2-[2-[4-(diethylamino)phenyl]vinyl]-1,3,3-trimethyl-3H-indolium acetate

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

Organ toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 407

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

Test material

Test material information

Constituent 1

Reference substance name:

2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs.

EC Number:

296-117-1

EC Name:

2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs.

Cas Number:

92257-28-8

Molecular formula:

C30H33N3

IUPAC Name:

N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine

Test material form:

liquid

Details on test material:

- Name of test material : 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl]azo]-, ar' and ar''-Me derivs.
- IUPAC name : N-(2-ethylhexyl)-1-[4-(2-phenyldiazen-1-yl)phenyl]naphthalen-2-amine
- Molecular formula : C30H33N3
- Molecular weight : 435.60 g/mol
- Smiles : CCCCC(CC)CNc1ccc2ccccc2c1Nc1ccc(N=Nc2ccccc2)cc1
- Substance type : Organic
- Physical state : Liquid

Test animals

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: (P) x wks; (F1) x wks: 6 to 8 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: Male - 169.52 g (= 100 %) and Female - 162.35 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.
identified by the picric acid marking. A group of animals in one cage was additionally identified by the label affixed to each cage. A label according to groups identified the cage and each label contained information on cage and study number. It also bear species, strain, sex and identification numbers of rats within it.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): Rodent feed, provided ad libitum from individual feeders on cage top
- Water (e.g. ad libitum): Water was from a local source and passed through the reverse osmosis membrane before use, ad libitum
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C (actual range, 19.3 °C to 22.5 °C)
- Humidity (%):30% to 70% (actual range, 45.1% to 58.7%).
- Air changes (per hr): 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: 06-12-2017
To: 02-03-2018

Administration / exposure

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

not specified

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was diluted with corn oil for preparation of solution(s).
The solution(s) of 2-Naphthalenamine, N-(2-ethylhexyl)-1-[[4-(phenylazo)phenyl] azo]-, ar’ and ar’’-Me derivs (CAS No. 92257-28-8) 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): upto 10 ml/kg body weight
- Lot/batch no. (if required):
- Purity:

Details on mating procedure:

Reproductive organ weigth and histopathology were examined.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

stability determined by UV

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Details on study schedule:

not specified

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

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
0 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:
- Rationale for animal assignment (if not random):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.
- Other:

Positive control:

No

Examinations

Parental animals: Observations and examinations:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: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:Not specified
- 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): Not specified
- Time schedule for examinations:Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the initiation of the dosing and at scheduled sacrifice.
- Dose groups that were examined: All dose group were examined.

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: Not specified
- Animals fasted: Yes, overnight
- How many animals: All 72 animals were examined.
- Parameters checked in table [No.?] were examined.Hemoglobin (g/dL), Red Blood Corpuscles (x 106 /µL), Hematocrit (%), Mean Corpuscular Volume (fL), Mean Corpuscular Hemoglobin (pg), Mean Corpuscular Hemoglobin Concentration (g/dL), Platelets (x 103 /µL), White Blood Corpuscles (x 103 /µL), Reticulocytes (%), Neutrophils (%), Lymphocytes (%), Eosinophils (%), Monocytes (%), Basophil (%) and 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.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, overnight
- How many animals: All 72 animals were examined.
- Parameters checked in table [No.?] were examined.: Total Protein (g/dL), Blood Urea Nitrogen (mg/dL), Urea Nitrogen (mg/dL) Calculated, Alanine Aminotransferase (U/L), Aspartate Aminotransferase (U/L), Alkaline Phosphatase (U/L), 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 (mmol/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
- Dose groups that were examined:All dose group were examined.
- Battery of functions tested: sensory activity, grip strength, motor activity, Visual Placing Response were examined.

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.

Oestrous cyclicity (parental animals):

not specified

Sperm parameters (parental animals):

not specified

Litter observations:

not specified

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes
after 28 consecutive days of oral administration, all surviving study rats were sacrificed on day 29 (Group I, III, IV, V). In addition all rats from reversal groups were sacrificed on day 43 (Group II and VI).

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.

Postmortem examinations (offspring):

not specified

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 was 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).

Reproductive indices:

not specified

Offspring viability indices:

not specified

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.

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

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.

Dermal irritation (if dermal study):

not specified

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 -
Animals from control and different dose groups exhibited normal body weight gain throughout the dosing period of 28 days. Reduced body weight gain of 5.48% was observed in male animals from 1000 mg/kg dose group and this effect was attributed to the biological variation within the test system and considered to be of no toxicological importance.
During the post-dosing recovery period, animals from 1000 mg/kg reversal group exhibited normal body weight gain when compared with that of control animals.
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 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:

no effects observed

Description (incidence and severity):

Male and Female -
Haematological investigations conducted at the end of dosing period on day 29 and at the end of recovery period on day 43, revealed following significant changes in the values of different parameters studied when compared with that of respective controls, however the increase/decrease in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent.
Male :
MCHC : Increased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05) and
HCT : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).
Female :
Platelets : Increased values were obtained for animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Hb, HCT and MCHC : Decreased values were obtained for animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Total RBC : Decreased values were obtained for animals from 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05) and
Total RBC : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Male and Female -
Biochemical investigations conducted at the end of dosing period on day 29 and at the end of recovery period on day 43, revealed following significant changes in the values of different parameters studied when compared with that of respective controls, however the increase/decreased in the values obtained was within normal biological and laboratory limits or the effect was not dose dependent.
Male :
Total Bilirubin : Elevated levels were observed in animals from 1000 mg/kg dose group, sacrificed on day 29 (p<0.05),
Creatinine : Elevated levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05),
Glucose : 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 500 mg/kg and 1000 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Globulin and Bile Acid : Decreased levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05),
Total Cholesterol : Elevated levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05) and
Glucose : Decreased levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).
Female :
Total Protein and Aspartate Aminotransferase : Elevated levels were observed in animals from 250 mg/kg and 500 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Total Bilirubin : Elevated 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),
Albumin : Elevated 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 and 500 mg/kg dose groups, sacrificed on day 29 (p<0.05),
Triglycerides : Decreased levels were observed in animals from 250 mg/kg dose group, sacrificed on day 29 (p<0.05) and
Total Protein : Elevated levels were observed in animals from 1000 mg/kg reversal dose group, sacrificed on day 43 (p<0.05).

Urinalysis findings:

no effects observed

Description (incidence and severity):

Male and Female -
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 23, 24, 25, 26 and 43) in male and female animals of different dose groups as compared to control group animals, except for higher volume of urine was observed in female animals from 250 mg/kg dose group (p<0.05). This higher volume of urine analyses were considered to be incidental and of no toxicological importance.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

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; minimal, multifocal tubular eosinophilic luminal secretion in the kidneys; minimal, multifocal brown pigmentation in spleen; minimal, diffuse dilatation of zona reticularis and/or minimal, multifocal vacuolation in zona fasciculata in the adrenals; minimal, luminal seminal coagulum in urinary bladder; minimal, luminal dilatation in the uterus; minimal, multifocal dilatation of Brunner’s gland in the duodenum; presence of persistent Rathke’s pouch in the pituitary in male and female animals from control and high dose group.

No histopathological changes were observed in reproductive organ of treated male and female rats as compared to control.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Functional Observations
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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

ophthalmological examination

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: No effect on reproductive organ weight and histopathology

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

other: not specified

Generation:

other: not specified

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: not specified

Remarks on result:

other: not specified

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

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	169.17	170.12	200.23	228.53	254.67
		±SD	8.50	8.68	8.97	8.90	8.24
II	0 (Reversal)	Mean	168.87	169.73	197.90	225.98	251.90
		±SD	8.94	8.82	9.33	8.34	7.82
III	250	Mean	169.37	170.28	200.75	226.62	253.32
		±SD	8.04	7.66	8.06	8.49	8.30
IV	500	Mean	169.67	170.67	199.43	227.15	253.32
		±SD	7.37	7.12	7.50	8.52	7.45
V	1000	Mean	170.12	170.92	197.05	219.97	242.32
		±SD	6.55	6.31	4.84	3.52	3.64
VI	1000 (Reversal)	Mean	169.93	171.15	199.50	224.07	248.75
		±SD	5.65	5.71	6.66	5.96	6.05

 

Group	Dose (mg/kg)		Weeks
Number			4	5	6
I	0	Mean	279.80
		±SD	9.06
II	0 (Reversal)	Mean	278.05	295.50	314.10
		±SD	9.40	11.63	9.91
III	250	Mean	277.17
		±SD	9.28
IV	500	Mean	279.68
		±SD	6.85
V	1000	Mean	264.47*
		±SD	4.66
VI	1000 (Reversal)	Mean	271.88	294.02	317.88
		±SD	7.01	6.14	6.80

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

Sex - Female

Group	Dose (mg/kg)		Weeks
Number			Day 0	Day 1	1	2	3
I	0	Mean	162.00	163.50	182.68	195.25	200.42
		±SD	9.85	9.92	12.09	13.39	14.17
II	0 (Reversal)	Mean	161.78	163.08	179.25	188.37	196.62
		±SD	10.42	10.09	17.80	20.61	24.43
III	250	Mean	161.60	162.28	179.13	196.15	208.33
		±SD	10.64	10.98	6.35	9.89	15.63
IV	500	Mean	163.15	163.93	178.10	191.40	202.67
		±SD	9.72	9.62	9.14	8.09	9.01
V	1000	Mean	162.98	163.78	180.02	198.17	207.32
		±SD	8.90	8.97	8.69	13.79	12.42
VI	1000 (Reversal)	Mean	162.57	163.35	178.02	196.28	206.73
		±SD	7.22	7.24	9.57	13.81	17.55

 

Group	Dose (mg/kg)		Weeks
Number			4	5	6
I	0	Mean	208.07
		±SD	12.94
II	0 (Reversal)	Mean	204.57	213.80	223.95
		±SD	23.71	23.21	21.65
III	250	Mean	215.58
		±SD	14.03
IV	500	Mean	210.32
		±SD	9.17
V	1000	Mean	216.13
		±SD	11.36
VI	1000 (Reversal)	Mean	213.83	221.57	229.22
		±SD	15.90	15.25	11.54

GROUP MEAN ABSOLUTE ORGAN WEIGHTS (g)

Sex : Male

Day : 29 and 43

GroupNumber	Dose (mg/kg)		Body Weight (g)	Brain	Liver	Kidneys	Adrenals	Testes	Prostate + Seminal Vesicle with Coagulation gland
I	0	Mean	258.267	1.837	9.101	1.971	0.045	2.852	1.649
		±SD	11.271	0.098	0.664	0.205	0.006	0.129	0.464
II	0 (Reversal)	Mean	294.200	1.879	11.420	2.480	0.050	2.882	1.784
		±SD	11.583	0.127	1.541	0.319	0.005	0.283	0.210
III	250	Mean	255.883	1.777	14.287	2.198	0.037	2.866	1.768
		±SD	9.429	0.105	1.364	0.126	0.005	0.160	0.209
IV	500	Mean	260.067	1.722	11.482	2.069	0.040	2.783	1.595
		±SD	5.421	0.137	1.318	0.152	0.005	0.226	0.164
V	1000	Mean	246.467	1.728	10.302	1.648	0.032	2.569	1.283
		±SD	2.295	0.065	1.134	0.169	0.005	0.323	0.067
VI	1000 (Reversal)	Mean	301.067	1.812	10.614	1.890	0.048	2.612	1.485
		±SD	7.816	0.037	0.800	0.360	0.008	0.509	0.170

 

GroupNumber	Dose (mg/kg)		Heart	Spleen	Thymus	Epididymides
I	0	Mean	1.101	1.428	0.296	0.926
		±SD	0.277	0.521	0.073	0.114
II	0 (Reversal)	Mean	1.320	1.159	0.356	0.933
		±SD	0.309	0.204	0.085	0.052
III	250	Mean	1.100	1.259	0.284	0.946
		±SD	0.040	0.169	0.035	0.118
IV	500	Mean	1.132	1.119	0.209	0.952
		±SD	0.075	0.147	0.034	0.132
V	1000	Mean	0.807	1.065	0.096	0.764
		±SD	0.073	0.370	0.019	0.095
VI	1000 (Reversal)	Mean	1.112	1.022	0.414	1.042
		±SD	0.104	0.132	0.115	0.100

Sex : Female

Day : 29 and 43

GroupNumber	Dose (mg/kg)		Body Weight (g)	Brain	Liver	Kidneys	Adrenals	Ovaries
I	0	Mean	196.583	1.778	5.758	1.273	0.055	0.087
		±SD	13.034	0.058	0.251	0.092	0.008	0.021
II	0 (Reversal)	Mean	210.850	1.715	7.461	1.497	0.047	0.097
		±SD	22.274	0.054	1.994	0.291	0.012	0.012
III	250	Mean	202.283	1.702	8.790	1.515	0.053	0.101
		±SD	15.398	0.121	1.055	0.147	0.010	0.016
IV	500	Mean	195.050	1.634	9.273	1.421	0.051	0.086
		±SD	9.819	0.096	0.984	0.147	0.006	0.021
V	1000	Mean	199.367	1.667	8.977	1.363	0.049	0.090
		±SD	13.093	0.085	1.103	0.230	0.009	0.018
VI	1000 (Reversal)	Mean	214.050	1.893	8.813	1.784	0.063	0.124
		±SD	11.406	0.089	0.904	0.132	0.014	0.020

 

Group No.	Dose (mg/kg)		Heart	Spleen	Thymus	Uterus
I	0	Mean	0.719	0.932	0.238	0.425
		±SD	0.085	0.170	0.066	0.188
II	0 (Reversal)	Mean	0.860	0.964	0.313	0.514
		±SD	0.078	0.270	0.060	0.087
III	250	Mean	0.798	0.950	0.215	0.379
		±SD	0.086	0.182	0.042	0.036
IV	500	Mean	0.832	0.824	0.216	0.317
		±SD	0.067	0.229	0.102	0.031
V	1000	Mean	0.737	0.991	0.358	0.370
		±SD	0.113	0.246	0.410	0.100
VI	1000 (Reversal)	Mean	0.975	0.931	0.388	0.488
		±SD	0.057	0.254	0.049	0.056

GROUP MEAN RELATIVE ORGAN WEIGHTS (%)

Sex : Male

Day : 29 and 43

GroupNumber	Dose (mg/kg)		Body Weight (g)	Brain	Liver	Kidneys	Adrenals	Testes	Prostate + Seminal Vesicle with Coagulation gland
I	0	Mean	258.267	0.712	3.524	0.762	0.017	1.105	0.640
		±SD	11.271	0.031	0.198	0.060	0.002	0.052	0.188
II	0 (Reversal)	Mean	294.200	0.639	3.879	0.843	0.017	0.980	0.608
		±SD	11.583	0.054	0.472	0.107	0.002	0.097	0.085
III	250	Mean	255.883	0.694	5.576*	0.859*	0.014*	1.121	0.690
		±SD	9.429	0.026	0.396	0.031	0.002	0.073	0.073
IV	500	Mean	260.067	0.662	4.416*	0.796	0.015	1.071	0.613
		±SD	5.421	0.056	0.497	0.071	0.002	0.098	0.062
V	1000	Mean	246.467	0.701	4.180*	0.669*	0.013*	1.044	0.520
		±SD	2.295	0.022	0.457	0.066	0.002	0.138	0.027
VI	1000 (Reversal)	Mean	301.067	0.602	3.526	0.629*	0.016	0.871	0.493*
		±SD	7.816	0.023	0.258	0.126	0.002	0.188	0.054

 

GroupNumber	Dose (mg/kg)		Heart	Spleen	Thymus	Epididymides
I	0	Mean	0.425	0.551	0.114	0.359
		±SD	0.094	0.192	0.026	0.049
II	0 (Reversal)	Mean	0.447	0.395	0.121	0.317
		±SD	0.093	0.071	0.028	0.015
III	250	Mean	0.430	0.491	0.111	0.369
		±SD	0.019	0.055	0.014	0.042
IV	500	Mean	0.436	0.430	0.080*	0.367
		±SD	0.033	0.053	0.012	0.057
V	1000	Mean	0.327*	0.432	0.039*	0.310
		±SD	0.029	0.151	0.008	0.039
VI	1000 (Reversal)	Mean	0.370	0.339	0.137	0.346
		±SD	0.035	0.040	0.036	0.029

Sex : Female

Day : 29 and 43

GroupNumber	Dose (mg/kg)		Body Weight (g)	Brain	Liver	Kidneys	Adrenals	Ovaries
I	0	Mean	196.583	0.907	2.935	0.650	0.028	0.044
		±SD	13.034	0.067	0.157	0.062	0.005	0.012
II	0 (Reversal)	Mean	210.850	0.822	3.548	0.720	0.023	0.047
		±SD	22.274	0.101	0.894	0.179	0.008	0.009
III	250	Mean	202.283	0.848	4.386*	0.755	0.026	0.050
		±SD	15.398	0.111	0.745	0.111	0.006	0.011
IV	500	Mean	195.050	0.839	4.756*	0.730	0.026	0.044
		±SD	9.819	0.060	0.458	0.080	0.002	0.011
V	1000	Mean	199.367	0.838	4.494*	0.680	0.024	0.045
		±SD	13.093	0.056	0.379	0.077	0.004	0.008
VI	1000 (Reversal)	Mean	214.050	0.886	4.131	0.837	0.029	0.058
		±SD	11.406	0.062	0.513	0.095	0.007	0.012

 

Group No.	Dose (mg/kg)		Heart	Spleen	Thymus	Uterus
I	0	Mean	0.366	0.473	0.122	0.222
		±SD	0.036	0.075	0.036	0.113
II	0 (Reversal)	Mean	0.411	0.468	0.150	0.245
		±SD	0.048	0.167	0.037	0.041
III	250	Mean	0.398	0.474	0.107	0.189
		±SD	0.062	0.109	0.021	0.030
IV	500	Mean	0.427	0.421	0.110	0.163
		±SD	0.041	0.109	0.050	0.014
V	1000	Mean	0.368	0.497	0.180	0.186
		±SD	0.037	0.126	0.206	0.050
VI	1000 (Reversal)	Mean	0.457	0.441	0.181	0.229
		±SD	0.045	0.147	0.022	0.027

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

SUMMARY OF HISTOPATHOLOGY FINDINGS

Sex : Male	Fates : All
Days : All

 

Dose		0 mg/kg	1000 mg/kg
Number of Animals :		6	6
		# AWA	# AWA
Adrenals  Dilatation  Vacuolation	# Ex	6 4 1	6 1 1
Aorta	# Ex	6 NAD	6 NAD
Brain (cerebrum, cerebellum and pons)	# Ex	6 NAD	6 NAD
Caecum	# Ex	6 NAD	6 NAD
	# Ex	6 NAD	6 NAD
Duodenum	# Ex	6 NAD	6 NAD
Epididymides	# Ex	6 NAD	6 NAD
Eyes	# Ex	6 NAD	6 NAD
Heart	# Ex	6 NAD	6 NAD
Ileum	# Ex	6 NAD	6 NAD
Jejunum	# Ex	6 NAD	6 NAD
Kidneys  Eosinophilic secretion	# Ex	6 2	6 2
Liver  Infiltration	# Ex	6 3	6 4
Lungs	# Ex	6 NAD	6 NAD

Dose		0 mg/kg	1000 mg/kg
Number of Animals :		6	6
		# AWA	# AWA
Mesenteric Lymphnodes	# Ex	6 NAD	6 NAD
Muscles - Skeletal muscle	# Ex	6 NAD	6 NAD
Oesophagus	# Ex	6 NAD	6 NAD
Pancreas	# Ex	6 NAD	6 NAD
Pharyngeal Lymphnodes	# Ex	6 NAD	6 NAD
Pituitary	# Ex	6 NAD	6 NAD
Prostate	# Ex	6 NAD	6 NAD
Rectum	# Ex	6 NAD	6 NAD
Sciatic Nerve	# Ex	6 NAD	6 NAD
Seminal Vesicles with  Coagulation Gland	# Ex	6 NAD	6 NAD
Skin with mammary Gland	# Ex	6 NAD	6 NAD
Spleen  Pigmentation	# Ex	6 1	6 1
Spinal Cord (Cervical, mid thoracic and lumbar)	# Ex	6 NAD	6 NAD
Sternum with bone marrow	# Ex	6 NAD	6 NAD

Dose		0 mg/kg	1000 mg/kg
Number of Animals :		6	6
		# AWA	# AWA
Stomach	# Ex	6 NAD	6 NAD
Testes	# Ex	6 NAD	6 NAD
Thymus	# Ex	6 NAD	6 NAD
Thyroid / Parathyroid	# Ex	6 NAD	6 NAD
Trachea	# Ex	6 NAD	6 NAD
Urinary bladder  Seminal coagulum	# Ex	6 1	6 2

Sex : Female	Fates : All
Days : All

 

Dose		0 mg/kg	1000 mg/kg
Number of Animals :		6	6
		# AWA	# AWA
Adrenals  Dilatation	# Ex	6 2	6 2
Aorta	# Ex	6 NAD	6 NAD
Brain (cerebrum, cerebellum and pons)	# Ex	6 NAD	6 NAD
Caecum	# Ex	6 NAD	6 NAD
Cervix	# Ex	6 NAD	6 NAD
	# Ex	6 NAD	6 NAD
Duodenum  Dilatation	# Ex	6 1	6 0
Eyes	# Ex	6 NAD	6 NAD
Heart	# Ex	6 NAD	6 NAD
Ileum	# Ex	6 NAD	6 NAD
Jejunum	# Ex	6 NAD	6 NAD
Liver  Infiltration	# Ex	6 3	6 3
Kidneys  Eosinophilic secretion	# Ex	6 1	6 2
Lungs	# Ex	6 NAD	6 NAD
Mesenteric Lymphnodes	# Ex	6 NAD	6 NAD

Dose		0 mg/kg	1000 mg/kg
Number of Animals :		6	6
		# AWA	# AWA
Muscles - Skeletal muscle	# Ex	6 NAD	6 NAD
Oesophagus	# Ex	6 NAD	6 NAD
Ovaries	# Ex	6 NAD	6 NAD
Pancreas	# Ex	6 NAD	6 NAD
Pharyngeal Lymphnodes	# Ex	6 NAD	6 NAD
Pituitary  Rathke’s pouch	# Ex	6 1	6 0
Rectum	# Ex	6 NAD	6 NAD
Sciatic Nerve	# Ex	6 NAD	6 NAD
Skin with mammary Gland	# Ex	6 NAD	6 NAD
Spleen  Pigmentation	# Ex	6 2	6 1
Spinal Cord (Cervical, mid thoracic and lumbar)	# Ex	6 NAD	6 NAD
Sternum with bone marrow	# Ex	6 NAD	6 NAD
Stomach	# Ex	6 NAD	6 NAD
Thymus	# Ex	6 NAD	6 NAD

Dose			0 mg/kg	1000 mg/kg
Number of Animals :			6	6
			# AWA	# AWA
Thyroid / Parathyroid		# Ex	6 NAD	6 NAD
Trachea		# Ex	6 NAD	6 NAD
Urinary bladder		# Ex	6 NAD	6 NAD
Uterus  Dilatation		# Ex	6 2	6 2
Vagina		# Ex	6 NAD	6 NAD

# AWA = Number of animals with abnormality in the group.

# Ex  = Number of animals examined

NAD = No Abnormality Detected

Applicant's summary and conclusion

Conclusions:

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 28-day Oral Toxicity study, Sprague Dawley male and female rats were treated with the test chemical in the concentration of 0, 250 mg/kg, 500 mg/kg and 1000 mg/kg body weight orally by gavage in corn oil. Two additional dose levels were added to the study as control 0 mg/kg (Rev.) and test item 1000 mg/kg (Rev.), in order to study the reversibility or delayed occurrence of symptoms, if any. No effect on survival, clinical sign, body weight, Feed intake, Ophthalmoscopic examination and functional observation battery of treated male and female rats at the end of dosing period of 28 days and the recovery period of 14 days. Similarly, in male animals at the end of the recovery period on day 43, revealed statistically significant increase in the values of MCHC and statistically significant decrease in the values of HCT at 1000 mg/kg and in female on day 29, revealed statistically significant increase in the values of Platelets at 250 mg/kg, 500 mg/kg and 1000 mg/kg, and statistically significant decrease in the values of Hb, HCT and MCHC at 250 mg/kg, 500 mg/kg and 1000 mg/kg, and Total RBC at 500 mg/kg and 1000 mg/kg, Haematological analysis in female animals conducted at the end of the recovery period on day 43, revealed statistically significant decrease in the values of Total RBC at 1000 mg/kg. In male and female animals at the end of the dosing period on day 29, revealed statistically significant increase in the values of Total Bilirubin at 1000 mg/kg and Creatinine at 250 mg/kg, in male, Total Protein and Aspartate Aminotransferase at 250 mg/kg and 500 mg/kg, Total Bilirubin at 250, 500 and 1000 mg/kg, and Albumin at 500 mg/kg in female rat. In addition, statistically significant decrease was observed in the values of Glucose at 500 mg/kg, Calcium at 500 mg/kg and 1000 mg/kg, Globulin and Bile Acid at 250 mg/kg in male rat and, Calcium at 250 and 500 mg/kg, and Triglycerides at 250 mg/kg in female rat. At the end of the recovery period on day 43 (Reversal groups) statistically significant increase were observed in the values of Total Cholesterol at 1000 mg/kg in male rat and Total Protein at 1000 mg/kg in female rat. In addition, statistically significant decrease was observed in the values of Glucose at 1000 mg/kg in male rat. The increase/decrease in the values of various parameters was marginal and within the normal biological and laboratory limits for hematology and clinical chemistry parameters. In Urine analysis conducted during 4th and 6th week of dosing period (on day 23, 24, 25, 26 and 43), revealed no abnormality attributable to the treatment except for higher volume of urine was observed in female animals from 250 mg/kg dose group and considered to be incidental and of no toxicological importance. In addition, at termination of dosing on day 29, male animals from 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 kidneys were observed in male animals from 250 mg/kg dose group, when compared with that of controls. Decreased relative weights of kidneys and heart weight at 1000 mg/kg in male as compared to controls. In addition, decreased relative weights of adrenals were observed in male animals from 250 and 1000 mg/kg dose groups when compared with that of controls. Decreased relative weights of thymus were observed in male animals from 500 mg/kg and 1000 mg/kg dose groups when compared with that of controls. Organ weight data of male animals sacrificed on day 43 from 1000 mg/kg reversal group, revealed decreased relative weights of kidneys and prostate + seminal vesicle with coagulation gland as whole when compared with that of controls.At termination of dosing on day 29, female animals from 250 mg/kg, 500 mg/kg and 1000 mg/kg dose groups revealed increased relative weights of liver. Organ weight data of female animals sacrificed on day 43 from 1000 mg/kg reversal group, was found to be comparable with that of controls. No effect on Epididymides and Testes of male rat and Ovaries, Uterus Dilatation and Vagina of female rats were observed as compared to control. 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 animals at 250, 500 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. Histopathological examination did not reveal any abnormality attributable to the treatment in reproductive organ. 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.