Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity: Oral
The low observed adverse effect level (LOAEL) was considered to be 50 mg/kg /day when Sprague Dawley rats were exposed daily to Solvent blue 4 (SBL) by oral route for 28 days.
Repeated dose toxicity: Inhalation
This end point was considered for waiver since exposure of humans via inhalation is highly unlikely taking into account the very low vapour pressure of α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (Solvent Blue 4). The vapour pressure value is 0.00000000000000267Pa at 25 deg C.
Particle size of this chemical is in the micrometer range and thus exposure to inhalable particulates (mostly nano sized) also appears unlikely. Also, workers in industry are normally mandated to wear personla protective equirments (like masks). Thus, this exposure is also unlikely and therefore based on exposure considerations; this end point was considered for waiver.
Repeated Dose toxicity: Dermal
Acute dermal toxicity study for Solvent Blue 4 indicates LD50 > 2000 mg/kg bw. Thus, it is considered that the chemical is unlikely to exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
Repeated Dose 28-Days Oral Toxicity Study of Solvent Blue 4 (SBL CAS No. 6786-83-0) in Sprague Dawley (SD) rats.
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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
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 solvent blue.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 50, 150 or 450 mg/kg/body weight/day
Basis:
actual ingested
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
Details on study design:
Details on study design
- Dose selection rationale: No data available
- Rationale for animal assignment (if not random): No data available
- Rationale for selecting satellite groups: No data available
- Post-exposure recovery period in satellite groups: Animals were randomized by body weight.
- Section schedule rationale (if not random): Animals were randomized by body weight.
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.
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):
no effects observed
Description (incidence and severity):
No change was observed in water consumption of treated male and female rat as compare to control.
Ophthalmological findings:
effects observed, treatment-related
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
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
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.
Details on results:
Clinical chemistry: When treated with 50 mg/kg/ body weight /day in male rat potassium and total proteins level were significantly increased, while in female rat a significant decrease were observed in the total bile acid level as compared to the control.
Organ weights: In male rats, relative organ weight of brain was significantly increased while no changes were observed in female organ weight when treated with 50 mg/kg/body weight/day as compare to control.
Gross pathology: 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 and white/pale patches on liver/liver congestion/dark reddish liver in male and female rat and blotted with gastric content were observed in male rat were observed as compare to control.

When treated with 150 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 in male and female and blotted with gastric content were observed in male rat were observed as compare to control. When treated with 50 mg/kg/body weight/day perineum stained bluish in color, alimentary canal including stomach and intestine stained bluish in color in male and female rat, Mild blue tinged patches and stomach blotted with gastric content were observed in male rat as compare to control.
Histopathology: No significant changes were observed in 50 mg/kg/body weight/day treated male and female rats as compare to control.
Dose descriptor:
LOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effect on survival, body weight, food consumptions, locomotor activity, hematology, clinical chemistry, organ weight and gross pathology.
Critical effects observed:
not specified

Low observed effect level (LOEL) forSolvent blue 4 (SBL) was considered to be 50 mg/kg body weight /day when administered orally by gavage.

Conclusions:
LOEL for Solvent blue 4 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 Solvent blue 4 (containing less than 0.1% Michler's Ketone) was evaluated in male and female Sprague Dawley rats. The test chemical was administered by oral gavage in the concentration of 0,50, 150 or 450mg/kg body weight/day. The results showed thatSolvent blue 4in treated rats had nasal discharge, red crust around nostrils and soft feces and vocalization on handling, and decreased body weight were also observed as compare to control. In male rat, there were increased levels of RBC, monocytes, potassium and total protein, while female rats showed decreased level of total bile acid as compare to control.In male rats, the relative organ weight of brain was significantly increased while no changes were observed in females when treated with 50 mg/kg/body weight/day as compare to control. At 50 mg/kg/body weight/day, the results showed a perineum stained bluish in color, alimentary canal including stomach and intestine stained bluish in color in both male and female rats. Mild blue tinged patches and stomach blotted with gastric content were also observed in male rats as compare to control. In addition, histopathology showed no significant changes at treatment with 50 mg/kg/body weight/day as compare to control.

Therefore, LOEL is considered to be 50 mg/kg/day when male and female Sprague Dawley rats were exposed daily toSolvent blue 4by oral route for 28 days.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable without restriction

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Additional information

Repeated dose toxicity: Oral The low observed adverse effect level (LOAEL) was considered to be 50 mg/kg /day when Sprague Dawley rats were exposed daily to Solvent blue 4 (SBL) by oral route for 28 days.

Repeated dose toxicity: Inhalation This end point was considered for waiver since exposure of humans via inhalation is highly unlikely taking into account the very low vapour pressure of α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (Solvent Blue 4). The vapour pressure value is 0.00000000000000267Pa at 25 deg C. Particle size of this chemical is in the micrometer range and thus exposure to inhalable particulates (mostly nano sized) also appears unlikely. Also, workers in industry are normally mandated to wear personla protective equirments (like masks). Thus, this exposure is also unlikely and therefore based on exposure considerations; this end point was considered for waiver.

Repeated Dose toxicity: Dermal Acute dermal toxicity study for Solvent Blue 4 indicates LD50 > 2000 mg/kg bw. Thus, it is considered that the chemical is unlikely to exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
OECD GLP experimental results

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
This end point was considered for waiver since exposure of humans via inhalation is unlikely taking into account the very low vapour pressure of α,α-bis[4-(dimethylamino)phenyl]-4-(phenylamino)naphthalene-1-methanol (Solvent Blue 4). Exposure to inhalable particulates is possible only for the workers in industry (where it is expected that they use the protective equipments where exposure to inhalable particulates is likely). Thus, this exposure also stands negated and therefore based on exposure considerations; this end point was considered for waiver.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Acute dermal toxicity study for Solvent Blue 4 indicates LD50 > 2000 mg/kg bw. Thus, it is considered that the chemical is unlikely to exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Justification for classification or non-classification

Based upon available information and the LOAEL value, it can be concluded that the Solvent Blue 4 with less than 0.1% Michler's Ketone is unlikely to cause toxic effects in low concentrations on repeated exposure