α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Repeated Dose 28-Days Oral Toxicity Study of test chemical in Sprague Dawley (SD) rats.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (Solvent Blue 4) (containing less than 0.1% Michler’s Ketone)

IUPAC Name:

α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (Solvent Blue 4) (containing less than 0.1% Michler’s Ketone)

Test material form:

solid

Details on test material:

- Name of test material: 1-Naphthalenemethanol, .alpha.,.alpha.-bis[4-(dimethylamino)phenyl]-4-(phenylamino)-
- Molecular formula: C33H33N3O
- Molecular weight: 487.644 g/mol
- Smiles notation: N(c1ccc(cc1)C(c1ccc(N(C)C)cc1)(c1c2c(c(Nc3ccccc3)cc1)cccc2)O)(C)C
- InChl: 1S/C33H33N3O/c1-35(2)27-18-14-24(15-19-27)33(37,25-16-20-28(21-17-25)36(3)4)31-22-23-32(30-13-9-8-12-29(30)31)34-26-10-6-5-7-11-26/h5-23,34,37H,1-4H3
- Substance type: Organic
- Physical state: solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Details on test animal
TEST ANIMALS
- Source: Central Animal Facility (CAF), NIPER, Sector-67, S.A.S. Nagar, 160 062, Punjab, India.

- Age at study initiation: 7 to 8 weeks old

- Weight at study initiation: Male: 196.26 - 241.83 g, Female : 182.80-213.04 g

- Fasting period before study: A fast of 2h before chemical administered.
- Housing: Animals were housed in sterilized solid bottom polypropylene cages with stainless steel grill tops with facilities for food, water bottles and bedding of clean paddy husk. The cages were suspended on stainless steel racks. Animals were identified by assigning a unique identification (ID) number written on the tail, also specified on individual cage tag.

- Diet (e.g. ad libitum): Standard laboratory sterile extruded pelleted rodent feed (Batch No. 0001642169) manufactured by Provimi Animal Nutrition India Pvt. Ltd., Bangalore, ad libitum.

- Water (e.g. ad libitum): Potable tap water filtered through Reviva Reverse Osmosis System (water filter cum purifier) of Eureka Forbes in polypropylene bottles with stainless steel sipper tubes. ad libitum.

- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3o C
- Humidity (%):30-70 %,
- Air changes (per hr): 25 ± 5 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: Males - From: 20.12.2014 to 16. 01.2015
Females: 22.12.2014 to 18.01.2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: Dose of the test item was freshly prepared prior to dosing on each day. The test item Solvent blue 4 (SBL) was administered to each rat at the dose levels of 50, 150 or 450 mg/kg in the dose volume of 10 ml/kg body weight. The test item was weighed on a weighing balance. Then,it was transferred to calibrated falcon tube. Some quantity of the corn oil was added initially and vortexed. The sufficient quantity of vehicle was added to make up the required volume of dose.


DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): No data available

- Storage temperature of food: No data available

VEHICLE
- Justification for use and choice of vehicle (if other than water): Chemical was insoluble in water, the corn oil was used as vehicle to deliver the desired dose levels as it is also recommended in the toxicological evaluation guidelines.

- Concentration in vehicle: 0, 50, 150 or 450 mg/kg body weight /day
- Amount of vehicle (if gavage): 10 ml/kg body weight
- Lot/batch no. (if required): No data available
- Purity: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Solubility of the test item was checked by visual inspection method, Homogeneity was tested for a portion from each layer, i.e. upper layer (between 10-8 ml marks), middle layer (between 6-4 ml marks) and bottom layer (between 2ml and bottom of the tube) was taken and tested by UV-spectroscopy and concentration of test substances was calculated by using the absorbance of the standard concentrations of the test chemical

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

0, 50, 150 or 450 mg/kg/body weight/day

No. of animals per sex per dose:

Total: 56
0 mg/kg/day: 7 male, 7 female
50 mg/kg/day: 7 male, 7 female
150 mg/kg/day: 7 male, 7 female
450 mg/kg/day: 7 male, 7 female

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule : Twice daily (morning and evening)
- Cage side observations checked in table [No.?] were included. : Mortality and morbidity were observed

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily for behavioral changes or reaction to treatment and detailed clinical signs were recorded weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: On day 1, 8, 15, 22, 28, and day 29 (before schedule sacrifice)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, weekly
- 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: No data available

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Weekly once during 28 days of treatment.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: In fourth week of treatment.

- Dose groups that were examined: All surviving animals were examined.

HAEMATOLOGY: Yes
Yes
- Time schedule for collection of blood: Blood was collected on completion of 28 days of treatment and prior to necropsy.
- Anaesthetic used for blood collection: Yes, slight anaesthesia used (identity not mentioned
- Animals fasted: Animals were fasted overnight.
- How many animals: All surviving male and female rats from each group.
- Parameters checked in table [No.?] were examined: Haemoglobin (Hb), RBC Count
Total and differential leucocyte count (TLC / DLC), Haematocrit (Hct / PCV), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC) and Platelet count were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected on completion of 28 days of treatment and prior to necropsy.
- Animals fasted: Animals were fasted overnight.
- How many animals: All surviving male and female rats from each group.
- Parameters checked in table [No.?] were examined: Sodium and Potassium, Glucose,
Total cholesterol, Blood urea, Creatinine, Total protein, Albumin, SGPT (Serum glutamic pyruvic transaminase) /ALT
SGOT (Serum glutamic oxaloacetic transaminase) /AST, Hormones analysis (testosterone and estrogen) and Total bile acids were examined.


URINALYSIS: No data available
- Time schedule for collection of urine: No data available
- Metabolism cages used for collection of urine: No data available
- Animals fasted: No data available
- Parameters checked in table [No.?] were examined.: No data available

NEUROBEHAVIOURAL EXAMINATION: No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Yes, locomotor activity was examined.

OTHER: Absolute and relative organ weight was measured.

Sacrifice and pathology:

Sacrifice and pathology
GROSS PATHOLOGY: Yes
A complete necropsy was carried out on all animals. Tissues were collected from Brain, Stomach, Large intestine, Small intestine, Liver, Kidneys, Adrenal gland(s), Spleen, Heart, Thymus, Lungs, Testis/Ovary, Uterus, Lymph nodes, Peripheral nerve (Sciatic), Bone marrow and Gross lesions, if any.
Tissues were preserved in 10% formal saline. However, testes, ovaries and uterus were first fixed in Bouin’s fixative for short duration then transferred to 10%
formal saline.


HISTOPATHOLOGY: Yes
Histological examination was conducted on tissues/organs from the control and the low-dose group animals.
The required tissues for histology were cut and stained with haematoxylin and eosin.
The histological examination was based on double blind analysis by using Olympus Trinocular Microscope, (Model BX-51) at magnification of 10x, 20x and 100x.

Organs examined:
Lung, Liver, Kidney, Heart, Spleen, Testis, Ovary, Adrenal gland, Large intestine, Brain, Sciatic nerve, Lymph nodes, Bone marrow, Stomach, Thymus and Small intestine were examined.

Statistics:

Statistical analysis was carried out by using Microsoft Excel and IBM SPSS statistics version- 20.0. All analyses and comparisons were evaluated at the 5 % level, statistically significant differences (p ≤ 0.05) indicated by appropriate notation. The focus was to examine the mean differences and their significance in control vs low-dose group only as there was total mortality observed in mid and high-dose groups in both the sexes. The statistical decision was taken by preparing the univariant GLM MODEL to check the significance between above mentioned groups. For multiple comparisons Turkey’s HSD test was applied.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

When treated with 450 mg/kg/body weight/day, all the male and female were dead on day 5 as compare to control. When treated with 150 mg/kg/body weight/day, all the male and female were dead on day 11 as compare to control. When treated with 50 mg/kg/body weight/day, all the male and female were survived throughout the treatment period as compare to control.

Mortality:

mortality observed, treatment-related

Description (incidence):

When treated with 450 mg/kg/body weight/day, all the male and female were dead on day 5 as compare to control. When treated with 150 mg/kg/body weight/day, all the male and female were dead on day 11 as compare to control. When treated with 50 mg/kg/body weight/day, all the male and female were survived throughout the treatment period as compare to control.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight: When treated with 50 mg/kg/body weight/day significant decrease was observed in treated male and female rat as compare to control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No change was observed in food consumption of treated male and female rat as compare to control.

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 abnormalities were found in the ophthalmological examination of 50 mg/kg/body weight /day dose group male and female as compare to control.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

When treated with 50 mg/kg/ body weight /day in male rat RBC count and percentage of monocytes increased significantly and no significant changes were observed in female rat as compare to control

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

When treated with 50 mg/kg/ body weight /day in male rat potassium and total proteins level were significantly increased and in female rat significant decrease were observed in the total bile acid level as compared to the control.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In male rats, relative organ weight of brain was significantly increased and no changes were observed in female organ weight when treated with 50 mg/kg/body weight/day as compare to control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

When treated with 450 mg/kg/body weight/day perineum stained bluish in color, alimentary canal including stomach and intestine stained bluish in color, enlargement of atria in heart, lung congestion and marked thinning of fore stomach wall observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No significant changes were observed in 50 mg/kg/body weight/day treated male and female rat as compare to control.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

Dose descriptor:

LOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

Remarks on result:

other: dose related effects observed

Target system / organ toxicity

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Applicant's summary and conclusion

Conclusions:

LOEL for test chemical was considered to be 50 mg/kg body weight /day when administered orally by gavage.

Executive summary:

In a 28 days repeated dose toxicity study, the effect of test chemical (containing less than 0.1% Michler's Ketone) was evaluated in male and female Sprague Dawley rats .TheSprague Dawley (SD) rats of 7-8 weeks old were divided into four groups of each sex and each group comprised of either 7 males or 7 females. Test chemical was administered at the dose levels of 50, 150 and 450 mg/kg/day for 28 consecutive days by oral route. Male animals were divided into four groups, group 1 served as control and group 2, 3 and 4 served as low, mid and high-dose group respectively. Female animals were divided into four groups, group 5 served as control and group 6, 7 and 8 served as low, mid and high-dose group respectively. Corn oil was used as vehicle for the present study.

In 450 mg/kg/day dose treatment group in male, the mortality was observed on the day 3, 4, 5, whereas in the 150 mg/kg/day dose treatment group in male rats the mortality was observed in the day 4, 5, 7, 8, and 11. In 450 mg/kg/day dose treatment group in females the mortality was seen on day 3, 4 and 5 and in the 150 mg/kg/day –dose treatment group in female animals’ mortality was found on day 5, 6, 7, 8, and 10 after the administration of test chemical. At the end of the 28 days study only male and female animals of control and 50 mg/kg/day dose group survived up to the completion of the treatment period. During the treatment period, detailed clinical signs, body weight, food and water consumption were recorded weekly. In the groups survived after 28 days of treatment, no abnormalities were found in the ophthalmological examination (control vs. low-dose treated group). However, a significant decrease was observed in the motor activity of treated female animals as compared to the control animals. After the completion of dosing period, blood was collected from all the surviving animals for hematology and clinical biochemistry analysis. All survived animals were sacrificed on the day of

study termination and gross lesions as well as the weight of different organs were recorded. All organs were fixed in 10 % formal saline, except the reproductive organs, which were fixed in Bouin’s fixative for short duration and thereafter shifted to 10 % formal saline.

In the terminally sacrificed and the animals died during treatment, the necropsy findings that were common to all the dosed treated groups include bluish stained perineum and alimentary canal which include stomach and intestine. The finding of stomach blotted with gastric content was common to all the treated group male animals and in one female animal of 450 mg/kg/day dose treated group. Mild blue tinged patches were observed in 50 mg/kg/day dose treated male rats. The enlargement of atria in heart, lung congestion and marked thinning of fore stomach wall was confined to 150 mg/kg/day and 450 mg/kg/day dose groups of both the sexes as observed after mortality. White / pale patches on liver / liver congestion / dark reddish liver were confined to 450 mg/kg/day dose group of male and female animals during the time of gross necropsy after mortality. In male rats, the relative weight of brain was significantly increased in 50 mg/kg/day -dose group as compared to control group. Hematology of male animals showed a significant elevation in the red blood cell count as well as the percentage of monocytes in the 50 mg/kg/day dose group as compared to control animals.

In male rats, significant elevation in the levels of potassium and total proteins was noticed with 50 mg/kg/day dose treatment as compared to control animals. In female rat, significant decrease in the level of total bile acid was noticed in the 50 mg/kg/day dose treated animals as compared to control animals. In the microscopic histopathological study there was no significant change observed in 50 mg/kg/day dose group animals as compared to control animals. Few microscopic findings were observed in control

as well as in 50 mg/kg/day dose treated animals, which include excess of lymphocytes in bronchi and alveoli of lungs, ileum and colon. These types of findings may be considered within the range of normal background lesions, which are seen in rats with the age groups used in the present study. Further, these types of changes were considered incidental in nature with carbon dioxide inhalation.Hence LOEL for test chemical was considered to be 50 mg/kg body weight /day when administered orally by gavage.