Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

EC Number:

276-538-7

EC Name:

Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Cas Number:

72252-58-5

Molecular formula:

C23H8Cr2N6O16S3.4Na

IUPAC Name:

tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana

- Females (if applicable) nulliparous and non-pregnant: [Yes]
- Age at study initiation: 6-7 weeks
- Weight at study initiation: Males: 178.25 to 207.13g ; Females: 138.86 to 160.73g

- Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in each group and sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week.
- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet – Pellet (Certified) manufactured by Envigo, P.O.Box 44220, Madison WI 53744-4220, was provided ad libitum to the rats.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: Start: 04 November 2016 End: 08 November 2016


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 and 24°C
- Humidity (%): 56 to 67 %
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 09 November 2016 To: 06 December 2016

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 3 hours) each day for a period of 28 consecutive days. Similarly, the vehicle was administered to rats in vehicle control/vehicle control recovery group once orally for 28 consecutive days.
The vehicle or the dose formulations were not administered to recovery groups for 14 days following the 28-day treatment period.
The dose formulation and the vehicle were administered at an equivolume of 10 mL/kg/day. The dose volume was calculated for individual animals on the first day of the treatment period and was adjusted according to the most recent body weights recorded during the treatment period.

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.1% carboxymethyl cellulose sodium salt (low viscosity) in Milli-Q® water was used as vehicle for dose formulation preparation as the test item forms a suspension in the vehicle and also the same vehicle was used in the dose range finding toxicity study (Study No. N3062).
- Concentration in vehicle: 1.0 g of Sodium carboxy methyl cellulose was added to about 900 mL of Milli-Q® water in a 1000 mL pre-marked beaker* and stirred on a magnetic stirrer. The final volume was made up to the mark with Milli-Q® water. After obtaining an uniform suspension, the suspension was stored at room temperature in the experimental room.
- Amount of vehicle (if gavage): 10 mL per kg body weight
- Lot/batch no. (if required): 051M0191V

STABILITY OF TEST MATERIAL
- Storage condition of test material: Ambient (+15 to +25°C)
- Stability under test conditions: The stability and homogeneity of the test item in the vehicle was established at 1 and 100 mg/mL under Advinus Study No. G11857. Based on the results, the test item was stable and homogenous in the vehicle up to 48 hours when stored at room temperature.
- Solubility and stability of the test substance in the solvent/vehicle: 0.1% carboxymethyl cellulose sodium salt (low viscosity) in Milli-Q® water was used as vehicle for dose formulation preparation as the test item forms a suspension in the vehicle and also the same vehicle was used in the dose range finding toxicity study (Study No. N3062).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during week 4 of the treatment period and analysed in-house. For each set, duplicate sample was drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Advinus Study No. G11857. One set of samples were analysed for concentration (a.i) analysis.
Dose formulations were considered acceptable as the overall mean results were within ± 15.0% of the theoretical concentration and the overall relative standard deviation (RSD) was less than 10.0%.
Sample prepared on 09 November 2016 was initially analysed on 10 November 2016, however due to poor chromatography results were not acceptable. Hence back up samples were analysed on 11 November 2016.

Duration of treatment / exposure:

The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 3 hours) each day for a period of 28 consecutive days. Similarly, the vehicle was administered to rats in vehicle control/vehicle control recovery group once orally for 28 consecutive days.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex per group

Control animals:

yes

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): Animals were randomly distributed to different groups by body weight stratification method using ProvantisTM software. Rats with extreme body weights were discarded. Grouping was done one day prior to start of treatment during acclimatization.

- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment, at the end of the treatment period for main groups and at the end of recovery period for recovery groups. Before examination, mydriasis was induced using a 1 % solution of Tropicamide.
- Dose groups that were examined: 0, 100, 300 and 1000 mg/kg per body weight

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery periods
- Anaesthetic used for blood collection: Yes (Isoflurane anaesthesia)
- Animals fasted: Yes
- How many animals: 60 (30 males + 30 females)

- Parameters checked in [Section 8.7.2 in main report] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery periods
- Animals fasted: Yes
- How many animals: 60 (30 males + 30 females)
- Parameters checked in [Section 8.7.4 in main report] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the treatment and recovery periods
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters checked in [Section 8.8 in main report] were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Section 8.9 in main report)

HISTOPATHOLOGY: Yes (Section 8.10 in main report)

Statistics:

Data captured using Provantis™ for the parameters body weight and organ weights; laboratory Investigations - Haematology (Coagulation tests PT and APTT which were entered retrospectively in ProvantisTM) and Clinical Chemistry were analyzed using built-in statistical tests.
Derived data like net body weight change, food consumption and organ weight ratios were also be analyzed using above mentioned methods.
The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights) were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA is performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test is found to be significant
The data pertaining to males and female rats was evaluated separately.
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated by the following symbols throughout the report:
+/-: Significantly higher/lower than the respective control group

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs observed both during the treatment and recovery period at all the treated groups in both sexes. However, the grey coloured faeces at 100 mg/kg bwt/day and black coloured faeces at 300 and 1000 mg/kg bwt/day doses in both sexes were observed during the treatment period. The colored feces was attributed to the nature of the test item and not an adverse findings.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities observed both during the treatment and recovery period at all the treated groups in both sexes.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and net body weight gains were not significantly different from the vehicle control group at all the tested doses in both sexes during the treatment and recovery period

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The mean food consumption was not altered by the treatment in any of the tested doses either during the treatment or recovery period in both sexes. However, incidence of significantly higher food intake was observed during Days 8-15 of treatment in the high dose recovery group. This change was considered incidental because of isolated occurrence.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmological examination was carried out with an ophthalmoscope prior to start of treatment, at the end of the treatment and recovery period did not reveal any abnormalities in the eyes of the experimental rats.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes observed in the haematological parameters analysed across the groups in both male and female rats.

All statistical significances observed in haematology parameters were considered incidental in nature, as the changes were of minimal magnitude and not dose progressive.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes observed in the biochemical parameters analysed across the groups in both sexes.
All differences observed in biochemical parameters between vehicle control and treatment groups, including the changes that reached statistical significance were considered as incidental due to lack of dose progression and/or the changes were of minimal magnitude.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related changes were observed in any of the urine parameters analyzed in both male and female rats.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage and Handling observations: No treatment-related abnormalities were observed in all the tested dose groups in both sexes.
Open field observations: No treatment-related abnormalities were observed in any of the doses tested in both sexes.
Sensory observations: No treatment-related abnormalities were observed in any of the groups in both sexes.
Motor Activity: The following statistically significant variations were observed in the motor activity of rats when compared to respective vehicle control group:
Males:
Lower: Stereotypic time at interval 1, 2, 3 and total stereotypic time, ambulatory time at interval 1, 2, 3 and total ambulatory time, horizontal counts at interval 1 and 3, total horizontal counts, ambulatory counts at interval 1 and 3 and total ambulatory counts at the high dose in the main toxicity group.

Females:
Higher: Ambulatory time at interval 1, 2 and total ambulatory time, horizontal counts at interval 1, 2 and total horizontal counts, ambulatory counts at interval 1 and total ambulatory counts in the high dose recovery group.

Lower: Ambulatory time, horizontal counts and ambulatory counts at interval 3 at the high dose in the main toxicity group.
The above observed statistical variations in the motor activity measurements are considered to be incidental as the there were no changes observed in the home cage or open field observations. Further there were no clinical signs observed during daily clinical observation.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in terminal fasting body weight, organ weights and their ratios in both male and female rats.
In males, at the end of recovery period the increase in absolute and relative weights of adrenals as well as seminal vesicles and coagulating glands at 1000 mg/kg/day were considered as incidental findings as similar changes were not observed at the end of treatment period.
All other changes in weights observed were considered incidental and not related to test item administration due to minimal magnitude and/or lack of dose progression.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Grossly, gray/green discoloration of kidneys at ≥ 300 mg/kg/day in both sexes, green discoloration of mandibular lymph nodes and mesenteric lymph node at ≥300 mg/kg/day in both sexes, gray/green discoloration of liver lobes at 1000 mg/kg/day in both sexes, multifocal green discoloration of glandular mucosa of stomach at 1000 mg/kg/day in both sexes, gray discoloration of testes at 1000 mg/kg/day in males, focal green discoloration of lungs at 1000 mg/kg/day in females and green intestinal content at ≥100 mg/kg/day in both sexes were observed at the end of treatment period.

At 1000 mg/kg/day, the test item-related changes observed in kidneys, liver, mandibular lymph nodes, mesenteric lymph node and testes persisted at the end of recovery period. In lungs, the finding was observed only at the end of treatment period in females whereas in males, the finding was observed only at the end of recovery period. In stomach, the finding was observed at the end of treatment period in males whereas in females, the finding was observed at the end of treatment and recovery period.

All these gross pathological changes were attributed to the physical appearance of test item and considered as test item-related non-adverse change.
Grossly observed dilated pelvis in kidneys and dilated uterus with cervix were considered as incidental changes and not related to test item administration.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopically dark deposition in lamina propria of jejunum at 1000 mg/kg/day in both sexes, pigment macrophages in lungs at 100 mg/kg/day in males and at ≥ 300 mg/kg/day in females, dark deposits in mandibular lymph nodes at ≥ 300 mg/kg/day in both sexes and dark deposits in mesenteric lymph nodes at ≥ 300 mg/kg/day in males and at 1000 mg/kg/day in females were observed at the end of treatment period. Dark deposits in lamina propria of jejunum (males and females), pigment macrophages in lungs (males) and dark deposits in mandibular and mesenteric lymph nodes (males and females) were also observed at 1000 mg/kg/day dose at the end of recovery period.
These changes were characterized by grayish to greenish coloured deposition either in lamina propria of jejunum or in macrophages of lungs, mandibular and mesenteric lymph nodes without any inflammatory reaction. These changes were attributed to the physical appearance of test item and considered as test item-related non-adverse change.
All other microscopic findings observed were considered incidental as the findings were distributed randomly across the groups and the incidences in treatment groups were similar to control groups.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

<= 1 000 mg/kg bw/day (nominal)

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

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

other: histopathology

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

To summarise, daily administration of test item to Wistar rats by oral gavage at the dose levels of 100, 300 and 1000 mg/kg/day did not cause any adverse clinical signs or mortalities. There were no test item-related changes in body weights, net body weight gains, food consumption and neurological findings. There were no test item-related changes observed in haematological, coagulation, clinical chemistry and urine parameters. Terminal fasting body weight and organ weights/their ratio were not affected in both sexes.
Grossly, green color intestinal content was observed in both sexes and microscopically single incidence of pigment macrophages in lungs was observed in male rats at 100 mg/kg/day. At 300 mg/kg/day, grossly, gray discoloration of kidneys, green discoloration of mandibular lymph nodes and mesenteric lymph node and green color intestinal content were observed in both sexes. Microscopically pigment macrophages in lungs in females, dark deposits in mandibular lymph nodes in both sexes and pigment deposits in mesenteric lymph nodes in males were observed. At 1000 mg/kg/day, grossly, gray/green discoloration of kidneys and liver lobes in both sexes, green discoloration of mandibular lymph nodes and mesenteric lymph node in both sexes, multifocal green discoloration of glandular mucosa of stomach in both sexes, gray discoloration of testes in males, focal green discoloration of lungs in females and green color intestinal content in both sexes were observed at the end of treatment period. These gross changes persisted in male and/or female except green intestinal content at the end of recovery period. Microscopically dark deposition in lamina propria of jejunum in both sexes, pigment macrophages in lungs in females, dark deposits in mandibular lymph nodes and mesenteric lymph nodes in both sexes were observed at the end of treatment period. At the end of recovery period dark deposition in lamina propria of jejunum (males and females), pigment macrophages in lungs (males) and dark deposits in mandibular and mesenteric lymph nodes (males and females) were also observed.
All the above gross and microscopic changes (without any inflammatory response) observed in different groups were attributed to the physical appearance of test item and considered as test item-related non-adverse changes
Hence, the evaluated No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg/day following oral gavage administration for 28 consecutive days to wistar rats under the test conditions and doses employed.

Executive summary:

The purpose of this repeated dose toxicity study was to evaluate the systemic toxicity profile of the test item in wistar rats when administered orally by gavage for 28 consecutive daysand also to assess the reversibilityof any effects following 14days recovery period. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a No Observed Adverse Effect Level (NOAEL).

The test item was weighed and suspended in vehiclei.e.,0.1% Carboxymethylcellulose Sodium salt (low viscosity) in Milli-Q^®water and administered to rats at the graduated dose levels of 100, 300, and 1000 mg/kg/day to low (G2), mid (G3), and high (G4)/ high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/ vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 10 mL/kg body weight. Each group in the experiment was comprised of five male and five female rats.

Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and and at the end of recovery period for recovery groups. The body weights and food consumption was measured during the course of the in-life phase. Neurological examinations were conducted towards the end of treatment for main groups and towards the end of recovery period for recovery groups.The clinical laboratory investigations such as haematology, coagulation, clinical chemistry and urine analysis were performed at termination.

All toxicity group rats in the experiment were subjected to detailed necropsy and the organ weights and their ratio were derived as percent fasting body weights. Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose group animals (G4). In addition, all gross lesions from all the animals were examined microscopically. Further, jejunum, lungs, mandibular lymph nodes and mesenteric lymph node showing test item-related histopathological changes in high dose (G4) group were examined in the respective lower dose groups and recovery groups.

Salient findings are provided below:

· Clinical Signs, Mortalities and Ophthalmological Examination:No clinical signs or mortalities or ocular abnormalities were observed throughout the experimental period.

· Neurological Findings:No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.

· Body Weights:The mean body weights were unaffected by the treatment in both the sexes at all the tested doses in both sexes.

·  Food Consumption:There were no significant differences observed in the food consumption at all the doses tested in both sexes throughout the experimental period.

·  Haematology, Coagulation, Clinical chemistry and Urine Parameters:No test item-related changes were observed in the haematological, coagulation, clinical chemistry and urine parameters.

·  Organ Weights:The terminal fasting body weights, organ weights and their ratios were not affected by test item administration.

Gross and Histopathology: At 100 mg/kg/day, grossly, green colored intestinal content was observed in both sexes at the end of treatment period. Microscopically single incidence of pigment macrophages in lungs was observed in male rats.

At 300 mg/kg/day, gray discoloration of kidneys, green discoloration of mandibular lymph nodes and mesenteric lymph node and green colored intestinal content were observed in both sexes. Microscopically pigmented macrophages in lungs in females, pigment deposits in mandibular lymph nodes in both sexes and pigment deposits in mesenteric lymph nodes in males were observed.

At 1000 mg/kg/day, grossly, gray/green discoloration of kidneys and liver lobes in both sexes, green discoloration of mandibular lymph nodes and mesenteric lymph node in both sexes, multifocal green discoloration of glandular mucosa of stomach in both sexes, gray discoloration of testes in males, focal green discoloration of lungs in females and green color intestinal content in both sexes were observed at the end of treatment period. These gross changes persisted in males and/or females except green colored intestinal content at the end of recovery period. Microscopically pigment deposition in lamina propria of jejunum in both sexes, pigment macrophages in lungs in females, pigment deposits in mandibular lymph nodes and mesenteric lymph nodes in both sexes were observed at the end of treatment period. Pigment deposition in lamina propria of jejunum (males and females), pigment macrophages in lungs (males) and pigment deposits in mandibular and mesenteric lymph nodes (males and females) were also observed at the end of recovery period.

All the above gross and microscopic changes (without any inflammatory response) observed in different groups were attributed to the physical appearance of test item and considered as test item-related non-adverse changes.

Hence, the evaluated No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg/day following oral gavage administration for 28 consecutive days to wistar rats under the test conditions and doses employed.