9,10-Anthracenedione, 1,4-diamino-, N,N'-bis(4-C7-17-branched alkylphenyl) derivs.

Endpoint summary

Description of key information

Repeated dose oral toxicity:

In a study, the test chemical has been investigated for repeated dose toxicity to a greater or lesser extent. Study based on in vivo experiments in rodents, i.e. most commonly in rats for the test chemical are summarized 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 found to be 1000 mg/kg body weight in male and female animals.

 

Based on this value it can be concluded that the substance is considered to not toxic as per the criteria mentioned in CLP regulation.

Repeated dose toxicity: Inhalation

According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the low vapour pressure of the substance  9,10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs (CAS no. 97862-23-2), which is reported as 1.05E-14Pa at 25 C. Also, the acute inhalation LC50 value is considered t be 5mg/L. Thus, exposure to inhalable dust, mist and vapour of the chemical substance9,10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs is highly unlikely. Therefore this endpoint for repeated dose toxicity by inhalation route of exposure is considered for waiver.

Repeated dose toxicity: Dermal

The acute toxicity value for 9,10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs (CAS no 97862-23-2) (as provided in section 7.2.3) is >2000 mg/kg b.wt.mg/kg body weight by a study report . Also, given the use of the chemica, repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that 10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs. shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that,10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs. shall exhibit repeated dose toxicity by the dermal route. Hence this end point for repeated dose toxicity by dermal route of exposure was considered for waiver.

Key value for chemical safety assessment

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:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

To assess toxicological profile of the test chemical, to determine target organ of toxicity, its reversibility and No Observed Adverse Effect Level (NOAEL) in the rat after 28 consecutive days of oral administration.

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

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):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

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):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item formulation analysis for concentration and stability were conducted

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

- 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):

Observations and examinations performed and frequency:

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.

Sacrifice and pathology:

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.

Statistics:

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.

Significance was calculated at 5% level and indicated in the summary tables as follows:

* = Significant than control at 95% level of confidence (p<0.05).

Clinical signs:

no effects observed

Description (incidence and severity):

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).

Mortality:

no mortality observed

Description (incidence):

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.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

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):

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.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

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.

Urinalysis findings:

no effects observed

Description (incidence and severity):

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.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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.

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

VIABILITY

	Dose (mg/kg)		Mortality
Group			Males		Females
Number	Male	Female	Absolute	Relative %	Absolute	Relative %
I	0	0	0/6	0	0/6	0
II	0 (Reversal)	0 (Reversal)	0/6	0	0/6	0
III	250	250	0/6	0	0/6	0
IV	500	500	0/6	0	0/6	0
V	1000	1000	0/6	0	0/6	0
VI	1000 (Reversal)	1000 (Reversal)	0/6	0	0/6	0

GROUP MEAN BODY WEIGHT (g)

Sex - Male

Group	Dose (mg/kg)		Weeks
Number			Day 0	Day 1	1	2	3
I	0	Mean	168.63	173.78	202.92	230.08	257.55
		±SD	8.31	7.71	7.23	6.90	8.53
II	0 (Reversal)	Mean	168.40	172.35	202.75	229.78	256.60
		±SD	8.23	8.48	10.66	12.30	11.64
III	250	Mean	168.57	172.20	200.13	227.10	257.23
		±SD	7.06	7.20	9.55	10.81	13.04
IV	500	Mean	168.73	172.07	201.25	226.55	256.32
		±SD	7.02	5.67	7.44	11.76	10.61
V	1000	Mean	169.53	173.27	200.77	225.07	254.22
		±SD	4.97	4.88	9.90	12.28	13.54
VI	1000 (Reversal)	Mean	169.52	174.18	203.98	228.50	256.08
		±SD	3.77	3.25	4.36	7.61	10.53

 

Group	Dose (mg/kg)		Weeks
Number			4	5	6
I	0	Mean	284.20
		±SD	9.64
II	0 (Reversal)	Mean	282.32	303.12	324.32
		±SD	11.48	13.87	13.70
III	250	Mean	282.90
		±SD	13.76
IV	500	Mean	281.05
		±SD	10.27
V	1000	Mean	278.03
		±SD	13.45
VI	1000 (Reversal)	Mean	281.20	298.12	320.67
		±SD	10.88	11.77	10.83

GROUP MEAN BODY WEIGHT (g)

Sex - Female

Group	Dose (mg/kg)		Weeks
Number			Day 0	Day 1	1	2	3
I	0	Mean	152.10	155.28	169.40	182.25	191.83
		±SD	6.46	6.96	8.75	12.85	14.03
II	0 (Reversal)	Mean	152.02	156.67	171.72	179.98	185.23
		±SD	7.15	7.49	9.15	11.72	12.40
III	250	Mean	151.80	155.80	171.80	182.17	191.63
		±SD	6.91	6.77	11.31	14.84	17.86
IV	500	Mean	152.75	156.37	166.67	177.98	185.85
		±SD	6.75	6.30	8.39	10.29	11.55
V	1000	Mean	153.05	155.60	166.47	172.42	179.30
		±SD	6.75	6.60	8.01	8.47	11.28
VI	1000 (Reversal)	Mean	151.87	154.93	167.40	175.65	181.03
		±SD	4.79	4.73	9.13	10.54	12.76

 

Group	Dose (mg/kg)		Weeks
Number			4	5	6
I	0	Mean	199.30
		±SD	14.30
II	0 (Reversal)	Mean	191.70	195.52	199.48
		±SD	13.48	13.31	14.18
III	250	Mean	196.75
		±SD	18.51
IV	500	Mean	190.50
		±SD	11.40
V	1000	Mean	183.32
		±SD	11.35
VI	1000 (Reversal)	Mean	186.37	189.88	193.00
		±SD	14.44	15.12	15.20

GROUP MEAN FEED CONSUMPTION (g/animal/day)

Sex - Male

Group	Dose (mg/kg)		Day
Number			1	8	15	22	28
I	0	Mean	18.35	19.75	20.77	21.58	23.33
II	0 (Reversal)	Mean	18.58	20.25	20.88	22.00	22.92
III	250	Mean	18.35	20.03	20.83	21.80	22.63
IV	500	Mean	18.78	20.32	21.10	22.05	22.95
V	1000	Mean	18.83	19.60	20.68	21.72	22.10
VI	1000 (Reversal)	Mean	18.93	20.00	21.07	22.13	22.50

 

Group	Dose (mg/kg)		Day
Number			36	42
I	0	Mean
II	0 (Reversal)	Mean	25.72	26.53
III	250	Mean
IV	500	Mean
V	1000	Mean
VI	1000 (Reversal)	Mean	24.75	25.50

GROUP MEAN FEED CONSUMPTION (g/animal/day)

Sex - Female

Group	Dose (mg/kg)		Day
Number			1	8	15	22	28
I	0	Mean	14.97	16.87	17.92	19.10	20.23
II	0 (Reversal)	Mean	15.15	16.90	18.38	18.52	19.72
III	250	Mean	15.12	16.48	17.60	18.72	19.27
IV	500	Mean	14.85	16.32	18.03	18.57	19.23
V	1000	Mean	15.03	16.62	17.88	18.08	18.43
VI	1000 (Reversal)	Mean	15.23	15.92	17.75	18.32	18.47

 

Group	Dose (mg/kg)		Day
Number			36	42
I	0	Mean
II	0 (Reversal)	Mean	22.23	22.80
III	250	Mean
IV	500	Mean
V	1000	Mean
VI	1000 (Reversal)	Mean	20.95	21.80

OPHTHALMOSCOPIC EXAMINATION

Sex : Male

Day : 0

Group Number	Dose  mg/kg	Ophthalmoscopic Finding	Total Number of Animals	Animal Number
I	0	No abnormality detected	6	1 - 6
II	0 (Reversal)	No abnormality detected	6	13 - 18
III	250	No abnormality detected	6	25 - 30
IV	500	No abnormality detected	6	37 - 42
V	1000	No abnormality detected	6	49 - 54
VI	1000 (Reversal)	No abnormality detected	6	61 - 66

 Sex : Female

Day : 0 

Group Number	Dose  mg/kg	Ophthalmoscopic Finding	Total Number of Animals	Animal Number
I	0	No abnormality detected	6	7 - 12
II	0 (Reversal)	No abnormality detected	6	19 - 24
III	250	No abnormality detected	6	31 - 36
IV	500	No abnormality detected	6	43 - 48
V	1000	No abnormality detected	6	55 - 60
VI	1000 (Reversal)	No abnormality detected	6	67 - 72

OPHTHALMOSCOPIC EXAMINATION

Sex : Male

Week: 4 and 6

Group Number	Dose  mg/kg	Ophthalmoscopic Finding	Total Number of Animals	Animal Number
I	0	No abnormality detected	6	1 - 6
II	0 (Reversal)	No abnormality detected	6	13 - 18
III	250	No abnormality detected	6	25 - 30
IV	500	No abnormality detected	6	37 - 42
V	1000	No abnormality detected	6	49 - 54
VI	1000 (Reversal)	No abnormality detected	6	61 - 66

 Sex : Female

Week: 4 and 6

Group Number	Dose  mg/kg	Ophthalmoscopic Finding	Total Number of Animals	Animal Number
I	0	No abnormality detected	6	7 - 12
II	0 (Reversal)	No abnormality detected	6	19 - 24
III	250	No abnormality detected	6	31 - 36
IV	500	No abnormality detected	6	43 - 48
V	1000	No abnormality detected	6	55 - 60
VI	1000 (Reversal)	No abnormality detected	6	67 - 72

SUMMARY OF CLINICAL OBSERVATIONS AND GENERAL APPEARANCE

Sex : Male

Group Number	Dose (mg/kg)	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days from - to	Mortality
I	0	Nil	6	1 - 6	1 - 28	0/6
II	0 (Reversal)	Nil	6	13 - 18	1 - 42	0/6
III	250	Test Item coloured faces	6	25 - 30	2 - 28	0/6
IV	500	Test Item coloured faces	6	37 - 42	2 - 28	0/6
V	1000	Test Item coloured faces	6	49 - 54	2 - 28	0/6
VI	1000 (Reversal)	Test Item coloured faces	6	61, 62 63, 64 65, 66	2 - 32 2 - 31 2 - 34	0/6

Sex : Female

Group Number	Dose (mg/kg)	Observed Signs	Total Number of Animals	Animal Nos.	Period of signs in days from - to	Mortality
I	0	Nil	6	7 - 12	1 - 28	0/6
II	0 (Reversal)	Nil	6	19 - 24	1 - 42	0/6
III	250	Test Item coloured faces	6	31 - 36	2 - 28	0/6
IV	500	Test Item coloured faces	6	43 - 48	2 - 28	0/6
V	1000	Test Item coloured faces	6	55 - 60	2 - 28	0/6
VI	1000 (Reversal)	Test Item coloured faces	6	67, 68 69, 70 71, 72	2 - 31 2 - 33 2 - 32	0/6

SUMMARY OF FUNCTIONAL OBSERVATIONAL

Sex   : Male
Day   : 28 and 42

 

Group Number		I	II	III	IV	V	VI
Dose		0 mg/kg	0 mg/kg Reversal	250 mg/kg	500 mg/kg	1000 mg/kg	1000 mg/kg Reversal
Number of animals observed		6	6	6	6	6	6
Number of animals within normal limit		6/6	6/6	6/6	6/6	6/6	6/6
Number of animals with significant deviation		0/6	0/6	0/6	0/6	0/6	0/6
Parameters
Arousal level : Apparently normal		6/6	6/6	6/6	6/6	6/6	6/6
Visual response : Orienting response		6/6	6/6	6/6	6/6	6/6	6/6
Touch response : Orienting response		6/6	6/6	6/6	6/6	6/6	6/6
Auditory response : Orienting response		6/6	6/6	6/6	6/6	6/6	6/6
Tail pinch response : Orienting response		6/6	6/6	6/6	6/6	6/6	6/6
Visual placing response : Early extension of forelimbs to reach for the screen		6/6	6/6	6/6	6/6	6/6	6/6
Air righting response : Lands with all feet on ground		6/6	6/6	6/6	6/6	6/6	6/6
Grip Strength (kg) - Mean ± SD		0.897 ±0.024	0.941 ±0.015	0.856 ±0.039	0.914 ±0.033	0.876 ±0.023	0.955 ±0.094
Motor Activity -	Interval ‘1’	540.00 ± 185.41	646.17 ± 169.53	544.00 ± 128.63	540.00 ± 152.49	533.33 ± 108.64	637.33 ± 221.89
Mean ± SD	Interval ‘2’	347.00 ± 119.52	307.50 ± 114.40	361.83 ± 139.58	374.17 ± 24.19	384.17 ± 88.01	335.83 ± 177.05
	Interval ‘3’	233.83 ± 109.03	229.83 ± 94.90	272.17 ± 62.09	254.50 ± 114.63	295.83 ± 103.33	254.83 ± 93.95

Sex   : Female
Day   : 28 and 42

Group Number		I	II	III	IV	V	VI
Dose		0 mg/kg	0 mg/kg Reversal	250 mg/kg	500 mg/kg	1000 mg/kg	1000 mg/kg Reversal
Number of animals observed		6	6	6	6	6	6
Number of animals within normal limit		6/6	6/6	6/6	6/6	6/6	6/6
Number of animals with significant deviation		0/6	0/6	0/6	0/6	0/6	0/6
Parameters
Arousal level : Apparently normal		6/6	6/6	6/6	6/6	6/6	6/6
Visual response : Orienting response		6/6	6/6	6/6	6/6	6/6	6/6
Touch response : Orienting response		6/6	6/6	6/6	6/6	6/6	6/6
Auditory response : Orienting response		6/6	6/6	6/6	6/6	6/6	6/6
Tail pinch response : Orienting response		6/6	6/6	6/6	6/6	6/6	6/6
Visual placing response : Early extension of forelimbs to reach for the screen		6/6	6/6	6/6	6/6	6/6	6/6
Air righting response : Lands with all feet on ground		6/6	6/6	6/6	6/6	6/6	6/6
Grip Strength (kg) - Mean ± SD		0.777 ± 0.025	0.860 ±0.036	0.820 ± 0.025	0.815 ± 0.046	0.818 ± 0.037	0.866 ±0.042
Motor Activity -	Interval ‘1’	595.17 ±81.83	680.33 ±365.26	594.83 ±57.27	578.50 ±118.58	561.50 ±241.16	657.67 ±147.05
Mean ± SD	Interval ‘2’	351.17 ±62.60	336.83 ±98.40	314.00 ±143.39	393.33 ±83.88	343.00 ±137.65	333.17 ±128.22
	Interval ‘3’	282.00 ±137.93	269.33 ±104.21	240.67 ±109.95	268.17 ±125.89	260.83 ±157.12	270.33 ±93.43

GROUP MEAN HAEMATOLOGY

Sex : Male

Day : 29 and 43

Group	Dose (mg/kg)		Hb	Total RBC	Rt	HCT	MCV	MCH	MCHC
Number			(g/dL)	(^6/µL)	(%)	(%)	(fL)	(pg)	(g/dL)
I	0	Mean	15.35	8.27	4.43	46.30	55.97	18.57	33.17
		±SD	0.77	0.33	1.11	2.18	2.49	0.72	0.60
II	0 (Reversal)	Mean	13.93	8.77	4.48	45.50	51.88	15.92	30.65
		±SD	0.44	0.47	0.56	1.74	1.17	0.58	0.55
III	250	Mean	15.72	8.46	4.85	47.33	56.03	18.60	33.20
		±SD	0.22	0.21	0.73	0.86	0.76	0.20	0.32
IV	500	Mean	15.42	8.27	4.47	46.77	56.55	18.65	32.97
		±SD	0.89	0.47	1.19	2.28	2.07	0.85	0.67
V	1000	Mean	15.25	8.24	4.45	46.20	56.12	18.52	32.98
		±SD	0.63	0.44	0.90	1.86	1.10	0.48	0.40
VI	1000 (Reversal)	Mean	13.35	8.25*	4.52	43.30	52.48	16.15	30.73
		±SD	0.61	0.21	0.71	1.68	1.21	0.48	0.40

 

 

Group	Dose (mg/kg)		Platelets	Total WBC	Differential %					Pt.
Number			(^3/ µL)	(^3/µL)	N	L	E	M	B	(Sec.)
I	0	Mean	441.17	13.33	17.17	81.50	1.00	0.33	0.00	20.17
		±SD	46.36	3.55	3.49	3.39	0.89	0.52	0.00	5.98
II	0 (Reversal)	Mean	409.33	10.42	16.33	81.83	0.67	1.17	0.00	19.17
		±SD	35.89	3.58	3.01	3.06	0.82	1.17	0.00	3.49
III	250	Mean	442.17	12.65	17.00	81.83	0.83	0.33	0.00	19.83
		±SD	12.64	0.96	3.16	3.19	0.75	0.52	0.00	5.27
IV	500	Mean	457.00	12.37	14.17	84.00	1.33	0.50	0.00	19.00
		±SD	38.76	3.11	2.14	2.90	0.82	0.55	0.00	6.00
V	1000	Mean	441.33	13.00	15.33	83.67	0.33	0.67	0.00	20.17
		±SD	21.73	0.93	2.88	2.50	0.52	0.82	0.00	6.01
VI	1000 (Reversal)	Mean	375.33	13.23	17.00	80.67	1.33	1.00	0.00	20.50
		±SD	91.67	1.93	2.61	2.42	1.21	0.63	0.00	5.47

Sex : Female

Day : 29 and 43

Group	Dose (mg/kg)		Hb	Total RBC	Rt	HCT	MCV	MCH	MCHC
Number			(g/dL)	(^6/µL)	(%)	(%)	(fL)	(pg)	(g/dL)
I	0	Mean	13.63	7.01	4.45	40.28	57.38	19.42	33.80
		±SD	1.46	0.54	0.99	3.87	2.39	1.09	0.55
II	0 (Reversal)	Mean	13.52	8.42	4.58	44.28	52.63	16.10	30.52
		±SD	0.59	0.33	0.53	1.79	2.36	0.85	0.39
III	250	Mean	14.30	7.65	4.70	43.63	57.03	18.72	32.78*
		±SD	0.43	0.32	0.65	1.12	2.14	0.94	0.42
IV	500	Mean	14.07	7.42	4.63	42.47	57.22	18.98	33.15
		±SD	0.73	0.29	1.01	1.84	2.70	1.05	0.56
V	1000	Mean	14.35	7.51	4.37	43.00	57.28	19.12	33.42
		±SD	0.40	0.33	1.05	1.24	2.16	0.69	0.70
VI	1000 (Reversal)	Mean	13.20	8.28	4.78	43.03	52.00	15.97	30.70
		±SD	0.48	0.37	0.77	1.48	1.29	0.23	0.33

 

Group	Dose (mg/kg)		Platelets	Total WBC	Differential %					Pt.
Number			(^3/ µL)	(^3/µL)	N	L	E	M	B	(Sec.)
I	0	Mean	349.83	14.42	17.50	81.17	0.83	0.50	0.00	20.83
		±SD	64.71	2.00	2.74	2.79	0.75	0.55	0.00	5.81
II	0 (Reversal)	Mean	437.00	13.12	16.83	81.33	0.67	1.17	0.00	21.50
		±SD	49.77	3.50	2.64	2.94	0.82	0.98	0.00	3.08
III	250	Mean	398.00	14.80	17.17	82.00	0.67	0.17	0.00	21.00
		±SD	66.51	1.24	2.93	2.90	0.82	0.41	0.00	4.98
IV	500	Mean	354.50	12.13	14.83	83.67	0.83	0.67	0.00	20.83
		±SD	81.89	3.80	1.72	1.03	0.75	0.82	0.00	4.71
V	1000	Mean	393.50	12.73	16.67	82.33	0.50	0.50	0.00	24.50
		±SD	76.11	3.33	3.44	3.14	0.55	0.84	0.00	4.85
VI	1000 (Reversal)	Mean	402.50	13.87	16.83	81.33	1.00	0.83	0.00	21.00
		±SD	88.49	2.95	2.32	1.51	1.26	0.75	0.00	6.16

Sex : Male

Day : 29 and 43

Group Number	Dose (mg/kg)	Animal Nos.	Cell Morphology
I	0	1 - 6	Normocytic, Normochromic
II	0 (Reversal)	13 - 18	Normocytic, Normochromic
III	250	25 - 30	Normocytic, Normochromic
IV	500	37 - 42	Normocytic, Normochromic
V	1000	49 - 54	Normocytic, Normochromic
VI	1000 (Reversal)	61 - 66	Normocytic, Normochromic

Sex : Female

Day : 29 and 43

Group Number	Dose (mg/kg)	Animal Nos.	Cell Morphology
I	0	7 - 12	Normocytic, Normochromic
II	0 (Reversal)	19 - 24	Normocytic, Normochromic
III	250	31 - 36	Normocytic, Normochromic
IV	500	43 - 48	Normocytic, Normochromic
V	1000	55 - 60	Normocytic, Normochromic
VI	1000 (Reversal)	67 - 72	Normocytic, Normochromic

GROUP MEAN CLINICAL BIOCHEMISTRY

Sex : Male

Day : 29 and 43

Group Number	Dose (mg/kg)		TotalProtein (g/dL)	BUN (mg/dL)	Urea (mg/dL)	ALT (U/L)	AST (U/L)	ALP (U/L)	Glucose (mg/dL)
I	0	Mean	6.25	15.33	33.43	50.67	86.33	175.00	96.17
		±SD	0.27	2.80	6.11	9.18	7.06	61.65	14.01
II	0 (Reversal)	Mean	6.18	17.00	37.06	43.00	102.17	80.67	95.33
		±SD	0.30	2.68	5.85	6.26	13.76	26.40	10.09
III	250	Mean	5.93	16.33	35.61	53.67	83.17	173.33	82.67
		±SD	0.21	2.58	5.63	9.61	14.55	47.11	5.54
IV	500	Mean	5.87	15.83	34.52	53.17	79.17	127.83	80.50*
		±SD	0.58	2.23	4.86	6.55	15.17	33.56	8.17
V	1000	Mean	5.85	13.83	30.16	55.00	80.67	169.33	84.83
		±SD	0.42	2.56	5.59	11.40	16.32	62.86	5.91
VI	1000 (Reversal)	Mean	6.73*	15.67	34.15	42.50	91.83	71.83	90.50
		±SD	0.22	2.16	4.71	7.20	16.81	13.53	11.15

 

Group Number	Dose (mg/kg)		Calcium(mmol/L)	Phospho-rous (mg/dL)	GGT (U/L)	Total Bilirubin (mg/dL)	Albumin (g/dL)	Globulin (g/dL)	Creatinine (mg/dL)
I	0	Mean	3.83	6.73	5.00	0.17	1.18	5.07	0.48
		±SD	0.15	0.54	0.89	0.02	0.16	0.37	0.04
II	0 (Reversal)	Mean	3.72	6.77	7.17	0.16	0.85	5.30	0.45
		±SD	0.07	0.34	1.33	0.01	0.08	0.26	0.02
III	250	Mean	3.37*	7.98	4.83	0.18	1.37	4.57	0.49
		±SD	0.17	0.84	0.75	0.02	0.12	0.23	0.02
IV	500	Mean	2.93*	6.90	5.00	0.16	1.29	4.58	0.50
		±SD	0.84	1.13	0.63	0.02	0.13	0.56	0.05
V	1000	Mean	3.28*	6.37	5.17	0.19	1.37	4.48	0.46
		±SD	0.12	1.24	1.47	0.03	0.20	0.35	0.10
VI	1000 (Reversal)	Mean	3.67	7.42	7.00	0.17	0.93	5.80*	0.49*
		±SD	0.09	0.61	0.63	0.02	0.12	0.34	0.04

Sex : Male

Day : 29 and 43

Group Number	Dose (mg/kg)		Sodium (mmol/L)	Potassium (mmol/L)	Chloride (mmol/L)	Total Cholesterol (mg/dL)	Triglycerides (mg/dL)	Bile Acids (µmol/L)
I	0	Mean	146.96	3.62	109.88	49.50	70.00	19.42
		±SD	1.71	0.11	0.53	12.58	16.84	13.66
II	0 (Reversal)	Mean	146.03	3.78	106.35	50.50	58.33	12.44
		±SD	1.48	0.24	1.83	6.02	11.62	4.97
III	250	Mean	150.20	4.05	112.83	44.33	64.67	20.36
		±SD	4.19	0.41	5.19	3.20	17.56	12.79
IV	500	Mean	151.18*	3.64	109.48	36.17*	46.00	13.93
		±SD	2.44	0.38	7.09	2.64	8.56	6.64
V	1000	Mean	145.87	3.59	108.93	50.33	59.33	16.77
		±SD	1.32	0.21	1.36	10.25	15.67	8.48
VI	1000 (Reversal)	Mean	146.80	3.94	106.83	56.67	104.00*	5.64*
		±SD	1.20	0.19	1.12	7.23	40.96	4.74

* = Significant at 95% level of confidence (p<0.05)

Sex : Female

Day : 29 and 43

Group Number	Dose (mg/kg)		TotalProtein (g/dL)	BUN (mg/dL)	Urea (mg/dL)	ALT (U/L)	AST (U/L)	ALP (U/L)	Glucose (mg/dL)
I	0	Mean	5.99	17.50	38.15	56.00	80.67	83.67	81.83
		±SD	0.39	2.07	4.52	26.83	8.48	23.44	4.96
II	0 (Reversal)	Mean	6.09	14.83	32.34	35.17	96.00	49.33	84.83
		±SD	0.27	1.72	3.75	5.46	19.46	20.58	4.36
III	250	Mean	5.95	14.50*	31.61*	50.00	120.67	87.33	85.17
		±SD	0.49	1.52	3.31	17.91	60.31	15.95	6.55
IV	500	Mean	6.16	15.00	32.70	45.17	99.17	87.00	80.67
		±SD	0.43	1.79	3.90	8.93	28.00	24.43	9.00
V	1000	Mean	5.98	14.67*	31.97*	51.17	91.50	109.50	90.50
		±SD	0.19	1.86	4.06	14.12	17.64	53.31	20.95
VI	1000 (Reversal)	Mean	6.55*	17.17	37.42	34.00	92.67	41.33	92.33*
		±SD	0.26	2.32	5.05	3.03	9.31	11.20	4.68

 

Group Number	Dose (mg/kg)		Calcium(mmol/L)	Phospho-rous (mg/dL)	GGT (U/L)	Total Bilirubin (mg/dL)	Albumin (g/dL)	Globulin (g/dL)	Creatinine (mg/dL)
I	0	Mean	3.56	5.90	5.67	0.17	1.41	4.58	0.47
		±SD	0.18	0.42	0.52	0.03	0.21	0.27	0.03
II	0 (Reversal)	Mean	3.67	6.53	6.17	0.17	0.90	5.18	0.50
		±SD	0.19	0.80	0.98	0.03	0.07	0.28	0.06
III	250	Mean	3.66	6.12	5.67	0.13	1.42	4.53	0.48
		±SD	0.15	0.73	1.03	0.03	0.28	0.40	0.04
IV	500	Mean	3.70	6.23	6.00	0.15	1.50	4.65	0.47
		±SD	0.15	0.80	1.79	0.03	0.16	0.37	0.05
V	1000	Mean	3.47	5.80	5.83	0.16	1.36	4.62	0.48
		±SD	0.13	0.68	1.17	0.02	0.17	0.16	0.08
VI	1000 (Reversal)	Mean	3.61	6.92	6.17	0.20	1.04*	5.53*	0.56
		±SD	0.12	0.80	0.75	0.02	0.08	0.21	0.03

Sex : Female

Day : 29 and 43

Group Number	Dose (mg/kg)		Sodium (mmol/L)	Potassium (mmol/L)	Chloride (mmol/L)	Total Cholesterol (mg/dL)	Triglycerides (mg/dL)	Bile Acids (µmol/L)
I	0	Mean	146.31	3.52	109.22	47.50	67.83	27.16
		±SD	1.25	0.13	1.10	6.53	18.54	20.16
II	0 (Reversal)	Mean	152.68	3.77	107.38	52.50	57.50	9.54
		±SD	1.18	0.34	1.32	9.33	22.02	2.86
III	250	Mean	145.89	3.62	107.74	48.67	77.50	18.61
		±SD	2.60	0.29	2.89	10.42	26.88	12.76
IV	500	Mean	148.45	3.71	107.01	50.00	68.17	20.25
		±SD	1.41	0.20	1.90	10.70	15.68	11.18
V	1000	Mean	147.45	3.78	112.05	49.17	58.33	10.51
		±SD	1.11	0.22	1.68	8.35	16.02	1.43
VI	1000 (Reversal)	Mean	148.61	3.81	107.81	64.67	58.50	11.80
		±SD	4.35	0.42	1.94	18.02	24.08	7.70

Sex   : Male 

Day   : 26, 27 and 43 

Group	Dose		Volume	Glucose	Bilirubin	Ketones	Sp.Gr.	Occult Blood
Number	mg/kg		(ml)	(mmol/L)	(mmol/L)	(mmol/L)	(g/L)	(caCELLS/µL)
I	0	Mean	6.600	-ve	-ve	-ve	1.018	-ve
		±SD	0.525	-ve	-ve	-ve	0.003	-ve
II	0 (Rev.)	Mean	6.517	-ve	-ve	-ve	1.017	-ve
		±SD	0.649	-ve	-ve	-ve	0.003	-ve
III	250	Mean	5.350	-ve	-ve	-ve	1.018	-ve
		±SD	1.232	-ve	-ve	-ve	0.003	-ve
IV	500	Mean	5.883	-ve	-ve	-ve	1.018	-ve
		±SD	1.177	-ve	-ve	-ve	0.003	-ve
V	1000	Mean	6.033	-ve	-ve	-ve	1.017	-ve
		±SD	1.221	-ve	-ve	-ve	0.003	-ve
VI	1000 (Rev.)	Mean	6.233	-ve	-ve	-ve	1.017	-ve
		±SD	0.596	-ve	-ve	-ve	0.003	-ve

                                                                 

Group	Dose		pH	Urobilinogen	Nitrite
Number	mg/kg			(mmol/L)
I	0	Mean	7.250	-ve	-ve
		±SD	0.274	-ve	-ve
II	0 (Rev.)	Mean	7.333	-ve	-ve
		±SD	0.258	-ve	-ve
III	250	Mean	7.250	-ve	-ve
		±SD	0.274	-ve	-ve
IV	500	Mean	7.250	-ve	-ve
		±SD	0.274	-ve	-ve
V	1000	Mean	7.250	-ve	-ve
		±SD	0.274	-ve	-ve
VI	1000 (Rev.)	Mean	7.333	-ve	-ve
		±SD	0.258	-ve	-ve

Sex   : Female 

Day   : 26, 27 and 43

Group	Dose		Volume	Glucose	Bilirubin	Ketones	Sp.Gr.	Occult Blood
Number	mg/kg		(ml)	(mmol/L)	(mmol/L)	(mmol/L)	(g/L)	(caCELLS/µL)
I	0	Mean	5.350	-ve	-ve	-ve	1.018	-ve
		±SD	0.712	-ve	-ve	-ve	0.003	-ve
II	0 (Rev.)	Mean	5.717	-ve	-ve	-ve	1.019	-ve
		±SD	1.132	-ve	-ve	-ve	0.002	-ve
III	250	Mean	5.017	-ve	-ve	-ve	1.018	-ve
		±SD	0.711	-ve	-ve	-ve	0.003	-ve
IV	500	Mean	5.067	-ve	-ve	-ve	1.018	-ve
		±SD	0.585	-ve	-ve	-ve	0.003	-ve
V	1000	Mean	5.350	-ve	-ve	-ve	1.018	-ve
		±SD	1.219	-ve	-ve	-ve	0.003	-ve
VI	1000 (Rev.)	Mean	5.933	-ve	-ve	-ve	1.018	-ve
		±SD	0.987	-ve	-ve	-ve	0.003	-ve

 

Group	Dose		pH	Urobilinogen	Nitrite
Number	mg/kg			(mmol/L)
I	0	Mean	7.333	-ve	-ve
		±SD	0.258	-ve	-ve
II	0 (Rev.)	Mean	7.083	-ve	-ve
		±SD	0.376	-ve	-ve
III	250	Mean	7.250	-ve	-ve
		±SD	0.274	-ve	-ve
IV	500	Mean	7.250	-ve	-ve
		±SD	0.274	-ve	-ve
V	1000	Mean	7.250	-ve	-ve
		±SD	0.274	-ve	-ve
VI	1000 (Rev.)	Mean	7.250	-ve	-ve
		±SD	0.274	-ve	-ve

 Sp.Gr. : Specific gravity          +ve : Positive   -ve : Negative

Qualitative Absent                            = 0 Trace                               = + Small amount of analyte     = ++ Moderate amount of analyte  = +++ Large amount of analyte     = ++++

Rev. = Reversal

Conclusions:

The 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 found to be 1000 mg/kg body weight in male and female animals.

Executive summary:

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 found 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:

subchronic

Species:

rat

Quality of whole database:

K1 data from study report

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

Endpoint conclusion:

no study available

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:

repeated dose toxicity: dermal, other

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Waiver

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose oral toxicity:

In a study, the test chemical has been investigated for repeated dose toxicity to a greater or lesser extent. Study based on in vivo experiments in rodents, i.e. most commonly in rats for the test chemical are summarized 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 found to be 1000 mg/kg body weight in male and female animals.

 

Based on this value it can be concluded that the substance is considered to not toxic as per the criteria mentioned in CLP regulation.

Repeated dose toxicity: Inhalation

According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the low vapour pressure of the substance  9,10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs (CAS no. 97862-23-2), which is reported as 1.05E-14Pa at 25 C. Also, the acute inhalation LC50 value is considered t be 5mg/L. Thus, exposure to inhalable dust, mist and vapour of the chemical substance9,10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs is highly unlikely. Therefore this endpoint for repeated dose toxicity by inhalation route of exposure is considered for waiver.

Repeated dose toxicity: Dermal

The acute toxicity value for 9,10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs (CAS no 97862-23-2) (as provided in section 7.2.3) is >2000 mg/kg b.wt.mg/kg body weight by a study report . Also, given the use of the chemica, repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that 10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs. shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that,10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs. shall exhibit repeated dose toxicity by the dermal route. Hence this end point for repeated dose toxicity by dermal route of exposure was considered for waiver.

Based on the data available for the target chemical, 9,10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs (CAS no. 97862-23-2) does not exhibit toxic nature upon repeated exposure by oral, inhalation and dermal route of exposure and hence is not likely to classify as a toxicant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the target chemical, 9,10-Anthracenedione, 1,4-diamino-, N,N'-bis (4-C7-17-branched alkylphenyl) derivs (CAS no. 97862-23-2) does not exhibit toxic nature upon repeated exposure by oral, inhalation and dermal route of exposure and hence is not likely to classify as a toxicant as per the criteria mentioned in CLP regulation.