Registration Dossier

Administrative data

Description of key information

In the repeated dose toxicity studies (28 days and 90 days), the adverse effects observed where not specific enough and were not considered conclusive for classification for any specific target organ toxicity at repeated doses as no specific organs could be identified.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2015-11-13 to 2016-05-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Selected due to its availability in requisie numbers. This strain has been used for non-clinical safety studies, recommended by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Taconic Biosciences, Inc. USA, through its representative Vivo Bio Tech Ltd. Telangana, India.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 weeks
- Weight at study initiation: the weight variation of animals used did not exceed ± 20% of the mean weight for each sex
* Mean body weight for male : 235g (min = 209 g - max = 256 g; n = 60)
* mean body weight for female 176g (min = 160 g - max = 193 g; n=60).

- Housing: Cage: sterilized solid bottom polypropylene [size 42 cm (L) X 29 cm (W)X 19cm (H)] with stainless steel grill tops, facilities for food and water bottle, and with bedding of clean and sterilized corn-cob. Cages were suspended on movable stainless steel racks arranged in such a way that every cage received almost same amount of light. For this purpose, cages arranged on the first row were shifted to the last row, those on the second row were shifted to the first row. Likewise all the cages were rotated. This kind of cage rotation was carried out every week thoughout the treatment and observation period.
Groups of 2/3 animals/sex/cage.

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 to 8 days

DETAILS OF FOOD AND WATER QUALITY:
Food : 'Altromin' brand extruded pelleted rat feed manufactured by M/s Altromin Spezialfutter GmbH & Co. KG, Germany, and supplied by ATNT laboratories Mumbai.
The feed is analysed for nutritional components and environmental contaminants once in a year. Results of dietary analysis were provided by the manufacturer for each batch of diet.
Water : Potable water filtered through 'Aquaguard' and treated by UV irradiation. Water is analyzed half yearly for potability and once in a year for various environmental contaminant.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30-70%
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES:
* For males:
-End of Treatment Period: from: 2015-11-13 To: 2016-02-17 and 2016-02-18
- End of Recovery Period: 2015-11-13 To: 2016-03-16
* For females:
-End of Treatment Period: from: 2015-11-13 To: 2016-02-19 and 2016-02-20
- End of Recovery Period: 2015-11-13 To: 2016-03-18
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions prepared freshly just before daily dosing: a define quantity of test item is suspended in corn oil and stirred.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on the solubility testing.
- Concentration in vehicle: varied in concentrations to allow a constant dosage volume.
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): Batch 146 (exp: august 2016),119 (exp: december 2015),165 (exp: May 2016).
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1 (control): 5 ml/kg corn oil
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G2 (control reversal): 5 mL/kg corn oil
Dose / conc.:
6.25 mg/kg bw/day (nominal)
Remarks:
G3
Dose / conc.:
12.5 mg/kg bw/day (nominal)
Remarks:
G4
Dose / conc.:
25 mg/kg bw/day (nominal)
Remarks:
G5
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
G6
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
G7 (reversal)
No. of animals per sex per dose:
Treatment groups (G1, G3, G4, G5, G6): 10
Reversal groups (G2; G7): 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: range finding study
- Rationale for animal assignment (if not random): random assignment
- Rationale for selecting satellite groups: 6.25,12.5,25 and 50 mg/kg body weight
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily (during treatment and recovery period), except for mortality/moribund twice daily (early and in afternoon).
- Cage side observations : moribund, mortality, abnormal behaviour and/or appearance.

CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily (during treatment and recovery period)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before initiation of the treatment and weekly thereafter (during treatment and recovery period)


BODY WEIGHT: Yes
- Time schedule for examinations: A
* At day 0, before initiation of treament
* Weekly and at necropsy (during treatment and recovery period)
* Before necropsy for all dead animals found


FOOD CONSUMPTION (not a feeding study)
- Time schedule: weekly (during treatment and recovery period)
- Fodd consumption for each animal determined using the amount of food offered and left in each cage in each group and the number of rats in each cage (without correction for spillage, considered minimal).


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at initiation and at termination of the treatment
- Dose groups that were examined:
* At initiation of treatment: all animals
* At termination of treatment: control group and 50 mg/kg dose group (G6)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after completion of 90 days of treatment (on day 91 and /or 92) and at the termination on the recovery period (on day 119)
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: yes, overnight
- How many animals: all rats
- Parameters checked (haemoglobin(Hb), packed cell volume (PCV), total red cell count (total RBC), total white cell count (total WBC), absolute RBC indices, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corspucular haemoglobin concentration (MCHC), platelet count, activated partial thromboplastin time (APTT) and prothrombin time (PT), differential WBC counts)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after completion of 90 days of treatment (on day 91 and /or 92) and at the termination on the recovery period (on day 119)
- Animals fasted: yes, overnight
- How many animals: all rats
- Parameters checked in table No 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: in the last week of treatment (day 85 to 89) and recovery period (day 114).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Thirtheen week of treatment
- Dose groups that were examined: all rats
- Functional observational battery suggested by Moser V.C. (1989). The neurological parameters in table No 3 were tested

IMMUNOLOGY: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY:Yes

At termination of the treatment period (day 91 and / or 92) and at the end of the recovery period (day 119), all surviving rats were sacrificed by exsanguinations under CO2 anesthesia and complete necropsies were carried out. All the found dead rats were also subjected to detailed necropsy examination.
All the tissues from all animals, were preserved in 10% neutral buffered formalin. Testes and eyes were collected in modified Davidson’s fixative. In addition, samples of any macroscopically abnormal tissues were preserved, along with samples of adjacent normal tissue where appropriate.

The following organs were weighted from all surviving animals: kidneys, liver, adrenals, testis, epididymides, uterus, thymus, spleen, brain, ovaries, heart. They were efully dissected and trimmed of any adherent tissue, as appropriate, and weighed wet as soon as possible to avoid drying.

The following organs and tissues were collected from all animals and fixed: gross, lesions, brain (cerebrum, cerebellum, midbrain), spinal cord, eye, pituitary, thyroid, parathyroid, spleen, thymus, adrenals, pancreas, trachea, lungs, heart, aorta, oesophagus, stomach, duodenum, jejunum, terminal ileum, colon, rectum, liver, kidneys, urinary bladder, prostale, seminal vesicle, epididymides, testes, ovaries, uterus, vagina, skin, sciatic nerve, bone marrow (smear), mammary gland (females).

Histopathological examination was conducted on the specified list of tissues including all macroscopically abnormal tissues of all animals from the vehicle control, 50 mg/kg dose level group and all the found dead animals. Due to high mortality rate (a total of 45%) observed in male and female rats treated at a dose of 50 mg/kg body weight, the data derived from the histopathology evaluation of the surviving animals or that of the found dead animals from this dose group was not sufficient to reach a meaningful interpretation about the probable effect of the test item in Wistar rats, as very few animals received a continuous oral administration of test article for 90 days.
Thus it was necessary to extend the histopathological evaluation to tissues from male and female rats treated with the test item at a dose of 25 mg/kg body weight as most of these animals had received the test article daily for a period of 90 days. The investigations were not extended to the lower dose groups and recovery groups in the absence of any test article related histopathological findings at 25 and 50 mg/kg dose levels.
Statistics:
Significance was judged at p < 0.05. See 'Any other information on materials and methods incl. tables' below, table No 4.
The suprscripts below are used for the results:
s+: significantly higher than the control group
s- : significantly lower than the control group
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Effects observed, treatment related:

* Blackish discoloration of feces
50 mg /kg: 10/15 male rats, 4/15 female rats
25 mg /kg: 6/10 male rats, 0/15 female rats
12.5 mg /kg: 5/10 male rats, 0/15 female rats
6.25 mg /kg: 1/10 male rats, 0/15 female rats

This is attributed to the dark color of the test item.

* Gasping
50 mg /kg: 6/15 male rats, 1/15 female rats
25 mg /kgt : 1/10 male rats, 0/10 female rats
12.5 mg/kg: 0/10 male rats, 0/10 female rats
6.25 mg /kg : 0/15 male rats, 0/15 female rats


Effects observed, non-treatment related:
* Salivation:
50 mg/kg: 2/15 male rats

* Bluish discoloration of perianal region:
50 mg/kg: 1/15 male rats

* Diarrheoa :
25 mg/kg: 1/10 male rats.

* hypoactivity
12.5 mg/kg: 1/10 male rats.

* Localized alopecia
25 mg/kg : 1/15 female rats.
12.5 mg/kg: 1/10 male rats.
6.25 mg/kg: 1/10 male rats.

Considered to be incidental findings

(See attached Table 2).
Mortality:
mortality observed, treatment-related
Description (incidence):
No mortality observed in the vehicle control group and during the subsequent recovery period of 28 days.

No treatment related mortality :
- in males: at and upto the dose level of 12.5 mg/kg
- in females: upto the dose level of 25 mg/kg

Treatment related mortality:
- in males: at 25 mg/kg (2/10 rats, at D36 and D89) and at 50 mg/kg (8/15 rats, at D14, D18, D25, D34, D42 and D64)
- in females: at 50 mg/kg (2/15 rats, at D31 and D36)

(See attached Table 1).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weight gain by male and female rats treated with the test article at and upto the dose of 50 mg/kg of body weight, was found to be comparable to that by the control rats throughout the treatment period and during the recovery period.

Althought statistically significant changes were observed, there were not considered to be a treament related change as these alterations were not dose dependent and therefore not considered to be of any toxicological significance

Significant decrease in body weight:
50 mg/kg: in females from D97 to 104 (-2.36%), during recovery period
25 mg/kg: in males and in females from D84 to D90 (-2.93% and -1.26% respectively)
6.25 mg/kg: in males from D77 to D84 (-1.00%)

Significant increase in body weight:
50 mg/kg: in males from D97 to D104 (+ 3.57%) and in females from D104 to D111 (+3.20%), during recovery period.

(See attached Tables 5 and 6).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
(not a feeding study) : the value of average daily food consumption by treated males and females remained comparable to those of the vehicle control group at the different dose levels

(see attached Table 7)
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The pretreatment and terminal ophtalmologic examinations did not reveal any remarkable and treatment related incidence of ocular abnormalities
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Non treatment related effect:
50 mg /kg (recovery) : statistically significant increase in eaosinophil count in male rats

No other effect in eosinophil count was observed at other doses for males and for females.
No other effect was observed for all the other haematological parameters for male and female rats treated with the test item up to 50 mg /kg.

The statistically significant increase in eosinophil count in male rats was not considered to be a treatment related change and not of biological significance due to lack of dose related effect.

(See attached Table 8).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Up to the dose level of 50 mg/kg bw no induced changes in the plasma levels of total protein, albumin, globulin, alanine, aminotransferase, aspartate aminotransferase, alkaline phosphatase, glucose, blood urea nitrogen, urea, creatinine, total cholesterol, total bilirubin, sodium, potassium, calcium, phosphorus and triglycerides in male and female rats.

Significant increase in plasma level of sodium
50 mg/kg: in males and females
25 mg/kg: in males

Significant decrease in plasma level of globulin
25 mg/kg: in females

Significant decrease in plasma level of total cholesterol
50 mg/kg (reversal): in females

Significant decrease in plasma level of calcium
50 mg/kg (reversal): in females

These significant differents were not considered to be a treatment related change due to lack of dose related effect and not considered to be biologically significant as the values were within biological limit.

(See attached Table 9)
Urinalysis findings:
no effects observed
Description (incidence and severity):
The data on urinalysis evaluated in the last week of treatment and recovery period did not indicate any abnormality due to treatment with the test item.
The data in test item treated and vehicle animals was found to be comparable.

(See attached Table 10)
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The neurological examinations (functional observations) conducted in the 13th week of the study did not reveal any remarkable and treatment related incidence onf neurological abnormalities.
No findings, indicative of a neurotoxic potential were encountered during these examinations.

(see attached Table 4)
Immunological findings:
not examined
Description (incidence and severity):
Not applicable
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The values of absolute and relative weights of kidneys , liver, adrenals, testes, epididymides, uterus, thymus,spleen, brain,ovaries and heart of male / female rats treated with the test article were found to be comparable to those of the vehicle control animals at termination of the treatment and also at the end of the recovery period.

Althought statistically significant changes were observed in a few organs weight at the end of treatments and recovery period, the changes observed were not considered to be treatment related due to lack of dose related effect and no coresponding (gross or histological) changes were observed in these organs in animals sacrified at the end of the treatement period.


Significant decrease in epididymides weight in gram
50 mg/kg: in males (mean of 1.17 g vs mean of 1.52 g in controls), during recovery period

Significant increase in uterus weight in gram
50 mg/kg: in females (mean of 0.73 g vs mean of 0.66 g in controls), during recovery period

(see attached Tables 11 and 12)
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item, at and up to the dose level of 50 mg /kg bw did not induce any remarkable and treatment related gross pathological alterations in any of the tissues of treated rats, as evident at the detailed necropsy examination carried out at termination of the study and at the end of the recovery period.

However few changes comprising of black colored ingesta in the stomach and intestine was noted in most of the found dead animals. This was attributed to the color of the test article. One male rat from the vehicle control group exhibited enlarged spleen which was confirmed microscopically as extramedullary haematopoiesis and congestion. Similarly one male rat treated at a dose of 12.5 mg/kg body weight exhibited multiple abscesses in lungs with adhesions to heart.
These changes were not considered to be treatment related.

AT TERMINAL SACRIFICE
NO ABNORMALITY DETECTED:
50 mg/kg (reversal): 4/4 male rats and 5/5 female rats
50 mg/kg: 3/3 male rats and 8/8 female rats
25 mg/kg: 8/9 male rats and 10/10 female rats
12.5 mg/kg: 9/10 male rats and 10/10 female rats
6.25 mg/kg: 10/10 male rats and 10/10 female rats
Control (reversal): 5/5 male rats and 5/5 female rats
Control: 9/10 male rats and 10/10 female rats

SPLEEN
Enlarged in size and increased in weight
Control: 1/10 male rats

LUNG
Right lobes, multiple abscess, adhesions with heart
12.5 mg/kg: 1/10 male rats


FOUND DEAD
NO ABNORMALITY DETECTED:
50 mg/kg (reversal): 1/1 male rats

STOMACH
Black coloured ingesta
50 mg/kg: 6/7 male rats and 2/2 female rats
25 mg/kg: 1/1 male rats

INTESTINE
Black coloured ingesta
50 mg/kg: 5/7 male rats and 1/2 female rats
25 mg/kg: 1/1 male rats

LUNG
Congestion, moderate
50 mg/kg: 1/7 male rats

LIVER
Congestion
50 mg/kg: 1/7 male rats

(see attached Table 13)

Neuropathological findings:
no effects observed
Description (incidence and severity):
No findings, indicative of a neurotoxic potential of the test item, were encountered during these examinations.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
ON SACRIFIED ANIMALS
NO ABNORMALITY DETECTED:
50 mg/kg: 0/3 male rats and 4/8 female rats
25 mg/kg: 2/8 male rats and 4/10 female rats
12.5 mg/kg: 9/10 male rats and 10/10 female rats
6.25 mg/kg: 10/10 male rats and 10/10 female rats
Control: 3/10 male rats and 3/10 female rats

LUNG
Emphysema in lungs
50 mg/kg : 1/3 male rats
25 mg/kg : 5/10 female rats

Multiple abscesses
12.5 mg/kg : 1/10 male rats

Adhesions (mild)
12.5 mg/kg : 1/10 male rats

Perivascular lymphotic aggregation
50 mg/kg : 1/8 female rats
25 mg/kg : 1/8 male rats
Control : 2/10 male rats and 7/10 female rats

LIVER
Portal lymphatic infiltration
Control : 1/10 male rats

KIDNEY
Tubular dilatation
Control : 1/10 male rats

OESOPHAGUS
Acanthosis
50 mg/kg : 1/8 female rats
Control : 1/10 male rats and 1/10 female rats

Hyperkeratosis
Control : 2/10 male rats and 1/10 female rats

SPLEEN
Increased haematopoiesis
Control : 1/10 male rats

Congestion
Control : 1/10 male rats

TRACHEA
Chronic tracheitis
25 mg/kg : 3/8 male rats
Control : 2/10 male rats

Inflammatory cell exudate in lumen
50 mg/kg : 1/8 female rats

COLON
Sub mucosal lymphoid hyperplasia
50 mg/kg : 1/3 male rats and 1/8 female rats
25 mg/kg : 1/8 male rats and 1/10 female rats

RECTUM
Sub mucosal lymphoid hyperplasia
50 mg/kg : 2/8 female rats
25 mg/kg : 1/8 male rats
Control: 1/10 male rats


PROSTATE
Sub mucosal lymphoid hyperplasia
50 mg/kg : 1/3 male rats

ON ANIMAL FOUND DEAD
NO ABNORMALITY DETECTED:
50 mg/kg: 0/7 male rats and 0/2 female rats
50 mg/kg (reversal): 0/1 male rats
25 mg/kg: 0/2 male rats

LUNGS
Emphysema
50 mg/kg : 7/7 male rats and 2/2 female rats
50 mg/kg (reversal): 1/1 male rats
25 mg/kg : 1/2 male rats

Foam cells in lungs
50 mg/kg : 2/7 male rats

Oedema in lungs
50 mg/kg : 5/7 male rats
50 mg/kg (reversal): 1/1 male rats
25 mg/kg : 2/2 male rats

Haemorrhages
50 mg/kg : 5/7 male rats and 1/2 female rats
50 mg/kg (reversal): 1/1 male rats
25 mg/kg : 1/2 male rats

LIVER
Congestion
50 mg/kg : 1/7 male rats and 2/2 female rats

TRACHEA
Inflammatory cell exudate in lumen
50 mg/kg : 4/7 male rats and 1/2 female rats

Chronic tracheitis
50 mg/kg : 2/7 male rats and 1/2 female rats
25 mg/kg : 1/2 male rats

Adhesions
50 mg/kg: 1/2 female rats

OESOPHAGUS
Acanthosis
25 mg/kg: 1/2 male rats

Hyperkeratosis
25 mg/kg: 1/2 male rats

(see attached Table 14)
Histopathological findings: neoplastic:
no effects observed
Details on results:
HISTOPATOLOGICAL EVALUATION OF TISSUES OF ANIMALS TREATED WITH VEHICLE AND TEST ITEM AT 50 mg/kg:

Histopathological examinations of the tissues of male and female rats from the vehicle control group and those treated at the 50 mg/kg dose group (surviving till termination) did not reveal any significant and treatment related histopathological alterations.
Few microscopic alterations observed were perivascular lymphocytic aggregation and a solitary incidence of emphysema in lungs. A solitary incidence of minimal portal lymphocytic infiltration in liver from one male rat treated with the vehicle control group.
Acanthosis and / or hyperkeratosis of minimal severity was observed in oesophagus of few male and female rats. This was seen in rats from vehicle control and treatment groups and was found to be comparable in incidence and severity. These were considered to be adaptive response to minimal repeated trauma to the oesophagus with oral gavage, hence were not considered to be test article related pathology. This was considered to be adaptive response to the trauma hence were not considered to be test article related pathology.
A solitary incidence of minimal dilatation of tubules in kidneys and increased extramedullary haematopoiesis and congestion in spleen was observed in one male rat treated from the vehicle control group. Minimal chronic tracheitis characterized by sub-mucosal infiltration of mononuclear cells was seen in trachea of two male rats from the vehicle control group and a solitary incidence of minimal inflammatory cell exudates in lumen of trachea was observed in female rat treated at the dose of 50 mg/kg body weight.
Chronic inflammatory cell infiltration in prostate was observed in one male rat treated at the dose of 50 mg/kg. However in absence of any other histopathological alteration in testes and epididymides this was considered to be incidental and of no toxicological significance. Few rats from the vehicle control and 50 mg/kg dose level showed sub-mucosal lymphoid hyperplasia of colon and rectum.

All these microscopic changes noticed in this study appeared to be incidental as their frequency was very low and not dose dependent.

ANIMALS TREATMENT WITH 20 mg/kg:HISTOPATOLOGICAL EVALUATION OF TISSUES OF ANIMALS TREATED WITH TEST ITEM AT 25 mg/kg:

Due to high mortality rate (a total of 45%) observed in male and female rats treated at a dose of 50 mg/kg, the data derived from the histopathology evaluation of the surviving animals or that of the found dead animals from this dose group was not sufficient to reach a meaningful interpretation about the probable effect of the test item, as very few animals this dose level received a continuous oral administration for 90 days. Thus it was necessary to extend the histopathological evaluation to tissues from male and female rats treated with the test item at a dose of 25 mg/kg as most of these animals had received the test article daily for a period of 90 days.

Therefore microscopic evaluation of all the tissues was conducted to reach a meaningful interpretation. The various histopathological findings observed in these male and female rats are as discussed below.

Out of eight male rats examined two rats did not show any histopathological alteration in any of the tissues examined. However few non specific microscopic alterations observed were perivascular lymphocytic aggregation in lungs; chronic tracheitis characterized by sub-mucosal infiltration of mononuclear cells in trachea and sub-mucosal lymphoid hyperplasia of colon and rectum. These were of minimal severity and their incidences were comparable to the control group rats.

Out of ten female rats treated at a dose of 25 mg/kg, four rats did not show any histopathological alteration in any of the tissues examined while five rats exhibited emphysema of minimal severity in lungs. A solitary incidence of minimal sub-mucosal lymphoid hyperplasia of colon was observed in a female rat.

It was also evident that the female rats exhibited decrease in body weight gain as compared to the control group as well as animals from other dose groups. It is well known that lowering of body weight is associated with emphysema as well as feed regurgitation (i.e the gastro-oesophageal reflux as in this case) apart from many other causes responsible for lowering of body weights. Also this decrease in body weight gain in female rats was found to be statistically significant as evident in last week of the study just prior to termination. However this histopathological alteration in form of emphysema in lungs and decrease in body weight gain were not considered to be an effect of the test article and hence was not considered to be a test article related effect.

HISTOPATHOLOGICAL EVALUATION OF TISSUES FROM FOUND DEAD ANIMALS

At the dose of 50 mg/kg 8/15 males and 3/15 females rats and at the dose of 25 mg/kg 2/10 male rats were found dead at various time points during the 90 day dosing period. The evaluation of tissues from these animals revealed pulmonary pathology comprising of minimal emphysema, scattered foam cells, oedema and hemorrhages in lungs. In general, the increased occurrence of scattered foam cells (alveolar macrophages) in lung after oral gavage was considered to be due to potential gastro-oesophageal reflux. The finding of black colored ingesta in the stomach and intestine observed during necropsy examination of the found dead animals could likely indicate reduced peristalsis promoting the reflux. However no correlating microscopic alterations were found in the stomach and the various segments of intestines examined suggestive of no effect of the test article.


Other factors like volume of the stomach contents at the time of the gavage; increased volume post dosing leading to increased pressure on the gastro-oesophageal junction causing subsequent reflux (even in rats with a very strong sphincter); significant increase in the weight of the gastric contents predisposing to delayed gastric emptying and / or increased gastric secretion triggering further reflux; viscous formulations along with high concentrations of the test article could have also contributed to the various pathological changes mentioned above.
Similarly oedema in lungs was also considered to be due to gavage related reflux associated with the cellular immune response to the reflux materials. Hemorrhages were also considered to be the effect of pulmonary insult due to reflux. Similarly changes in trachea comprised of inflammatory cell exudate in lumen, chronic trachitis and solitary incidence of adhesions in the mucosa were highly indicative of gavage related reflux associated changes.

Also a solitary incidence of congestion in liver was observed in one male rat and was considered to be a postmortem changes in absence of any other hepatic pathology. No other tissues from the found dead animals exhibited any microscopic alterations. Minimal to mild autolytic changes were present in the tissues evaluated from the found dead animals. Thus histopathological changes particularly seen in lungs and trachea in found dead animals were not considered to be the effect of the test article but were due to gastro-oesophageal reflux resulting into aspiration of the test article in respiratory system.
Acanthosis and hyperkeratosis of minimal severity observed in oesophagus of one found dead male rat treated at a dose of 25 mg/kg body weight was considered to be adaptive response to the likely trauma during gavage and hence was not considered to be effect of test article.

HISTOPATHOLOGICAL EVALUATION OF GROSS FINDINGS
A male rat treated at a dose of 12.5 mg/kg exhibited multiple abscesses in right lobes of lungs along with adhesions to heart. This was microscopically confirmed as multiple abscesses and adhesions and was considered to be incidental and not the effect of the test article.
The only gross finding observed during the necropsy examination of the found dead animals was black colored ingesta in the stomach and/or intestines. However no correlating microscopic alterations were found in the stomach and the various segments of intestines examined suggestive of no effect of the test article.

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 12.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
mortality
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality
Key result
Critical effects observed:
not specified
Conclusions:
The present study involved daily oral administration of THE test item to groups of Wistar rats, 10 per sex per dose, at the doses of 6.25,12.5, 25 and 50mg/kg of bw for 90 days to evaluate its toxicity and additional 5 animals per sex at the dose of 50 mg/kg bw to evaluate reversibility of toxicity, if any. The findings of this study were as follows:

* death of male rats - 2/10 at mg/kg and 8/15 at 50 mg/kg; death of female rats at 50 mg/kg

* no incidence of treatment related clinical abnormalities in vehicle control group; some treatment related clinical signs observed were - blackish discoloration of the feces, diarrhea, hypoactivity, gasping and blackish discoloration of perianal region at 25 mg/kg and 50 mg/kg bw.

* no incidence of treatment related ocular toxicity and neurotoxicity, at and upto the dose of 50 mg/kg bw

* no adverse effect on body weight gain by the surviving male and female rats treated at and upto the dose of 50 mg/kg bw

* no effect on average daily food consumption by the surviving male and female rats treated at and upto the dose of 50 mg/kg bw

* no effect on the haematologicval parameters of surviving male and female rats treated at and upto the dose of 50 mg/kg bw

* no effect on the chemical chemistry and urinalysis parameters of surviving male and female rats treated at and upto the dose of 50 mg/kg bw

* no significant alterations in the absolute and relative organ weights

* no test article related gross and microscopic pathological alterations in the tissues of male and female rats treated at and upto the level of 25 mg/kg and 50 mg/kg bw.

Based on the findings of this study, the No-Observed-Adverse-Effect-Level (NOAEL) of the test item in Wistar rats, following oral administration for 90 days was found to be 12.5 mg/kg bw in male rats and 25 mg/kg bw in female rats.
Executive summary:

Repeated Dose 90 Days Oral Toxicity Study of the test item in Wistar rats was performed in compliance with the Organization for Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals (No. 408, Section 4: Health Effects) on conduct of ‘Repeated Dose 90-Day Oral Toxicity Study in Rodents’ (Adopted: 21 September, 1998) and in accordance with the Study Plan (Study No. P/15666/SOR-90/15).

Groups of ten male and ten female Wistar rats were administered the test item by oral gavage daily at the doses of 6.25, 12.5, 25 and 50 mg/kg body weight for 90 days and were sacrificed on day 91 and / or 92 to evaluate its toxicity.

A concurrent control group of ten males and ten females receiving the vehicle, i.e. corn oil at 5 ml/kg was also maintained for 90 days.

Additionally, groups of five rats per sex which had received the vehicle at 5 ml/kg and the test item at 50 mg/kg body weight, were further observed for a period of 28 days following the 90 days treatment, for assessment of reversibility, persistence or delayed occurrence of toxicity.

The rats were examined daily for signs of toxicity, morbidity and mortality. They were subjected to detailed clinical examination before initiation of the study and weekly thereafter during the treatment period, recovery period. Ophthalmoscopic examination was conducted on all the animals before initiation of the treatment and on vehicle control and 50 mg/kg dose group animals at termination of treatment. In the thirteenth week of treatment, animals were additionally examined for assessment of sensory reactivity, assessment of grip strength and motor activity. Body weight and food consumption were recorded weekly. Laboratory investigations were performed on blood and urine at termination of the treatment and at the end of recovery period. All surviving animals sacrificed terminally were subjected to a detailed necropsy and weights of kidneys, liver, adrenals, testes, epididymides, uterus, thymus, spleen, brain, ovaries and heart were recorded. Histopathological evaluation was performed on all tissues : [brain, spinal cord, eyes, pituitary, thyroid, parathyroid, spleen, thymus, adrenals, pancreas, trachea, lungs, heart, aorta, oesophagus, stomach, duodenum, jejunum, terminal ileum (with peyer’s patch), colon, rectum, liver, kidneys, urinary bladder, prostate, seminal vesicle, epididymides, testes, ovaries, uterus with cervix, skin, vagina, sciatic nerve, bone marrow (smear), mammary gland (females), mesenteric lymph node, axillary lymph node and salivary glands] in all rats from the vehicle control, 25 mg/kg and 50 mg/kg dose groups. Animals found dead during the course of the study were also subjected to detailed necropsy and histopathological evaluations of aforementioned tissues.

 

There was no incidence of any treatment related mortality amongst the rats treated with test article at and upto the dose level of 12.5 mg/kg in both the sexes and at 25 mg/kg in females. However in males, at the dose of 25 mg/kg 2/10 rats were found dead on days 36 and 89, at the dose of 50 mg/kg 8/15 rats were found dead on days 14, 18, 25, 34, 42 and 64. In females at the dose of 50 mg/kg 2/15 rats were found dead on days 31 and 36. No mortality was observed in vehicle control group. During the subsequent recovery period of 28 days there was no incidence of any mortality in vehicle control and 50 mg/kg dose group.

The daily cageside clinical examination and weekly detailed examination of female rats at and upto the dose level of 25 mg/kg body weight and the vehicle control group did not reveal any remarkable treatment related clinical signs. However, at the dose level of 50 mg/kg dose group, daily general clinical examination revealed some treatment related clinical signs in few animals, which includes blackish discoloration of the feces and gasping in both the sexes. Whereas, salivation and bluish discoloration of perianal region were observed only in some of the male rats. In male rats there were single incidences each of diarrhoea and gasping at the dose level of 25 mg/kg and hypoactivity at the dose level of 12.5 mg/kg. Blackish discoloration of feces was observed in few animals in all the test article treated male rats. These incidences of blackish discoloration of feces and subsequent perianal region in the treated rats can be attributed to the dark black color of the test article.

Localized alopecia observed in one male rat each at the dose levels of 6.25 and 12.5 mg/kg and one female rat at the dose level of 25 mg/kg, was considered to be incidental findings.

Ophthalmological examination did not reveal any treatment related ocular abnormalities. Also, the observations on sensory reactivity, grip strength and motor activity conducted in the thirteenth week of treatment did not reveal any neurotoxic potential of the test article.

Body weight gain of surviving rats was not affected in male and female rats treated at and up to the dose of 50 mg/kg and were found to be comparable to that by the vehicle control group throughout the treatment period and also during the recovery period.

The test article did not have any adverse effect on the average daily food consumption by the surviving male and female rats treated at any of the dose levels.

The haematological parameters of haemoglobin, packed cell volume, total RBC count, total and differential WBC counts, absolute RBC indices, platelet count, activated partial thromboplastin time and prothrombin time of male and female rats treated with the test article were found to be comparable to those of the vehicle control animals at termination of the treatment and also at the end of the recovery period.

 

The test article did not alter the plasma levels of total protein, albumin, globulin, alanine aminotransferase, asparate aminotransferase, alkaline phosphatase, glucose, creatinine, calcium, total cholesterol, phosphorous, total bilirubin, blood urea nitrogen, urea, sodium, potassium and triglycerides in both the sexes.

The data on urinalysis indicated no adverse effect due to the treatment.

The values of absolute and relative weights of kidneys, liver, adrenals, testes, epididymides, uterus, thymus, spleen, brain, ovaries and heart of male or female rats treated with test article, were found to be comparable with those of the vehicle control group at the end of treatment period and also at the end of the recovery period.

No treatment related gross pathological changes were noted. However in most of the found dead animals there was presence of black colored ingesta in stomach and intestines. This was not considered to be treatment related and was attributed to the discoloration due to the color of the test article. Histopathological examination was performed on all enlisted tissues of the vehicle control, 25 mg/kg and 50 mg/kg dose group animals, where the changes in 25 mg/kg and 50 mg/kg dose group were incidental or comparable to the vehicle control group and not the effect of test article. The histopathological changes observed in found dead animals were not related to treatment.

Based on the findings of this study, the No-Observed-Adverse-Effect-Level (NOAEL) of the test item in Wistar rats, following oral administration for 90 days was found to be 12.5 mg/kg body weight in male rats and 25 mg/kg body weight in female rats.  

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
12.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Good quality database.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Sub-acute repeated toxicity test

During the test, the adverse effects observed are a statistical significant decrease in the body weight in the reversal group at 100 mg/kg bw (highest tested dose) and in G4 (75 mg/kg/day) and G5 (100 mg/kg/day) males on day 28 as compare to their respective control. In females this decrease was statistically different in high dose (G5) on Premating day 14 and gestion day 0 as compared to control.

Animals of G4 (75 mg/kg/day), G5 (100 mg/kg/day) & G7 (100 mg/kg/day reversal group) exhibited dullness and piloerection at various intervals and statistically significant decrease in grip strength and locomotor activity of G4, G5 and G7 groups as compare to their respective control groups.

Mortality observed in the highest tested group (i.e. 100 mg/kg bw), one male in G5, one male in G7 and one female in G5 are considered as treatement related, but without any modification of the haematological or biochemical parameters.

No specific organs could be identified.

Sub-chronic repeated toxicity test

During the test, the adverse effects observed are blackish discoloration of feces and subsequent perianal regions in the treated rats, which is attributed to the dark black color of the test item.

Other clinical treatment related signs was gasping in both the sexes, and salivation in some of the male rats. In male rats, there were sigle incidences of diarrhea and gasping at 25 mg/kg bw and hypoactivity at 12.5 mg/kg bw. Localized alopecia observed in one male rat each at the dose of 6.25 mg/kg bw and 12.5 mg/kg bw and on female at the dose of 25 mg/kg bw was considered to be incidental findings.

Mortality observed in the highest tested group, i.e. 50 mg/kg bw (eight males and two females) and at 25 mg/kg bw (two males) is considered as treatement related, but without any modification of the haematological or biochemical parameters. During the recovery period, there was no incidence of any mortality at 50 mg/kg bw.

No specific organs could be identified.

Selection of repeated dose toxicity via oral route - systemic effects endpoint:

The NOAEL from the 90-days repeated dose toxicity study of 12.5 mg/kg bw/day was selected.

Justification for classification or non-classification

As stated above, no specific organs can be identified in the subacute and subchronic studies performed. So Solvent Black 46 is not classified for any specific target organ toxicity at repeated doses.