Males:
After 14 days before the start of the mating and after the end of the mating period, the amount was measured twice a week for 2 consecutive days and converted into the daily dose. From the evening of the day before necropsy it was fasted.

Females:
For 14 days before the start of the mating, the amount was measured twice a week for 2 consecutive days and converted into the daily dose. During the pregnancy period, cumulative amounts for 0, 4, and 11 days of gestation were measured for 2 consecutive days from the 0th, 7th, 14th, and 19th gestation days, respectively, and converted into the daily amount, respectively.
- 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: Males: on the day after the final administration
- Anaesthetic used for blood collection: Males: Yes, sodium pentobarbital
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. Males: Prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen concentration were determined

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Males: on the day after the final administration
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined.
Males: GOT and GPT , γGTP , total protein (TP), urea nitrogen (BUN) , creatinine Jaff , total bilirubin (T-Bil) , glucose, inorganic phosphorus (IP) by Molybdenum blue method and Ca. Na and K, and Cl were also measured. The amount of albumin was calculated.

URINALYSIS: No data
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. No data

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,
Males: Gross observation of organs and tissues was conducted after further exsanguination by exsanguinating the animals collected. After removing the liver, kidney, thymus, testis and epididymis, the weight was measured, and the adrenal glands, brain, heart and spleen were further excised.

HISTOPATHOLOGY: Yes,
Males: testis and epididymis were fixed in Bouin's solution, and other organs / tissues were fixed in 10% neutral buffered formalin solution.

Male/Female: Paraffin embedded specimens were prepared for each organ and tissue in all fixed cases according to a conventional method. For the control group and 1000 mg / kg group, HE stained tissue specimens were prepared and histopathologically examined.

Other examinations:

Females:
Sex cycle:
Every day from observation start date to mating confirmation day was observed once. In addition, when the estrus period was observed for 2 consecutive days, it was counted as 1 time.

Observation of delivery status: After daily observation up to 4th day of nursing, on day 4 of nursing exsanguinated by ether anesthesia from the abdominal aorta, necropsy was done and the number of lesion traces and corpus luteums were counted. The liver, kidney, thymus and ovary were excised and weighed and fixed in 10% neutral buffered formalin solution together with adrenal glands, brain, heart and spleen.

Treatment of mother animals whose whole newborn died:
Including a maternal animal in which all newborns died [including those in the case of stillborn infants (1 case of 8 mg / kg group)] was found
to be bleeding from the abdominal aorta immediately under ether anesthesia and necropsied, The number and corpus luteum number were
counted. The liver, kidney, thymus and ovary were excised and weighed and fixed in 10% neutral buffered formalin solution together with adrenal glands, brain, heart and spleen.

Male and female in the same group of approximately 12 weeks old, to whom the test substance was administered over 14 days, were mated
together in a one to one
combination. The mating period was set as continuous living crossing until confirmed mating up to the 14th day, but mating of all cases was confirmed by 8th day after living together. The mating confirmation went at almost a fixed time every morning. A female whose sperm or vaginal plug was confirmed in the vaginal plaque was regarded as a copulating animal and the day was counted as the 0th day of pregnancy.

Statistics:

The statistical method of the measured value was the multiple comparison test and chi square test, and the significant difference test was carried out between the control group and each administration group of the test substance. In any of the tests, less than 5% of significance level was considered significant, and it was divided into less than 5% (p <0.05) and less than 1% (p <0.01). The newborn baby set the average of one stomach as one unit.

Multiple comparison test:
Weight, food consumption, number of estrous cycles, number of copulation days, pregnancy period, implantation number, number of corpus
luteums, implantation rate, total number of births, number of stillbirths, delivery rate, birth rate, survival rate on 4th day of nursing, number of neonates , Sex ratio, appearance rate of outer table abnormality, organ weight (absolute weight and relative weight), hematology examination results and blood biochemical examination results.
In the test, we test homodispersity by the Bartlett method, perform variance analysis by the one way
method in the case of equal variance, and if significant, compare the intergroup
comparison with the control group by the Dunnett method (if the number is equal) or Scheff was carried out by method (if the number of cases is not equal). On the other hand, when it is not recognized as equal variance, the analysis by oneway
method using the ranking (Kruskal Wallis test) is carried out, and if it is significant, the intergroup comparison with the control group is done by
the Dunnett method or Scheff Method.

Chi square test:
Mating rate, conception rate and birth rate

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

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:

not specified

Other effects:

not specified

Details on results:

Clinical signs and mortality
Clinical signs:
No abnormal symptoms were observed in the control group and the administration group of 200 mg / kg or less.

Males: In the 1000 mg / kg group, salivation was observed after administration in 1 to 12 animals until the final administration day after 7th day of administration. This symptom was seen from 2 to 3 minutes after administration but disappeared about 10 minutes after administration. No other abnormal symptoms were observed.

Females: In the 1000 mg / kg group, salivation was observed in 0 to 6 cases after 7 days of administration before the mating, 3 to 12 cases during
gestation period, and salivation after administration in all cases during the feeding period. This symptom was seen from 2 to 3 minutes after administration but disappeared about 10 minutes after administration.

Body weight and weight gain: No significant changes were noted

Food consumption and compound intake
Males: There was no significant difference between the control group and the 1000 mg/kg group except that a significant high level was observed on the 6th day of administration.

Females: No significant changes were noted

Food efficiency: No data available

Water consumption and compound intake: No data available

Opthalmoscopic examination: No data available

Haematology Males: No significant changes were noted

Clinical chemistry
Males: In the 1000 mg / kg group, total bilirubin showed a significant high value as compared with the control group. The other test items were almost the same as those of the control group in each administration group, and no significant difference was observed.

Urinanalysis: No data available

Neurobehaviour: No data available

Organ weights
Males: In the 1000 mg / kg group, absolute liver weight and relative weight were both significantly lower than the control group. There was no significant difference between any organ weight and the control group in the administration group of 200 mg / kg or less.

Females: No significant changes were noted

Gross pathology: No significant changes were noted

Histopathology
Males:
Liver: Very mild necrosis lesion was found in the control group and one in the 1000 mg / kg group, very slight to mild granulomas in the control group and 6 to 8 cases in the 1000 mg / kg group, very mild to mild cell collection Cough was seen in 3 to 5 cases of control group and 1000 mg / kg group.

Heart: Very mild to moderate cell infiltration was seen in the control group and 1 to 4 cases of 1000 mg / kg group.

Kidney: Very mild renal tubular basification was observed in the control group and 12 cases in the 1000 mg / kg group, with very mild cell infiltration in the control group and 12 cases in the 1000 mg / kg group. No marked changes were observed in other test sites (thymus, spleen, testis, epididymis, adrenal glands and brain).

Females:
Liver: Very mild necrosis was found in 2 of the control group.

Heart: Very mild cell infiltration was found in one case of 1000 mg / kg group.

Kidney: Very mild Ca deposition was found in the tubule in the 1000 mg / kg group.

Spleen: Very mild to moderate extramedullary hematopoiesis was found in 11-12 cases of control group and 1000 mg / kg group.

No marked changes were observed in other test sites (thymus, ovary, adrenal gland and brain).

Effect levels

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant changes were noted

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Absolute and relative organ weights of male rats in combined repeat dose and reproductive/developmental screening toxicity test of  

2-amino-1-napthalenesulphonic acid bu oral administration

Group	Control	2-amino-1-napthalenesulphonic acid
Dose (mg/Kg)	0	8	40	200	1000
Body weight (g)	494.7±28	480.2± 38.2	493.9± 26.6	485.6± 21.4	478.2± 26.4
Thymus (mg)	356.11± 82.33	328.28± 72.93	349.02± 101.54	290.50± 45.01	305.75± 91.68
Liver (g)	13.097± 1.668	12.369± 1.334	13.451± 1.885	12.503± 1.067	11.477± 1.082*
Kidney (g)	2.953± 0.244	3.017± 0.222	2.977± 0.254	3.029± 0.258	3.032± 0.169
Testes (g)	3.339± 0.164	3.399± 0.230	3.527± 0.26	3.322± 0.245	3.379± 0.141
Epididymides (g)	1.271± 0.012	1.325± 0.122	1.305± 0.114	1.322± 0.083	1.279± 0.070

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) in Crj: CD (SD), (SPF) rats for 2-Amino-1-naphthalenesulfonic acid is considered to be 200 mg/Kg bw.

Executive summary:

Combined repeated dose repro-devp. Screen was performed for the test chemical 2-Amino-1-naphthalenesulfonic acid to evaluate its toxic nature upon repeated application by the oral route of exposure.

 

The test compound was administered to Crj: CD (SD), (SPF) male and female rats at dose levels of 0, 8, 40, 200 or 1000 mg/Kg bw.

 

The animals were observed for clinical signs, mortality, hematological and blood chemistry parameters, changes in body weight and alteration in gross and histopathology if any.

Changes in organ weight (liver) in males and histopathological findings in females were noted at 1000 mg/Kg bw, no significant changes were noted at 200 mg/Kg bw in male and female animals. Therefore, the No Observed Adverse Effect Level (NOAEL) in Crj: CD (SD), (SPF) rats for 2-Amino-1-naphthalenesulfonic acid is considered to be 200 mg/Kg bw.