9,10-Anthracenedione, 1,4-diamino-, N,N'-mixed 2-ethylhexyl and hexyl and octyl derivs.

Endpoint summary

Description of key information

Repeated dose toxicity: Oral

The No Observed Adverse Effect Level (NOAEL) of the test chemical in the rat via oral route of exposure is considered to be 1000 mg/kg body weight in male and female animals.

Repeated dose toxicity: Inhalation

Test chemical has very low vapor pressure of 9.7058E-10 Pa (7.279e-12 mmHg), so the potential for the generation of inhalable vapours is very low, also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore this end point for repeated dose toxicity by inhalation route of exposure was considered for waiver.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. Given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that test chemical shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that test chemical shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: 2. Sprague Dawley; 3/4. Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

2. TEST ANIMALS
- Source:National Institute of Biosciences, Pune
- Females (if applicable) nulliparous and non-pregnant:[yes
- Age at study initiation:6 to 8 weeks
- Weight at study initiation: Male - 168.90 g (= 100 %), Female - 152.26 g (= 100 %)
- Fasting period before study:
- Housing: The rats were housed in polycarbonate cages with paddy as bedding.
After allocation to respective dose groups rats were housed 2/sex/cage.
- Diet (e.g. ad libitum): Rodent feed, ad libitum
- Water (e.g. ad libitum): Water was provided ad libitum from individual bottles attached to the cages. Water was from a local source and passed through the reverse osmosis membrane before use
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30% to 70%
- Air changes (per hr): The animal room was independently provided with at least ten air changes per hour of 100% fresh air that has been passed through the HEPA filters
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room

IN-LIFE DATES:
From: 07-12-2017
To: 02 -03- 2018

3. - Age at study initiation of dosing: 10 weeks old

4. TEST ANIMALS
- Source: Charles River Co., Ltd. (Atsugi city, Kanagawa Prefecture)
- Age at study initiation: 6 weeks
- Weight at study initiation: 131 to 146 g for males and 112 to 129 g for females
- Fasting period before study: No data
- Housing: The animals were raised in the rearing room (W 9.8 × D 8.2 × H 2.5 m, 200.9 m ^ 3) of the barrier system. Using a washing type rearing machine (W 674.2 × D 80.0 × H 175.5 cm) of Tokyo
Giken service (Fuchu-shi, Tokyo), a metallic front and bed network breeding cage (W 20.0 × D 28.2 × H 18.0 cm, one animal was housed in a breeding cage space of 10, 152 cm ^ 3). The breeding cages were replaced every other biweekly, and the feeders were changed once a week.
- Diet (e.g. ad libitum): Radiation sterilized improved NIH release rat / mouse feed produced by Oriental Yeast Co., Ltd. (Chuo-ku, Tokyo) was ingested ad libitum
- Water (e.g. ad libitum): No data
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1 ° C
- Humidity (%): 55 ± 5%
- Air changes (per hr): 150 ~ 300 lux, 12 hours (7 am lights a day, 7 pm off)
- Photoperiod (hrs dark / hrs light): 20 times / Hour

IN-LIFE DATES: From: To: No data

Route of administration:

oral: gavage

Vehicle:

other: 2. Corn oil; 3. 0.5 % Methylcellulose aqueous solution; 4.

Details on oral exposure:

2. PREPARATION OF DOSING SOLUTIONS:The test item was diluted with corn oil for preparation of solution(s). The solution(s) of the test chemical were made at volumes suitable for daily use for 28 days. The solution(s) were prepared at concentrations of 0, 25, 50 and 100 mg/ml such that dosage of 0 (vehicle), 250, 500 and 1000 mg/kg body weight respectively were administered.

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): corn oil
- Concentration in vehicle: 0 (vehicle), 250, 500 and 1000 mg/kg body weight
- Amount of vehicle (if gavage): 10 ml/kg body weight.
- Lot/batch no. (if required): No data
- Purity: No data

3. PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved in 0.5 % Methylcellulose aqueous solution to give dose level of 0, 40, 200 or 1000 mg/Kg/day

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): 0.5 % Methylcellulose aqueous solution
- Concentration in vehicle: 0, 40, 200 or 1000 mg/Kg/day
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data

4. PREPARATION OF DOSING SOLUTIONS: A predetermined amount of the test substance was precisely weighed every specified dose (100, 300 and 1,000 mg / kg), suspended in a 0.5% sodium
carboxymethyl cellulose aqueous solution using an agate mortar, and 0.1% (w / v) Tween 80 was added and sufficiently mixed to prepare the dose

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): 0.5% carboxymethylcellulose sodium / water solution containing 0.1% Tween 80
- Concentration in vehicle: 0,100, 300, 1000 mg/kg/day
- Amount of vehicle (if gavage): 0.5 % CMC Na solution with 0.1% Tween 80

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

2. The test item formulation analysis for concentration and stability were conducted
3. No data
4. Although it was confirmed that the administration solution was stable for 2 weeks after storage in the refrigerator, preparation was carried out once a week in this test. Concentration analysis of
the administration solution was performed on the preparations of administration 1 and 4 weeks for all groups, and it was in the range of 99.0 to 104% of the prescribed concentration and was
appropriately prepared.

Duration of treatment / exposure:

2. 28 days
3. Male: 42 days / - Female: 41 - 45 days (from 14 days before mating to day 4 of lactation)
4. 28 days

Frequency of treatment:

Daily

Remarks:

0, 250, 500 or 1000 mg/Kg/day / 2

Remarks:

0, 40, 200 or 1000 mg/Kg/day / 3

Remarks:

0, 100, 300, 1000 mg/kg/day / 4

No. of animals per sex per dose:

2. Total: 72
0 mg/kg bw: 6 male, 6 female
250 mg/kg bw: 6 male, 6 female
500 mg/kg bw: 6 male, 6 female
1000 mg/kg bw: 6 male, 6 female

Reversal group
500 mg/kg bw: 6 male, 6 female
1000 mg/kg bw: 6 male, 6 female

3. Test group:
0 mg/Kg/day: 7 males and 12 females
40 mg/Kg/day: 12 males and 12 females
200 mg/Kg/day: 12 males and 12 females
1000 mg/Kg/day: 7 males and 12 females

Recovery group:
0 mg/Kg/day: 5 males and 5 females
1000 mg/Kg/day: 5 males and 5 females

4. Total no of animals: 60

Test group
0 mg/kg/day-5 male and 5 female
100 mg/kg/day-5 male and 5 female
300 mg/kg/day-5 male and 5 female
1000 mg/kg/day-5 male and 5 female

Recovery group:
0 mg/kg/day-5 male and 5 female
1000 mg/kg/day-5 male and 5 female

Control animals:

yes, concurrent vehicle

Details on study design:

2. - Dose selection rationale: Dose were selected basd on Dose Range Finding study:
1) All the male and female animals from control and all the treated dose groups up to 1000 mg/kg survived throughout the dosing period of 7 days.
2) Male and female animals from control and all the treated dose groups exhibited normal body weight gain at the end of the dosing period of 7 days.
3) Daily clinical observations revealed test item coloured faces in male and female animals from different dose groups during the dosing period of 7 days.
4) Gross pathological examination revealed small and large intestine with test item coloured ingesta with test item coloured stomach mucosa in male and female animals from different dose groups.
Based on these results, the 28 day study dose levels were finalized as 0 mg/kg, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight and animals were exposed to the treatment every day for a period of 28 days.
- Rationale for animal assignment (if not random): A total of 72 animals (36 males + 36 females) were selected and randomly distributed into six groups with 6 animals/sex/group for main groups and 6 animals /sex /group for reversal group.
The animals of uniform body weight were selected. The individual body weights of the animals did not exceed ± 20% of group mean body weight. The group means body weights of all the groups were approximately equal..
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random):

3. No data

4. - Dose selection rationale: Preliminary studies with 2 weeks dosing for dose setting were performed at 4 doses of 0, 200, 600 and 1,800 mg / kg / day.
As a result, dead animals were not found in any of the administration groups, and no changes that could be attributed to administration of the test substance were observed in body weight, feed intake, pathological autopsy findings and organ weight. Therefore, the maximum dose in the 28-day repeated dose test was set to the limit dose of 1,000 mg / kg, divided by the common ratio 3 below, and the medium dose was set to 300 mg / kg and the low dose to 100 mg / kg. A control group to which only carrier (0.5% carboxymethylcellulose sodium aqueous solution containing 0.1% Tween 80) was administered was provided.
- Rationale for animal assignment (if not random): The animals were grouped by the random extraction method
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups: Yes, recovery group was included in the 0 and 1000 mg/Kg/day group
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): No data
- Other: No data

Positive control:

No data

Observations and examinations performed and frequency:

2. CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice daily
- Cage side observations checked in table [No.?] were included.: Viability observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on the day of randomization, day of first dosing, weekly thereafter and a fasting body weight at scheduled sacrifice on day 29 and day 43.

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:The eyes of all the animals were examined prior to the initiation of the dosing and at scheduled sacrifice. Eye examination was carried out by using a HEINE mini 2000 ophthalmoscope were employed for evaluation after the induction of mydriasis with 1% solution of tropicamide sulfate.
- Dose groups that were examined: All 72 animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of dosing period on day 29 and at termination of recovery period on day 43.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All 72 animals
- Parameters checked in table [No.?] were examined.: Hb (Hemoglobin (g/dL)), RBC (Red Blood Corpuscles (x 106 /µL)), HCT (Hematocrit (%)), MCV (Mean Corpuscular Volume (fL)), MCH (Mean Corpuscular Hemoglobin (pg)), MCHC (Mean Corpuscular Hemoglobin Concentration (g/dL)), Platelets (x 103 /µL), WBC (White Blood Corpuscles (x 103 /µL)), Rt.(Reticulocytes (%)), N (Neutrophils (%)), L (Lymphocytes (%)), E (Eosinophils (%)), M (Monocytes (%)), B (Basophil (%)) and Pt. (Prothrombin time (sec.)) were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of dosing period on day 29 and at termination of recovery period on day 43.
- Animals fasted: Yes
- How many animals:All 72 animals
- Parameters checked in table [No.?] were examined.: Total Protein (g/dL), Blood Urea Nitrogen (mg/dL), Urea Nitrogen (mg/dL) Calculated, ALT : Alanine Aminotransferase (U/L), AST, Aspartate Aminotransferase (U/L), ALP : Alkaline Phosphatase (U/L), GGT : Gamma Glutamyl Transferase (U/L), Glucose (mg/dL), Calcium (mmol/L), Phosphorous (mg/dL), Albumin (g/dL), Total Bilirubin (mg/dL), Creatinine (mmol/L), Total Cholesterol (mg/dL), Triglycerides (mg/dL), Globulin (g/dL) Calculated, Sodium (mmol/L), Potassium (mmol/L), Chloride (mmol/L) and
Bile acid (µmol/L) were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of dosing period and on reversal group rats at termination of recovery period on day 43.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined.: Volume , Appearance, Colour, pH , Specific Gravity, Proteins, Glucose, Ketones,Bilirubin, Urobililogen, Occult Blood and Nitrite were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the exposure period of 28 days and towards the end of the recovery period on day 42, sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) assessment of grip strength and motor activity assessment were conducted for all the animals
- Dose groups that were examined: All 72 animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other:Arousal level, Sensory Activity, Visual Placing Response, Air righting response, Grip Strength, Motor Activity,

IMMUNOLOGY: Not specified
- Time schedule for examinations:Not specified
- How many animals:Not specified
- Dose groups that were examined:Not specified
- Parameters checked in table [No.?] were examined.Not specified

OTHER: Organ Weights were examined.

3. CAGE SIDE OBSERVATIONS: Yes
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. No data

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

BODY WEIGHT: Yes
- Time schedule for examinations: No data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. TP, Alb, 2-Glob

URINALYSIS: Yes
- 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: Yes
- 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

IMMUNOLOGY: No data
- Time schedule for examinations: No data
- How many animals: No data
- Dose groups that were examined: No data
- Parameters checked in table [No.?] were examined. No data

OTHER: No data

4. CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed every morning, afternoon twice (once on holidays) every day
- Cage side observations checked in table [No.?] were included. the presence or absence of intoxication symptoms, behavior abnormalities, the presence of dead animals and the presence or absence of dead animals, etc. were recorded.

DETAILED CLINICAL OBSERVATIONS: Not specified
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: once a week from the start of administration until the end of recovery test.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes, Feed intake (g / week) was calculated by measuring the remaining amount fed once a
week.

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 and at the end of the recovery period
- Anaesthetic used for blood collection: Yes, ether anaesthesia was given to animals
- Animals fasted: Yes, for 16 hrs
- How many animals: All animals
- Parameters checked in table [No.?] were examined. The hematocrit value (HCT: corrected from the volume of total erythrocyte), the average red blood cell volume (WBC: dark field plate method), r
ed blood cell count (RBC: dark field plate method), hemoglobin amount (HGB: cyanomethemoglobin method) (Calculated from MCV: RBC, HCT), mean red blood cell hemoglobin amount (calculated fro
m MCH: HGB, RBC), mean red blood cell hemoglobin concentration (calculated from MCHC: HGB, HCT), platelet count (PLT: dark field plate method) (Flow cytochemistry method) was measured using
a blood automatic analyzer THMS. For calculating the reticulocyte (RC) ratio, whole blood was stained with Capilot, and a blood smear sample was prepared and stored. In addition, the prothrombin ti
me (guick 1-step method) and the activated partial thromboplastin time (clotting method) were measured for blood plasma of blood citrate-added blood using an automatic blood coagulation measuring apparatus KC-40.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of administration and at the end of the recovery period
- Animals fasted: Yes, for 16 hrs
- How many animals: All animals
- Parameters checked in table [No.?] were examined. By using serum, total protein (Buret method), albumin (BCG method), A / G ratio (calculated value), blood sugar (hexokinase method), neutral fat
(enzymatic method), total cholesterol (enzymatic method), urea EKTACHEM 700N (Kodak Company, USA) is used for nitrogen (BUN: urease modification method), calcium (alizarin method), inorganic
phosphorus (molybdenum blue method), sodium (electrode method), potassium (electrode method) and chlorine Glutamic acid oxaloacetate transaminase (GOT: Karmen modification method), glutamic
acid pyruvic acid transaminase (GPT: Wroblewski and LaDue improved method), γ-glutamyl transpeptidase (γ-GTP: enzymatic method) and alkaline phosphatase (ALP : Bessey-Lowry-Brock Impro
vement Method) was measured with Centrifi Chem ENCORE II

URINALYSIS: Yes
- Time schedule for collection of urine: Prior to hemotological examination, urine was collected using a urine collector for 24 hours (from 10 am until the next morning at 10 a.m.)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked in table [No.?] were examined. Urine volume, color tone and turbidity were inspected, and then urine density meter UR - S , Itabashi-ku, Tokyo) was used to measure the urin
e specific gravity, the urine was centrifuged, the sediment was stained by the Sternheimer method, and examined by a microscope. The pH, occult blood, ketone bodies, sugar, protein, bilirubin and
urobilinogen were measured using N-Martistic SG test paper and CLINITEK 200

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

IMMUNOLOGY: No data
- Time schedule for examinations: No data
- How many animals: No data
- Dose groups that were examined: No data No data
- Parameters checked in table [No.?] were examined. No data

OTHER: No data

Sacrifice and pathology:

2. GROSS PATHOLOGY: Yes, All the rats surviving at the end of the treatment were sacrificed and gross lesions were noted.

HISTOPATHOLOGY: Yes, From each rat, samples or the whole of the tissues listed below were preserved. All tissues were fixed in 10% neutral buffered formalin except, eyes and testes of all animals were preserved in Davidson’s solution for 24 hours and transferred to 10% neutral buffered formalin.
Procedure for preparation of slides of tissues of various organs from the rats of various dose groups were performed as per the standard operating procedures of Indian Institute of Toxicology, Pune.
Following tissue samples of organs from control and animals treated at different dose groups were preserved and those from control and treated at the highest dose level of 1000 mg/kg were subjected to histopathological examination.
Adrenals, Aorta, Brain (cerebrum, cerebellum and pons), Caecum, Cervix, Colon, Duodenum, Epididymides, Eyes, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Mesenteric Lymphnodes, Muscles - Skeletal muscle, Oesophagus, Ovaries, Pancreas, Pharyngeal Lymphnodes, Pituitary, Prostate, Rectum, Sciatic Nerve, Seminal Vesicles with coagulation gland, Skin with Mammary Gland, Spleen, Spinal Cord (Cervical, mid thoracic and lumbar), Sternum with bone marrow, Stomach, Testes, Thymus, Trachea, Thyroid / Parathyroid, Urinary Bladder, Uterus, Vagina.

3. GROSS PATHOLOGY: Yes, no detailed data available

HISTOPATHOLOGY: Yes, no detailed data available

4. GROSS PATHOLOGY: Yes, Pathologic dissection was conducted by ether anesthetizing the animal s at the end of the administration and at the end of the recovery period and killing them to exsang
uination. Macroscopic abnormalities were recorded on pathological autopsy findings recording sheets . Weight was measured for brain, liver, kidney, spleen, adrenal gland, testis and ovary, and the organ weight / body weight ratio was calculated.

Organs / tissues that showed a change in the above weighted organ and pituitary, eyeball, thyroid (including parathyroid gland), heart, lung, stomach, bladder, bone marrow (femur) and macroscopic
findings were 10% And fixed with formalin solution

HISTOPATHOLOGY: Yes, Histopathological examination was performed on the heart, liver, spleen, kidney and adrenal glands of the control group and high dose group among the fixed organs /
tissues. Thin cut specimens were prepared according to a conventional method, and stained with hematoxylin and eosin, followed by a microscopic examination.

Other examinations:

No data

Statistics:

2. Raw data was processed and analyzed for reporting group means and standard deviations with significance between the controls and treated groups, using SYSTAT 13 validated statistical software supplied by Starcom Information Technology Limited, Bangalore developed by Systat Software, Inc. USA. All the parameters characterized by continuous data such as body weight, feed consumption (calculated as gram per animal), organ weight, relative organ weight, haematological and clinical chemistry data were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. Where the data does not meet the homogeneity of variance, Student’s t-test were performed to calculate significance.

3. No data

4. The body weight, feed intake, hematology test value, biochemical test value, urine test value (only urine volume and urine specific gravity), organ weight and organ weight / body weight ratio of each test group are determined according to the automatic discrimination method shown below , Bartlett' s equidistance test was first carried out. In the case of equal variance, a one-way ANOVA is performed, and if the variance is significant, Dunnett's multiple comparison test if the number of samples in each group is the same, and Duncan's multiple range test if the number of samples in each grou p is different, And significant differences between each dose group were tested. In Bartlett's equality variance test, Kruskal-Wallis rank test was carried out in case of unequal variance, and significant difference between control group and each dosing group was tested by nonparametric Dunnett's multiple comparison test in case of significance. For dose correlation, significant differences were tested using Jonckheere's trend test. The significance level was carried out with a one-sided test of 5 and 1%

Clinical signs:

no effects observed

Description (incidence and severity):

2. Male -
Group I (Control, 0 mg/kg): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days (animal nos.1 to 6).
Group II (Control, 0 mg/kg, Reversal): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days and during the post-dosing recovery period (animal nos.13 to 18).
Group III (250 mg/kg): Test item coloured faeces were observed in all animals (animal nos.25 to 30, with onset from day 2) during the dosing period of 28 days.
Group IV (500 mg/kg): Test item coloured faeces were observed in all animals (animal nos.37 to 42, with onset from day 2) during the dosing period of 28 days.
Group V (1000 mg/kg): Test item coloured faeces were observed in all animals (animal nos.49 to 54, with onset from day 2) during the dosing period of 28 days.
Group VI (1000 mg/kg, Reversal): Test item coloured faeces were observed in all animals (animal nos.61 to 66, with onset from day 2) throughout the dosing period of 28 days and during the post-dosing recovery period (upto day 34).

Female -
Group I (Control, 0 mg/kg): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days (animal nos.7 to 12).
Group II (Control, 0 mg/kg, Reversal): No clinical signs of toxicity were observed in the animals throughout the dosing period of 28 days and during the post-dosing recovery period (animal nos.19 to 24).
Group III (250 mg/kg): Test item coloured faeces were observed in all animals (animal nos.31 to 36, with onset from day 2) during the dosing period of 28 days.
Group IV (500 mg/kg): Test item coloured faeces were observed in all animals (animal nos.43 to 48, with onset from day 2) during the dosing period of 28 days.
Group V (1000 mg/kg): Test item coloured faeces were observed in all animals (animal nos.55 to 60, with onset from day 2) during the dosing period of 28 days.
Group VI (1000 mg/kg, Reversal): Test item coloured faeces were observed in all animals (animal nos.67 to 70, with onset from day 2) throughout the dosing period of 28 days and during the post-dosing recovery period (upto day 33).

3. Colored feces were noted in male and female rats treated with 40, 200 or 1000 mg/Kg/day

4. No significant effect were observed at the dose level of 100, 300, 1000 mg/kg/day of treated group and recovery group as compared to control.

Mortality:

no mortality observed

Description (incidence):

2. All animals from control and different dose groups survived throughout the dosing period of 28 days and the post-dosing recovery period of 14 days.
4. No mortality was observed at the dose level of 100, 300, 1000 mg/kg/day of treated and recovery group as compared to control.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2. Male and Female -
Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days.
During the post-dosing recovery period, animals from 1000 mg/kg reversal group exhibited normal body weight gain when compared with that of respective control animals.

3. There was no difference between the groups during the administration period and the recovery period in both males and females, and there was no difference in body weight gain during the administration period (0 to 4 weeks) and during the recovery period (4 to 6 weeks)

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

2. Male and Female -
Animals from control and different dose groups exhibited normal feed consumption at the end of the dosing period of 28 days.
Animals from control reversal and high reversal dose groups exhibited normal feed consumption at the end of the recovery period of 14 days.

3. In males, there was no difference between the groups during the administration period and recovery period, and no difference was observed in total intake between 0 and 4 weeks. In females, food intake decreased in 1 week of administration in 300 and 1,000 mg / kg group compared with control group, but in both groups after 2 weeks of administration there was no difference from control group. Regarding the total food intake for 0 to 4 weeks, there was no difference between the 1,000 mg / kg group and the control group, but the 300 mg / kg group decreased in comparison with the control group.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

2. No ocular abnormalities were observed on ophthalmological examination in the animals during pre-exposure and at the end of the respective termination.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

2. Male :
Total RBC : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).
Female :
MCHC : Decreased values were obtained for animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05).
however the decrease in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent.

3. a) Results at the end of administration
In males, white blood cell count decreased in 100 mg / kg group compared with control group. In females, the ratio of neutrophils decreased and the proportion of lymphocytes increased in the 100
mg / kg group.

b) Result at end of recovery period
There was no difference in males between the control group and 1,000 mg / kg group. In females, MCHC decreased in the 1,000 mg / kg group.

In the test results at the end of the administration period and at the end of the recovery test, there was no difference between the groups in the prothrombin time and activated partial thromboplastin time in both males and females.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

2. Male :
Sodium : Elevated levels were observed in animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05), Glucose and Total Cholesterol : Decreased levels were observed in animals from 500 mg/kg dose group, sacrificed on day 29 (p<0.05), Calcium : Decreased levels were observed in animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05), Total Protein, Globulin, Creatinine and Triglycerides : Elevated levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05) and Bile Acid : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).

Female :
Blood Urea Nitrogen and Urea Nitrogen : Decreased levels were observed in animals from 250 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05) and Total Protein, Glucose, Albumin and Globulin : Elevated levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05). however the increase/decreased in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent.

3. Decrease in the TP, decrease in the Alb and increase in the percentage of alpha 2-Glob (Male) in 1000 mg/Kgday treated animals. Though albumin level (mg/dL) was significantly decreased in males of the high dose group, A/G ratio was not affected.

4. a) Results at the end of administration
In males, A / G increased in all test substance-administered groups, and GPT and γ-GTP increased in the 1,000 mg / kg group as compared with the control group. As another change, blood glucose decreased in the 100 mg / kg group.

In females, γ-GTP decreased in all test substance-administered groups, and potassium decreased in the 300 mg / kg group.

b) Results at the end of the recovery period
Chlorine increased in the 1,000 mg / kg group of males and calcium decreased in the female 1,000 mg / kg group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

2. No statistically significant variation was observed in the urine analyses conducted at the end of the dosing period in week 4 and 6 (on day 26, 27 and 43) in male and female animals of different dose groups as compared to control group animals.

3. Abnormal color of urine was observed in males and females treated with 200 or 1000 mg/Kg/day

4. a) Results at the end of administration
There was no difference between the groups in all the test items for both males and females.

b) Results at the end of the recovery period
There were no difference in test item between 1,000 mg / kg group and control group in both males and females.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

2. Before commencement of treatment: In home cage observation, rat from different dose groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closer. During handling observation, handling of rats did not reveal any abnormality from different dose groups and control group.
In the open field observation, rat did not reveal any abnormality from different dose groups and control group.

During treatment:
In home cage observation, rat from different dose groups and control group revealed normal behavior, alterations, vocalization, respiration and palpebral closer.
During handling observation, handling of rats did not reveal any abnormality from different dose groups and control group.
In the open field observation, rat did not reveal any abnormality from different dose groups and control group.
Detailed clinical observation did not reveal any abnormality in all groups during the dosing period of 28 days and during the post-dosing recovery period.

Sensory Reactivity Observations:
All animals from control and different dose groups showed normal arousal level, visual response, touch response, auditory response, tail pinch response and visual placing response. Normal air righting reflex was observed in all animals from control and different dose groups in week 4.

Grip Strength:
Grip strength values observed in male and female animals for control and different dose groups were comparable.

Motor Activity:
Motor activity values observed in male and female animals for control and different dose groups were comparable.

3. Colored aqueous content in the alimentary tract in 40 mg/Kgday treated male animals. Colored aqueous content in the alimentary tract (Male/Female), Mucosal discoloration of alimentary tract (Male) in 200 mg/Kg/day treated animals Colored aqueous content in the alimentary tract (Male/Female), Mucosal discoloration of alimentary tract (Male/Female) in 1000 mg/Kg/day treated animals

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

2. Male -
In comparison with controls at the end of dosing on day 29, organ weight data of animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups revealed increased relative weights of liver (p<0.05). In addition, increased relative weights of heart (p<0.05) were observed in animals from 500 mg/kg dose group.
In comparison with controls at the end of post-dosing recovery period on day 43, organ weight data of animals from 1000 mg/kg reversal group was found to be comparable.
Female -
In comparison with controls at the end of dosing on day 29, organ weight data of animals from 500 mg/kg and 1000 mg/kg dose groups revealed increased relative weights of liver (p<0.05).
In comparison with controls at the end of post-dosing recovery period on day 43, organ weight data of animals from 1000 mg/kg reversal group revealed decreased relative weights of kidneys, adrenals and ovaries (p<0.05).

Although significant changes in the values of organ weight were observed in male and female animals from different dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance.

4. a) Results at the end of administration
No differences were found between the control group and the test substance administration group for all weighing organs in both males and females.

b) Results at the end of the recovery period
The adrenal relative weight increased in the 1,000 mg / kg group of male and the splenic phase versus weight increased in the female 1,000 mg / kg group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

2. Gross pathological examination revealed test item coloured perianal region externally and test item coloured stomach mucosa in male and female animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups. Gross pathological examination in male and female animals from control, control reversal and 1000 mg/kg reversal dose groups did not reveal any abnormality.

4. At necropsy at the end of the administration and at the end of the recovery test, no macroscopic abnormalities increased in the test substance-administered group compared with the control group in both males and females.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

2. No treatment related histopathological changes were evident in male and female animals from control and high dose groups. Incidental, physiological and congenital histopathological changes which were covered in the background historical data of the pathology from control and high dose groups includes minimal, focal to multifocal periportal mononuclear cells infiltration in liver; focal mononuclear cells infiltration and/or minimal, multifocal tubular eosinophilic secretion and/or tubular dilatation and/or cystic dilatation of tubule in the kidneys; minimal, multifocal brown coloured pigmentation in the spleen; minimal, diffuse dilatation of zona reticularis and/or minimal, multifocal vacuolation in zona fasciculata and/or presence of accessory adrenocortical tissue in the adrenals; minimal, luminal seminal coagulum in urinary bladder; minimal, luminal dilatation in the uterus; presence of persistent Rathke’s pouch in the pituitary; presence of ultimobranchial cyst in the thyroid; multifocal, focal to multifocal alveolar histiocytosis in the lungs; diffuse mucification of epithelium in vagina; in male and female animals from control and high dose group.

4. Histopathology was performed on the control group dissected at the end of the treatment and on the sexes of the 1,000 mg / kg group. As a result, no difference was observed between both groups in both males and females. The main findings including the control group were small basolateral and granular nests in liver cells, basophilization and calcification of tubular epithelial cells of the kidneys, vacuolation of bundle bands of the adrenal gland, etc

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

other: No effect observed

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Conclusions:

The No Observed Adverse Effect Level (NOAEL) of the test chemical in the rat via oral route of exposure is considered to be 1000 mg/kg body weight in male and female animals.

Executive summary:

Data available for the various test chemicals was reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

In a repeated dose toxicity study, Sprague-Dawley male and female rats were treated with the test chemical in the concentration of 0, 250, 500 and 1000 mg/kg bw orally by gavage for 28 days. All the male and female animals from control and different dose groups up to 1000 mg/kg survived throughout the dosing period of 28 days and the recovery period of 14 days. Male and female animals from control and different dose groups exhibited normal body weight gain at the end of the dosing period of 28 days and the recovery period of 14 days. Feed intake of animals from control and different dose groups was found to be comparable throughout the dosing period of 28 days and the recovery period of 14 days. Ophthalmoscopic examination, conducted prior to and at the end of dosing period on animals from control and different dose groups did not reveal any abnormality. Test item coloured faeces were observed in male and female animals from different dose groups during the dosing period of 28 days and the recovery period of 14 days. Detailed clinical observations conducted at weekly interval (upto 6th week) did not reveal any abnormality in all male and female animals from control and different dose groups during the dosing period of 28 days and the recovery period of 14 days. Similarly, Towards the end of the exposure period in week 4, functional observation battery such as sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) revealed no abnormalities attributable to the treatment. Grip strength and Motor activity values observed in male and female animals for control and different dose groups were comparable. At the end of the dosing period on day 29, no statistically significant changes in the values of various hematological parameters and at the end of the recovery period on day 43, statistically significant decrease in the values of Total RBC at 1000 mg/kg, male. In female animals conducted at the end of the dosing period on day 29, statistically significant decrease in the values of MCHC at 250 mg/kg, female and at the end of the recovery period on day 43, no statistically significant changes in the values of various parameters were observed. The decrease in the values of various parameters was marginal and within the normal biological and laboratory limits. In male and female animals conducted at the end of the dosing period on day 29, statistically significant increase in the values of Sodium at 500 mg/kg in male. In addition, statistically significant decrease was observed in the values of Glucose and Total Cholesterol at 500 mg/kg, Calcium at 250 mg/kg, 500 mg/kg and 1000 mg/kg in male and Blood Urea Nitrogen and Urea Nitrogen at 250 and 1000 mg/kg in female. At the end of the recovery period on day 43 (Reversal groups) statistically significant increase were observed in the values of Total Protein, Globulin, Creatinine and Triglycerides at 1000 mg/kg in male and Total Protein, Glucose, Albumin and Globulin at 1000 mg/kg in female. In addition, statistically significant decrease was observed in the values of Bile Acid at 1000 mg/kg in male rats. The increase/decrease in the values of various parameters was marginal and within the normal biological and laboratory limits. Urine analysis conducted during 4th and 6th week of dosing period (on day 26, 27 and 43) no abnormality was attributable to the treatment. In addition, at termination of dosing on day 29, male animals at 250 500 and 1000 mg/kg dose groups revealed increased relative weights of liver when compared with that of controls. In addition, increased relative weights of heart were observed in male animals at 500 mg/kg dose group as compared to controls. In male animals sacrificed on day 43 at 1000 mg/kg reversal group, was found to be comparable with that of controls. At termination of dosing on day 29, at 500 and 1000 mg/kg dose groups revealed increased relative weights of liver and sacrificed on day 43 from 1000 mg/kg reversal group, revealed decreased relative weights of kidneys, adrenals and ovaries in female when compared with that of controls. Although significant changes in the values of organ weight were observed in male and female animals from different dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance. Test item coloured perianal region externally and test item coloured stomach mucosa in male and female at 250, 500 and 1000 mg/kg dose groups. In male and female animals from control, control reversal and 1000 mg/kg reversal dose groups did not reveal any abnormality. Although significant gross pathological observations were noted in male and female animals from different dose groups, no related histopathological changes were observed. Histopathological examination did not reveal any abnormality attributable to the treatment. Therefore, No Observed Adverse Effect Level (NOAEL) of the test chemical in the Sprague Dawley rat via oral route, over a period of 28 days was considered to be 1000 mg/kg body weight in male and female animals.

In another study, combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (OECD TG422) was performed to determine the toxic nature of the test chemical. The study was performed using Crl:CD (SD) male and female rats. Recovery group was also included in the study. The test chemical was dissolved in 0.5 % methylcellulose aqueous solution and used at dose level of 0, 40, 200 or 1000 mg/Kg/day. The treated animals were observed for mortality, clinical signs, changes in body weight, food consumption, urinalysis, hemotology, clinical chemistry and were subjected to gross and histopathology. No mortality was noted and no effects were observed in clinical signs, functional battery observations, body weight and food consumption changes, hematology, organ weights and histopathology. Colored feces (40, 200 and 1000 mg/Kg/day) and abnormal urine color (200 and 1000 mg/Kg/day) was noted in the treated animals. Decrease in the TP, decrease in the Alb and increase in the percentage of alpha 2-Glob (Male) in treated animals of 1000 mg/Kg/day. Though albumin level (mg/dL) was significantly decreased in males of the high dose group, A/G ratio was not affected. Colored aqueous content in the alimentary tract in 40 mg/Kgday treated male animals. Colored aqueous content in the alimentary tract (Male/Female), Mucosal discoloration of alimentary tract (Male) in 200 mg/Kg/day treated animals. Colored aqueous content in the alimentary tract (Male/Female), Mucosal discoloration of alimentary tract (Male/Female) in 1000 mg/Kg/day treated animals. Based on the observations of the study, the no observed adverse effect level (NOAEL) for the test chemical is considered to be 1000 mg/Kg/day when male and female rats were exposed to the test chemical in combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (OECD TG422). 

Another 28 days repeated dose toxicity study for the test chemical was assessed for its possible toxic nature. For this purpose subacute study was performed according to guideline for 28-Day Repeat Dose Toxicity Test of Chemicals (Japan). The test substance was exposed to Crj:CD (SD) male and female rats by oral gavage at the concentration of 0,100, 300, 1000 mg/kg/day for 28 days . Recovery group animals were also included in the study from 0 and 1000 mg/Kg/day. Animals were observed for clinical signs, changes in body weight, food consumption, hematology, clinical chemistry, urine analysis, organ weight, gross and histopathology. As a result of clinical observation, no abnormal animals were observed in either group of males and females during the administration period and recovery period, and no death cases were observed. Body weights did not differ between groups throughout the administration period and recovery period in both males and females. In the males, no difference was observed in the consumption of food during the administration period and the recovery period, and in females, in the 300 and 1,000 mg / kg group, the feeding amount decreased in one week after administration compared to the control group, but after 2 weeks, no significant changes were noted. The food intake of 0 to 4 weeks were almost unchanged between the 1,000 mg / kg group and the control group, so it was not thought that it was a change to the effect of the test substance. As a result of the blood morphological examination, a decrease in white blood cell count observed in the 100 mg / kg group of males at the end of administration, a decrease in the ratio of neutrophils observed in the female 100 mg / kg group, an increase in lymphocyte ratio were all dose - correlated changes. Also, with regard to the reduction of MCHC observed in the female 1,000 mg / kg group at the end of the recovery period, it was a slight change not observed at the end of administration, and the hematocrit and hemoglobin levels were not different from the control group It was not considered a meaningful change from. As a result of the blood coagulation test, no difference was observed between the control group of male and female and the test substance administered group at any examination time, and it was considered that there was no influence of administration of the test substance. As a result of biochemical examination, the increase in A / G observed in all male test substance-administered groups tended to be rather low in the control group (background value 1.38 ± 0.15, n = 35), 100 mg / kg group was minor and there was no dose correlation, GPT and γ-GPT in the 1,000 mg / kg group were extremely minor changes, both considered to be toxicologically meaningless change It was done. For females, γ-GPT decreased in all test substance-administered groups, potassium decreased in the 300 mg / kg group, but the former was a minor change and the latter was a dose-uncorrelated change. In addition, the examination at the end of the recovery period showed an increase in chlorine in the male 1,000 mg / kg group, a decrease in calcium in the female same dose group, but a slight change. As a result of the urine test, there was no item for which differences between groups were observed in both males and females, and there was no effect of administration of the test substance. As a result of organ weight measurement, in the measurement at the end of administration, there was no difference between the groups for both the actual weight and the relative weight for all weighing organs for both males and females. At the end of the recovery period, the increase in brain weight and adrenal relative weight was observed in the male 1,000 mg / kg group, and the increase in splenic phase versus weight was observed in the female 1,000 mg / kg group, but both were slight Since it was a change and it was not observed at the end of administration, it was not considered to be a toxicologically important change. As a result of pathological examination, lesions suggested by administration of the test substance, neither macroscopically nor histologically, were observed in both males and females. No significant effects were observed in treated animals compared to control. Therefore the no observed adverse effect level (NOAEL) for the test chemical was considered to be 1000mg/kg/day when Crj:CD (SD) male and female rats were exposed by oral gavage route of exposure for 28 days .

Based on the abave detailed studies available, the No Observed Adverse Effect Level (NOAEL) of the test chemical in the rat via oral route of exposure is considered to be 1000 mg/kg body weight in male and female animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data is Klimisch 2 and is from reviewe article/handbook

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation, other

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Quality of whole database:

Waiver

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Quality of whole database:

Waiver

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: Oral

Data available for the various test chemicals was reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

In a repeated dose toxicity study, Sprague-Dawley male and female rats were treated with the test chemical in the concentration of 0, 250, 500 and 1000 mg/kg bw orally by gavage for 28 days. All the male and female animals from control and different dose groups up to 1000 mg/kg survived throughout the dosing period of 28 days and the recovery period of 14 days. Male and female animals from control and different dose groups exhibited normal body weight gain at the end of the dosing period of 28 days and the recovery period of 14 days. Feed intake of animals from control and different dose groups was found to be comparable throughout the dosing period of 28 days and the recovery period of 14 days. Ophthalmoscopic examination, conducted prior to and at the end of dosing period on animals from control and different dose groups did not reveal any abnormality. Test item coloured faeces were observed in male and female animals from different dose groups during the dosing period of 28 days and the recovery period of 14 days. Detailed clinical observations conducted at weekly interval (upto 6th week) did not reveal any abnormality in all male and female animals from control and different dose groups during the dosing period of 28 days and the recovery period of 14 days. Similarly, Towards the end of the exposure period in week 4, functional observation battery such as sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) revealed no abnormalities attributable to the treatment. Grip strength and Motor activity values observed in male and female animals for control and different dose groups were comparable. At the end of the dosing period on day 29, no statistically significant changes in the values of various hematological parameters and at the end of the recovery period on day 43, statistically significant decrease in the values of Total RBC at 1000 mg/kg, male. In female animals conducted at the end of the dosing period on day 29, statistically significant decrease in the values of MCHC at 250 mg/kg, female and at the end of the recovery period on day 43, no statistically significant changes in the values of various parameters were observed. The decrease in the values of various parameters was marginal and within the normal biological and laboratory limits. In male and female animals conducted at the end of the dosing period on day 29, statistically significant increase in the values of Sodium at 500 mg/kg in male. In addition, statistically significant decrease was observed in the values of Glucose and Total Cholesterol at 500 mg/kg, Calcium at 250 mg/kg, 500 mg/kg and 1000 mg/kg in male and Blood Urea Nitrogen and Urea Nitrogen at 250 and 1000 mg/kg in female. At the end of the recovery period on day 43 (Reversal groups) statistically significant increase were observed in the values of Total Protein, Globulin, Creatinine and Triglycerides at 1000 mg/kg in male and Total Protein, Glucose, Albumin and Globulin at 1000 mg/kg in female. In addition, statistically significant decrease was observed in the values of Bile Acid at 1000 mg/kg in male rats. The increase/decrease in the values of various parameters was marginal and within the normal biological and laboratory limits. Urine analysis conducted during 4th and 6th week of dosing period (on day 26, 27 and 43) no abnormality was attributable to the treatment. In addition, at termination of dosing on day 29, male animals at 250 500 and 1000 mg/kg dose groups revealed increased relative weights of liver when compared with that of controls. In addition, increased relative weights of heart were observed in male animals at 500 mg/kg dose group as compared to controls. In male animals sacrificed on day 43 at 1000 mg/kg reversal group, was found to be comparable with that of controls. At termination of dosing on day 29, at 500 and 1000 mg/kg dose groups revealed increased relative weights of liver and sacrificed on day 43 from 1000 mg/kg reversal group, revealed decreased relative weights of kidneys, adrenals and ovaries in female when compared with that of controls. Although significant changes in the values of organ weight were observed in male and female animals from different dose groups, no related gross pathological and histopathological findings were seen, hence these findings were considered to be of no toxicological importance. Test item coloured perianal region externally and test item coloured stomach mucosa in male and female at 250, 500 and 1000 mg/kg dose groups. In male and female animals from control, control reversal and 1000 mg/kg reversal dose groups did not reveal any abnormality. Although significant gross pathological observations were noted in male and female animals from different dose groups, no related histopathological changes were observed. Histopathological examination did not reveal any abnormality attributable to the treatment. Therefore, No Observed Adverse Effect Level (NOAEL) of the test chemical in the Sprague Dawley rat via oral route, over a period of 28 days was considered to be 1000 mg/kg body weight in male and female animals.

In another study, combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (OECD TG422) was performed to determine the toxic nature of the test chemical. The study was performed using Crl:CD (SD) male and female rats. Recovery group was also included in the study. The test chemical was dissolved in 0.5 % methylcellulose aqueous solution and used at dose level of 0, 40, 200 or 1000 mg/Kg/day. The treated animals were observed for mortality, clinical signs, changes in body weight, food consumption, urinalysis, hemotology, clinical chemistry and were subjected to gross and histopathology. No mortality was noted and no effects were observed in clinical signs, functional battery observations, body weight and food consumption changes, hematology, organ weights and histopathology. Colored feces (40, 200 and 1000 mg/Kg/day) and abnormal urine color (200 and 1000 mg/Kg/day) was noted in the treated animals. Decrease in the TP, decrease in the Alb and increase in the percentage of alpha 2-Glob (Male) in treated animals of 1000 mg/Kg/day. Though albumin level (mg/dL) was significantly decreased in males of the high dose group, A/G ratio was not affected. Colored aqueous content in the alimentary tract in 40 mg/Kgday treated male animals. Colored aqueous content in the alimentary tract (Male/Female), Mucosal discoloration of alimentary tract (Male) in 200 mg/Kg/day treated animals. Colored aqueous content in the alimentary tract (Male/Female), Mucosal discoloration of alimentary tract (Male/Female) in 1000 mg/Kg/day treated animals. Based on the observations of the study, the no observed adverse effect level (NOAEL) for the test chemical is considered to be 1000 mg/Kg/day when male and female rats were exposed to the test chemical in combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (OECD TG422). 

Another 28 days repeated dose toxicity study for the test chemical was assessed for its possible toxic nature. For this purpose subacute study was performed according to guideline for 28-Day Repeat Dose Toxicity Test of Chemicals (Japan). The test substance was exposed to Crj:CD (SD) male and female rats by oral gavage at the concentration of 0,100, 300, 1000 mg/kg/day for 28 days . Recovery group animals were also included in the study from 0 and 1000 mg/Kg/day. Animals were observed for clinical signs, changes in body weight, food consumption, hematology, clinical chemistry, urine analysis, organ weight, gross and histopathology. As a result of clinical observation, no abnormal animals were observed in either group of males and females during the administration period and recovery period, and no death cases were observed. Body weights did not differ between groups throughout the administration period and recovery period in both males and females. In the males, no difference was observed in the consumption of food during the administration period and the recovery period, and in females, in the 300 and 1,000 mg / kg group, the feeding amount decreased in one week after administration compared to the control group, but after 2 weeks, no significant changes were noted. The food intake of 0 to 4 weeks were almost unchanged between the 1,000 mg / kg group and the control group, so it was not thought that it was a change to the effect of the test substance. As a result of the blood morphological examination, a decrease in white blood cell count observed in the 100 mg / kg group of males at the end of administration, a decrease in the ratio of neutrophils observed in the female 100 mg / kg group, an increase in lymphocyte ratio were all dose - correlated changes. Also, with regard to the reduction of MCHC observed in the female 1,000 mg / kg group at the end of the recovery period, it was a slight change not observed at the end of administration, and the hematocrit and hemoglobin levels were not different from the control group It was not considered a meaningful change from. As a result of the blood coagulation test, no difference was observed between the control group of male and female and the test substance administered group at any examination time, and it was considered that there was no influence of administration of the test substance. As a result of biochemical examination, the increase in A / G observed in all male test substance-administered groups tended to be rather low in the control group (background value 1.38 ± 0.15, n = 35), 100 mg / kg group was minor and there was no dose correlation, GPT and γ-GPT in the 1,000 mg / kg group were extremely minor changes, both considered to be toxicologically meaningless change It was done. For females, γ-GPT decreased in all test substance-administered groups, potassium decreased in the 300 mg / kg group, but the former was a minor change and the latter was a dose-uncorrelated change. In addition, the examination at the end of the recovery period showed an increase in chlorine in the male 1,000 mg / kg group, a decrease in calcium in the female same dose group, but a slight change. As a result of the urine test, there was no item for which differences between groups were observed in both males and females, and there was no effect of administration of the test substance. As a result of organ weight measurement, in the measurement at the end of administration, there was no difference between the groups for both the actual weight and the relative weight for all weighing organs for both males and females. At the end of the recovery period, the increase in brain weight and adrenal relative weight was observed in the male 1,000 mg / kg group, and the increase in splenic phase versus weight was observed in the female 1,000 mg / kg group, but both were slight Since it was a change and it was not observed at the end of administration, it was not considered to be a toxicologically important change. As a result of pathological examination, lesions suggested by administration of the test substance, neither macroscopically nor histologically, were observed in both males and females. No significant effects were observed in treated animals compared to control. Therefore the no observed adverse effect level (NOAEL) for the test chemical was considered to be 1000mg/kg/day when Crj:CD (SD) male and female rats were exposed by oral gavage route of exposure for 28 days .

Based on the above detailed studies available, the No Observed Adverse Effect Level (NOAEL) of the test chemical in the rat via oral route of exposure is considered to be 1000 mg/kg body weight in male and female animals.

Repeated dose toxicity: Inhalation

Test chemical has very low vapor pressure of 9.7058E-10 Pa (7.279e-12 mmHg), so the potential for the generation of inhalable vapours is very low, also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore this end point for repeated dose toxicity by inhalation route of exposure was considered for waiver.

Repeated dose toxicity: Dermal

The acute dermal toxicity value for test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. Given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that test chemical shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that test chemical shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Considering the above discussion and applying the weight of evidence approach with no toxic effects noted, the test chemical is not likely to be toxic as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Considering the above discussion and applying the weight of evidence approach with no toxic effects noted, the test chemical is not likely to be toxic as per the criteria mentioned in CLP regulation.