Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity: Oral

No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg body weight, when rats were treated with the given test chemical via oral route.

Repeated dose toxicity: Inhalation

A short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment.7-Diethylamino-4-methylcoumarin (CAS no 91-44-1) has very low vapor pressure of 0.00257 Pa. (1.92765825e-5 mmHg), so the potential for the generation of inhalable vapours is very low, also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore this end point for repeated dose toxicity by inhalation route of exposure was considered for waiver.

Repeated dose toxicity: Dermal

A short-term toxicity study does not need to be conducted because exposure of humans via dermal in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment. Also, the acute dermal toxicity value for test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. Given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that test chemical shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that test chemical shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
experimental data from various test chemicals
Justification for type of information:
Data is summarized based on the available information from various test chemicals.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: as mentioned below
Principles of method if other than guideline:
WoE report is based on 2 repeated dose toxicity studies i.e. WoE-2 and WoE-3.
GLP compliance:
not specified
Limit test:
no
Species:
other: 2.rat 3.mouse
Strain:
other: 2.wistar 3.SPF rats of the Carworth Farm E strain
Sex:
male/female
Details on test animals and environmental conditions:
2.TEST ANIMALS
- Source: Animals were procured from a CPCSEA approved Vendor [Gentox Bio Services Pvt. Ltd. (CPCSEA Registration No.: 1242/RCBT/S/08/CPCSEA)]
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) x wks; (F1) x wks: 12 - 13 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: Before the animals are brought in, the study room and cages were cleaned and disinfected. During the study, the floor of the experimental room and work tops were swept and mopped with disinfectant solution every day or as on requirement. Cages were cleaned at regular intervals.
A total 2-3 rats/sex were housed in Polycarbonate cages (size 37 [cm] x 21 [cm], height 20 [cm]). Cage rotation was carried out weekly during study period except during mating and during gestation and lactation only for females.
Sterilized corn cob produced from pure corn, dried and free from dust, procured from approved supplier, was used as bedding material. It was renewed as often as necessary to keep the animals dry and clean.
Bedding material of batch No. SPAR-33/2016 and SPAR-34/2016 (Sparconn Life Sciences Bangalore) was used in this study.
All the animals were identified by temporary tail marking with indelible ink and cage cards. Following allocation to the study, each animal was uniquely identified by micro toe pad tattooing and colour coded cage cards labelled with study no., study type, test system, sex, dose, group, animal number, dosing start date, experimental start and completion date. Pups were identified with body marking with permanent non-toxic marker pen
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): conventional laboratory pelleted diet, ad libitum
- Water (e.g. ad libitum): Aqua guard filtered drinking water in bottles was offered ad libitum.
- Acclimation period:7 days and 8 days prior to test item administration for Dose Range Finding Study (DRF) and Main Study respectively.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.00 to 24.90 °C
- Humidity (%): 39.90 to 65.50%.
- Air changes (per hr):12 hours light and 12 hours dark
- Photoperiod (hrs dark / hrs light):Air changes were about minimum 12 times per hour and filtered adequately.
3.no data
Route of administration:
other: 2.oral:gavage 3.oral: feed
Details on route of administration:
2.The Oral route is recommended by the regulatory guideline.
Vehicle:
other: 2.corn oil 3.not specified
Details on oral exposure:
2.PREPARATION OF DOSING SOLUTIONS: The test item was weighed and dissolved in a vehicle (corn oil) to achieve desired concentration of test item. Dose formulation was freshly prepared daily. At the time of dosing, dose formulation was kept on the magnetic stirrer to maintain the homogeneity of test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was used as a vehicle based on the solubility testing. The Stability and homogeneity of the test item in the vehicle was analyzed by validated method.
- Concentration in vehicle: 0, 250, 500 and 1000 mg/kg body weight
- Amount of vehicle (if gavage): 1.0 ml/100g body weight
- Lot/batch no. (if required): Lot nos.: MR020816, A611001 and A1701001
- Purity:
3.Details on oral exposure
PREPARATION OF DOSING SOLUTIONS: No data available

DIET PREPARATION
- Rate of preparation of diet (frequency): Daily
- 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): No data available
- Concentration in vehicle: 0, 0.1, 0.3 and 1 % (equivalent to 0, 150, 500 and 1500 mg/kg body weight/day)
- Amount of vehicle (if gavage): No data available
- 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:
2.The analytical method for the determination of 2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3yl]benzoxazole-5-sulphonamide was validated by analysis of test item, reference item and blank test vehicle. The validation was cover the aspects namely specificity, linearity, precision (% RSD) and accuracy (% recovery).
Specificity

The reference item solution, test item solution, diluting solvent and blank test vehicle were injected onto HPLC-UV using instrument parameters mentioned below.

3.No data
Duration of treatment / exposure:
2.63 days
3.Total: 32 weeks
F0: 9 weeks
F1: 21 months for male mice and 23 months for female mice
Frequency of treatment:
2.Daily
3.Daily
Remarks:
2. Doses / Concentrations:
0,250,500,1000 mg/kg bw/day
Remarks:
3. Doses / Concentrations:
0, 0.1, 0.3 and 1 % (equivalent to 0, 150, 500 and 1500 mg/kg body weight/day)
Basis:
nominal in diet
No. of animals per sex per dose:
2.Total:124
0 mg/kg bw: 13 male, 13 female
250 mg/kg bw: 13 male, 13 female
500 mg/kg bw: 13 male, 13 female
1000 mg/kg bw: 13 male, 13 female
Recovery Group
0 mg/kg bw: 5 male, 5 female
1000 mg/kg bw: 5 male, 5 female
3.Total: 520
0 % : 65 male, 65 female
0.1% : 65 male, 65 female
0.3 % : 65 male, 65 female
1 % : 65 male, 65 female
Control animals:
yes, concurrent vehicle
Details on study design:
2. - Rationale for animal assignment (if not random): The dose levels 250, 500 and 1000 mg/kg body weight were selected for the Main Study based on the results of Dose Range Finding (DRF).- Dose selection rationale:


3.Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included: Mortality was observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: No data available

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data No data available
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No 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: Yes / No / No data No data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data No data available
- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data No data available
- Time schedule for examinations: No data available
- Dose groups that were examined: No data available

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 3, 6, 12 and 18 months, and on all surviving animals at the end of the experiment
- Anaesthetic used for blood collection: Yes (identity) / No / No data No data available
- Animals fasted: Yes / No / No data No data available
- How many animals: 20 animals of high dose group and control group
- Parameters checked in table [No.?] were examined: Haemoglobin, haematocrit and red blood cell counts, reticulocyte count, white blood cells, lymphocyte and total leucocyte counts and polymorph count were examined.

CLINICAL CHEMISTRY: Yes / No / No data No data available
- Time schedule for collection of blood: No data available
- Animals fasted: Yes / No / No data No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined. No data available

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

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

OTHER:
Absolute and relative organ weights were examined.

Liver, lungs, kidney, heart and caecal weights were examined.
Positive control:
2.Not specified
3.no data
Observations and examinations performed and frequency:
2.CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Once daily

BODY WEIGHT: Yes
- Time schedule for examinations:Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), day 4 post-partum and before terminal sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes , Males and females were weighed during randomization, on the first day of dosing, at least weekly thereafter, and at termination. During pregnancy, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), day 4 post-partum and before terminal sacrifice.

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: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
-

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood:hematological parameters were analyzed at the end of the treatment and recovery periods using auto analyzer
- Anaesthetic used for blood collection: Yes,Isoflurane anaesthesia
- Animals fasted: Yes
- How many animals: five males and five females, randomly selected from each group, just prior to necropsy.
- Parameters checked in table [No.?] were examined.Parameters were given below
Total Erythrocyte Count (RBC)
Hematocrit (HCT)
Mean Corpuscular Volume (MCV)
Hemoglobin (HGB)
Mean Corpuscular Hemoglobin (
Mean Corpuscular Hemoglobin Concentration (MCHC)
Platelet Count (PLT)
Total Leukocyte count (WBC)
Prothombin Time (PT)
Activated Partial Thromboplastin time (aPTT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Clinical parameter were analyzed at the end of the treatment and recovery periods using auto analyzer
- Animals fasted: Yes
- How many animals:five males and five females, randomly selected from each group, just prior to necropsy.
- Parameters checked in table [No.?] were examined.Parameters were given below

Parameters
Glucose (Glu)
Cholesterol (Chol)
Triglycerides (TRIG)
Alanine amino transferase (ALT)
Aspartate amino transferase (AST)
Calcium
Albumin (Alb)
Total Protein (TP)
Creatinine (Crea)
Phosphorus
Urea
Sodium (Na)
Potassium (K)
Blood urea nitrogen (BUN) –
Globulin (Glob)
Alb/ Glb (A:G)
Bile acids

URINALYSIS: Not specified


NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity / other: All were observed.

IMMUNOLOGY: Not specified


OTHER:
Organ weight; Organs of five males and females, randomly selected from each groupwere trimmed of adherent tissue/fat and weighed, prior to preservation in fixative. Testes and epididymides of all adult males were weighed. Organs were kept in normal saline till they weighed. Organs from the found dead animals during the study was not weighed.

3.Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: No data
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weekly
- Anaesthetic used for blood collection: Cardiac puncture
- Animals fasted: No data
- How many animals: No data available

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: No data

URINALYSIS: Yes
- Time schedule for collection of urine: at week 12
Sacrifice and pathology:
2.GROSS PATHOLOGY: Yes ,At scheduled sacrifice date, all rats of main and recovery groups were euthanized by over dose of carbon dioxide followed by exsanguination. The animals were examined externally in unopened condition. This was followed by opening of the carcasses and topographic examination of different organs. This included careful examination of the external surface of the body, all orifices, cranial, thoracic and visceral cavities and their contents. Simultaneously gross lesions examination was performed in accordance with the Standard Operating Procedure (SOP) of the Laboratory. Number of implatation sites in uterus and number of corpora lutea of all pregnant females were counted during necropsy examination.
Similarly, necropsy of terminally sacrificed and found dead pups during study period were conducted and gross pathological observations were recorded.

HISTOPATHOLOGY: Yes,The following tissues were preserved in 10 % neutral buffered formalin (NBF) (except eyes, which was fixed using Modified Davidson’s fluid; testes and epididymis, which were fixed in Bouin’s fluid for approximately 24 hours and subsequently preserved in 10 % NBF) for subsequent histopathological examination.
Adrenals* Pancreas
Aorta Peyer's Patches
Bone (femur) with joint # Pituitary
Brain (cerebrum,cerebellum,mid brain)* Prostate and Seminal vesicle with coagulating glands as a whole
Cecum Rectum
Colon Salivary glands
Duodenum Sciatic Nerve
Epididymides * Skeletal muscle
Eyes with optic nerve Skin
Gross lesion (if any) Spinal Cord (cervical, mid-thoracic and lumbar)
Heart * Spleen *
Ileum Sternum with marrow #
Jejunum Stomach
Kidneys * Testes *
Liver * Thymus*
Lungs $ Thyroid with Parathyroids
Mammary glands Trachea
Mesenteric and Mandibular lymph node Urinary Bladder
Oesophagus Uterus
Ovaries with oviduct* Cervix with Vagina

#: decalcified prior to sectioning
$: inflated and then immersed in 10 % NBF
Full histopathology was carried out on the preserved organs (ovaries, uterus, cervix with vagina, testes, epididymides, prostate, seminal vesicle with coagulating glands) of all animals and all tissues of five males and females, randomly selected from each group animals in the control and high dose groups.
Other examinations:
No data
Statistics:
2.Raw data was analysed using statistical software “Sigma Plot 11.0” (Supplied by Cranes Software International Ltd. Bangalore). The mean and standard deviation was calculated using the software and all data was summarized in tabular form. All continuous data (body weight, feed consumption, Functional Observational Battery parameters, hematology, clinical chemistry, absolute and relative organ weights, maternal and pup parameters etc.) were checked for normality using Shapiro Wilk test. All homogenous data was analysed using ANOVA and data showing significance in their variances was subjected to Dunnett’s t-test. All heterogeneous data was analysed using F test and Student’s t-test, Dunn’s Test, Kruskal-Wallis, ANOVA on ranks.

3.GROSS PATHOLOGY: Yes
Non-neoplastic and neoplastic lesions were examined.
Organs and tissues from intermediate, low-dose, high-dose and control groups were examined.

HISTOPATHOLOGY: Yes
Organ examined:
Mammary gland, Liver, lungs, kidney, heart and caecal were examined.

3.Statistical analysis were peformed by using Student’s t-test for survival, body weight, food consumption and clinical sign.
Clinical signs:
no effects observed
Description (incidence and severity):
2.No significant effect were observed at doses 250, 500 and 1000 mg/kg body weight in treated group compare to control.

3.effects observed, treatment-related
Mortality: When treated with 1 %, slightly increased in mortality were observed in male rat as compared to control. Clinical signs: No treatment-related behavioural or clinical findings were observed.
Mortality:
no mortality observed
Description (incidence):
2.No mortality were observed at doses 250, 500 and 1000 mg/kg body weight in treated group compare to control.
3.mortality observed, treatment-related
Mortality: When treated with 1 %, slightly increased in mortality were observed in male rat as compared to control. Clinical signs: No treatment-related behavioural or clinical findings were observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
2.No significant effect were observed at doses 250, 500 and 1000 mg/kg body weight in treated group compare to control.

3.effects observed, treatment-related
Significantly reduced were observed in 1 % treated male rat as compared to control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
2.No significant effect were observed at doses 250, 500 and 1000 mg/kg body weight in treated group compare to control.
3.effects observed, treatment-related
Food consumption: No effect was observed on Food consumption of treated rat as compared to control. Compound intake: No data available
Food efficiency:
not specified
Description (incidence and severity):
2.No significant effect were observed at doses 250, 500 and 1000 mg/kg body weight in treated group compare to control.
3.not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
2.At the end of treatment period revealed statistically significant decreased of aPTT in male rats of group G4 treated at 1000 mg/kg, statistically significant increased of aPTT in female rats of group G2 treated at 250 mg/kg while statistically significant decreased in Lymphocyte Count in female rats of group G3 treated at 500 mg/kg. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes.
During treatment free recovery period, statistically significant increase was observed in PT of group G4-R male rats treated at 1000 mg/kg and statistically significant decreased was observed Hematocrit and Hemoglobin in G4-R male rats treated at 1000 mg/kg, while statistically significant decreased of Monocyte in female rats of group G4-R at 1000 mg/kg when compared to respective control group. The observed variations in PT, Hematocrit, Hemoglobin and monocyte were considered to be of no toxicological significance, of a minimal in nature and occurred in the absence of clear dose related response

3.effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
2.At the end of treatment period revealed, statistically significant increase in Chloride of G3 male rats treated at 500 mg/kg, Sodium of G4 male rats treated at 1000 mg/kg. Statistically significant decrease in bile acid of group G2, and G4 male rats treated at 250 mg/kg and 1000 mg/kg respectively while statistically significant decrease in Triglyceride of group G3 female rats treated at 500 mg/kg.
During treatment-free recovery period revealed statistically significant increase in Potassium in G4-R female rats treated at 1000 mg/kg while statistically decrease in Bile acid in G4-R female rats treated at 1000 mg/kg was observed when compared to respective control group. The observed variations in Potassium and Bile acid was considered incidental and not test item related as it was observed only in single sex, inconsistent, not dose dependent and further is not evidenced by histopathological observations

3.not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
2.At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to respective control group except, statistical significant increase absolute and relative adrenal weight in G2 male rat group compared to main control group. Statistical significant decrease relative testes and Epididymis weight in G4 male group rat was observed when compared to G1 rats. Statistical significant increased relative heart weight in G4-R male rats was observed when compared to G1-R rats
3.effects observed, treatment-related
Gross pathological findings:
no effects observed
Description (incidence and severity):
2.No treatment related changes were noted in gross (external and internal) pathological examination of all male and female rats in all the groups.
3.effects observed, treatment-related
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
2.Microscopic examination of control group and rats treated at 250, 500 and 1000 mg/kg revealed varying degree of pathological changes in different organs. This includes Liver: focal mild to multifocal mild mononuclear cell infiltration (Male: G1:1/5, G4:3/5; Female: focal minimal to multifocal minimal mononuclear cells infiltration (Female: G1: 0/5; G4: 3/5) and minimal chronic changes and hydropic degeneration were observed in (Female: G1: 0/5; G4: 1/5). Kidneys: Unilateral: focal minimal to multifocal mild mononuclear cells infiltration (Male: G1:2/5; G4:1/5; Female: focal minimal to multifocal minimal mononuclear cells infiltration Female: G1:1/5: G4:1/5); Lungs: focal mild to multifocal mild mononuclear cells infiltration (male: G1: 2/5: G4:1/5); and BALT hyperplasia in (male: G1: 2/5); Female: focal minimal to multifocal minimal mononuclear cells infiltration (Female: G1: 4/5; G4: 3/5). Adrenals: Vacuolar, degeneration multifocal, mild (male: G1:1/5), and Female: diffuse vacuolations (Female: G1:1/5); Intestine: GALT hyperplasia: focal minimal to multifocal mild (Male: G1: 2/5; G4: 2/5); Female: GALT hyperplasia: focal minimal to multifocal minimal (Female: G1: 1/5; G4: 1/5). Pituitary Gland: cyst minimal (Male: G4:1/5); Female: serosanginous fluid cavity, mild (Female: G4:1/5). Testes: Male: Retention of sperm: focal minimal to multifocal moderate (G1:0/13, G2:4/13, G3:0/13, G4:2/13); Exfoliation of round spermatid: focal minimal to focal mild (G1:1/13, G2:0/13, G3:1/13, G4:3/13); Seminiferous tubules: Vacuolar degeneration: focal minimal to multifocal mild (G1:0/13, G2:4/13, G3:0/13, G4:3/13); Seminiferous tubules: Necrosis: diffuse, marked (G4:1/13); Seminiferous tubules: Atrophy: diffuse, minimal (G4:1/13); Leydig cell: Vacuolar degeneration: minimal to mild(G1:1/13, G2:0/13, G3:2/13, G4:0/13): Epididymis: Necrosis: diffuse, marked (G4:1/13); Epididymis: decrease in sperm: diffuse, marked (G4:1/13); Epididymis: Exfoliation of epithelium (G4:1/13). Female: Ovary: Vacuolar degeneration: segmental minimal (G4:1/13); Uterus: Perimetrium and myometrium vacuolations: minimal (G1R:1/5).
Lesions observed in liver, kidneys, lungs, adrenals, intestine, pituitary gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item

3.Neoplastic and non-neoplastic lesions were observed. Neoplastic lesions that occurred in both control and treatment group and non-neoplastic that occurred only in the treatment group were considered to be dose independent.
Histopathological findings: neoplastic:
not specified
Description (incidence and severity):
3.Neoplastic and non-neoplastic lesions were observed. Neoplastic lesions that occurred in both control and treatment group and non-neoplastic that occurred only in the treatment group were considered to be dose independent.
Details on results:
3.Haematology
Significant reducetion were observed in haemoglobin, haematocrit and red blood cell counts of 1 % treated male and female rat at 3 months and in male rat at end of the study as compared to control.
Significant reduction in reticulocyte count and an increase in the total number of white blood cells in male and female rat were observed at 12 months.After 21 months, there was a highly significant reduction in the lymphocyte and total leucocyte counts in males and the polymorph count was significantly elevated but their distribution was random, and appeared unrelated to treatment.

Organ weights:
Significant reduction in the relative and absolute liver weights of the males and absolute weight of female rat at 0.3 % was observed.
Increases in relative and absolute kidney weights of females at 0.3 % dose and absolute kidney weight of female at 1 % dose group was observed.
When treated with 1 %, in female increased in relative heart and caecalweights were observed as compared to control.

Gross pathology:
Non-neoplastic lesions which frequently occurred in both control and treatment groups (mainly involving the lungs, kidneys or liver) or were isolated occurrences, were not considered to be related to the administration of Patent Blue V.

Histopathology:
Squamous cell carcinoma (1 animal in each dose), osteosarcoma (1 animal at each dose), nephroblastoma (1 male at intermediate dose), adrenocortical adenoma (1 male at lowest and intermediate doses, 2 males at highest dose), adrenocortical carcinoma (1 male at highest dose), adenocarcinoma of the thymus (1 female at intermediate dose), adenoma of mammary gland (1 female at intermediate and highest doses).
These were mainly isolated findings, of commonly occurring tumours showing no dose-response relationship.

Details on results:
The observed changes were mainly isolated findings, of commonly occurring tumours showing no dose-response relationship. Based on these arguments, the Panel concluded that these neoplastic lesions were not indicative of a carcinogenic effect.
Dose descriptor:
NOAEL
Effect level:
1 000 other: mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant effect were observed at this dose
Remarks on result:
other: No toxic effect were observed.
Critical effects observed:
not specified

2.

Mortality and Morbidity

Sex: Male

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Observation During Study Period

G1

Control

0

13

NMM

G2

Low

250

13

NMM

G3

Mid

500

13

NMM

G4

High

1000

13

NMM

G1-R

Control- Recovery

0

5

NMM

G4-R

High- Recovery

1000

5

NMM

 

Sex: Female

Group

Treatment

Dose (mg/kg b.wt.)

No. of Animals/

Group

Observation During Study Period

G1

Control

0

13

NMM

G2

Low

250

13

NMM

G3

Mid

500

13

NMM

G4

High

1000

13

01/13*

G1-R

Control -Recovery

0

5

NMM

G4-R

High- Recovery

1000

5

NMM

Keys:NMM = No mortality and morbidity observed, No.= Number, *= One female found dead during clinical sign observation on Gestation Day 24.

Summary of Days of Conception and Pregnancy Index (%)

Group(N)

G1(13)

G2(13)

G3(13)

G4(13)

Dose (mg/kg b.wt.)

0

250

500

1000

No. of females showed evidence of copulation

13

12

13

12

No. of females concieved between Days 1-5 of cohabitation

10

7

10

8

No. of females concieved after Day 5 of cohabitation

3

5

3

4

Females achieved pregnancy

13

12

13

12

Pregnancy Index (%)

100.00

92.31

100.00

92.31

Key:N= number of dams in a group, No. = Number

Post-natal Loss (%) and Pups Survival Index (%)

Group(N)

G1(13)

G2(12)

G3(11)

G4(10)

Dose(mg/kg bwt)

0

250

500

1000

Parameter

Mean

SD

Mean

SD

Mean

SD

Mean

SD

No. of Live Births

10.54

1.13

9.67

1.61

10.00

3.00

9.70

1.95

No. of alive pups at Post-natal Day 4

10.31

0.95

8.42

3.15

9.55

2.81

9.50

2.17

Post-natal Loss (%)

1.99

3.80

11.41

29.11

3.99

6.18

2.67

5.84

Fetal Survival Index at Post-natal Day 4 (%)

98.01

3.80

88.59

29.11

96.01

6.18

97.33

5.84

Keys:SD= Standard Deviation, N= number of dams in a group

Note:Group G2 one female was found non-pregnant. Group G3 two females were showed all the pups still births and in Group G4 one female was non-pregnant, one female was found dead and one females showed all the pups still births. 

Mean Gestational Lengthand Litter size

G1(13)

G2(12)

G3(11)

G4(11) 

0

250

500

1000

Mean

SD

Mean

SD

Mean

SD

Mean

SD

22.23

0.60

19.69

6.10

22.00

1.53

18.54

8.33

10.54

1.13

9.92

1.78

9.38

3.33

9.27

2.23

Keys:SD= Standard Deviation, N= number of dams in a group

Note:Group G2 one female was found non-pregnant. Group G3 two females were showed all the pups still births and in Group G4 one female was non-pregnant and one female was found dead.

Mean Pups Body Weight, Sex Ratio and Gross Observation

Group

G1

G2

G3

G4

Dose(mg/kg bwt)

0

250

500

1000

Mean Pups Weight

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Day 0/1

5.88

0.25

13

5.68

0.37

12

5.87

0.49

11

5.75

0.25

10

Day 4

8.71

0.41

13

8.48

0.93

11

8.50

1.02

11

8.83

0.78

10

Group(n)

G1(13)

G2(12)

G3(11)

G4(10)

Pups Body Weight gain

(%)

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Day 0/1-Day 4

48.09

5.55

13

49.23

15.97

11

44.01

6.89

11

53.52

10.37

10

Group

(Number of Litter size)

G1(137)

G2(119)

G3(122)

G4(97)

 Sex Ratio at birth

(Male/Female)

81/56

56/60

56/54

58/39

 Sex Ratio at Day 4

(Male/Female)

80/54

47/55

52/53

57/38

Gross Observations

NAD

NAD

NAD

NAD

Keys:SD= Standard Deviation, N= number of dams with live pups on Day 0/1, n= number of dams with alive pups on Day 4, NAD = No Abnormality Detected

Conclusions:
No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg body weight, when rats were treated with the given test chemical via oral route.
Executive summary:

Data available from the various sources was reviewed to determine the toxic nature of the given test chemical. The studies are as mentioned below:

In a Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity study, Wistar male and female rats were treated with test chemical in the concentration of 0, 250, 500 and 1000 mg/kg bw orally by gavage for aprrox 63 days. Statistically significant decrease were observed in number of rears (250, 500 and 1000 mg/kg bw at pre-treatment and 1000 mg/kg bw at Week 1), number of urine pools (250 mg/kg bw at Week 2) in male as compared to control. In recovery male, statistically significant increase in number of urine pools at week 4 in recovery at 1000 mg/kg bw as compared to recovery control. In female, statistically significant decrease was observed in number of fecal bolus (1000 mg/kg bw recovery at Week 1) as compared to control recovery group. The above changes observed were inconsistent/ biologically insignificant and not dose dependant hence considered as incidental and not attributed to the effect of test item administration. One female was found dead on day 41 at 1000 mg/kg bw due to gavaging error. Statistically significant decrease was observed in percent body weight change of 250, 500 and 1000 mg/kg bw on day 1-20 as compared to control group. Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. These changes observed were inconsistent, hence not considered as effect of the test item administration. Statistically significant decrease in feed consumption was observed in 250, 500 and 1000 mg/kg bw female on gestation day 14-20 as compared to the control. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration. No effect on Eye was observed in treated rats as compared to control. Similarly, At the end of treatment period revealed statistically significant decreased of aPTT in male rats at 1000 mg/kg, statistically significant increased of aPTT in female rats at 250 mg/kg while statistically significant decreased in Lymphocyte Count in female rats at 500 mg/kg. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment free recovery period, statistically significant increase was observed in PT of recovery male rats treated at 1000 mg/kg and statistically significant decreased was observed Hematocrit and Hemoglobin in recovery male rats treated at 1000 mg/kg, while statistically significant decreased of Monocyte in female recovery rats at 1000 mg/kg when compared to respective control group. The observed variations in PT, Hematocrit, Hemoglobin and monocyte were considered to be of no toxicological significance, minimal in nature and occurred in the absence of clear dose related response. At the end of treatment period revealed, statistically significant increase in Chloride of male rats treated at 500 mg/kg, Sodium of male rats treated at 1000 mg/kg. Statistically significant decrease in bile acid of male rats treated at 250 mg/kg and 1000 mg/kg respectively while statistically significant decrease in Triglyceride of female rats treated at 500 mg/kg. During treatment-free recovery period revealed statistically significant increase in Potassium in female rats treated at 1000 mg/kg while statistically decrease in Bile acid in female rats treated at 1000 mg/kg was observed when compared to respective control group. The observed variations in Potassium and Bile acid was considered incidental and not test item related as it was observed only in single sex, inconsistent, not dose dependent and further is not evidenced by histopathological observations. The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration. At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to respective control group except, statistical significant increase absolute and relative adrenal weight in male rat at 250 mg/kg bw as compared to main control group. Statistical significant decrease relative testes and Epididymis weight in male rat at 1000 mg/kg bw was observed when compared to control rats. Statistical significant increased relative heart weight in male recovery rats was observed when compared to control rats. External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality. Focal mild to multifocal mild mononuclear cell infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration and minimal chronic changes and hydropic degeneration in female liver, Unilateral: focal minimal to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Kidneys, focal mild to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Lungs and focal minimal to multifocal mild GALT hyperplasia in male and focal minimal to multifocal minimal GALT hyperplasia in female intestine at 250 and 1000 mg/kg bw, Vacuolar, degeneration multifocal, mild in male and diffuse vacuolations in female Adrenals at 250 mg/kg bw, minimal cyst in male and mild serosanginous fluid cavity in female Pituitary Gland, focal minimal to multifocal moderate Retention of sperm, focal minimal to focal mild Exfoliation of round spermatid, focal minimal to multifocal mild Seminiferous tubules Vacuolar degeneration, marked diffuse Necrosis of Seminiferous tubules, minimal diffuse Atrophy Seminiferous tubules, minimal to mild Vacuolar degeneration of Leydig cell, marked diffuse Necrosis of Epididymis, marked diffuse decrease in Epididymis sperm and Epididymis of Epididymis in male rat and segmental minimal Vacuolar degeneration of Ovary and minimal Perimetrium and myometrium vacuolations of Uterus in female rats were observed at different doses. Lesions observed in liver, kidneys, lungs, adrenals, intestine, pituitary gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item. In addition, no reproductive toxicity were observed in treated rats such as Gestational length, Litter size, No. of live births, Post-implantation loss, Post-natal loss, Pregnancy index, Pups sex ratio, survival, body weight on PND 4 and gross pathology of treated rats were observed as compared to control. One females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period at 250 and 1000 mg/kg bw. Therefore, No Observed Adverse Effect Level (NOAEL) of the test chemical is considered 1000 mg/kg body weight when Wistar male and female rats orally by gavage for Approx 63 days.

In another study, Combined repeated dose & carcinogenicity study was performed in male and female mice were treated with test chemical at a concentration of 0, 0.1, 0.3 and 1 % (equivalent to 0, 150, 500 and 1500 mg/kg body weight/day) orally. Slightly increased in mortality, significant decrease in body weight of male mice and significant reduction in haemoglobin, haematocrit, red blood cell counts and reticulocyte count, increase in the total number of white blood cells in male and female rat were observed at 1 % dose group as compared to control. Increase in relative heart and caecal weights were observed in female at 1 % dose group. In addition, non-neoplastic lesions of lungs, kidneys and liver were observed. Adrenocortical adenoma and carcinoma, adenoma of mammary gland were noted and were found to be dose independent. The observed changes were mainly isolated findings, of commonly occurring tumours showing no dose-response relationship. Therefore, NOAEL was considered to be 0.3 % (500 mg/kg body weight /day) when male and female mice were treated with test chemical orally for 32 weeks.

Thus based on the above studies, the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg / kg body weight and hence is not likely to classify as per the criteria mentioned in CLP regulation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Data is Klimicsh 2 and from experimental study report

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: inhalation, other
Data waiving:
other justification
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Waiver

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, other
Data waiving:
other justification
Justification for data waiving:
a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment
other:
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
Waiver

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: Oral

Data available from the various sources was reviewed to determine the toxic nature of the given test chemical. The studies are as mentioned below:

In a Repeated Dose Oral Toxicity Study in combination with Reproduction/ Developmental Toxicity study, Wistar male and female rats were treated with test chemical in the concentration of 0, 250, 500 and 1000 mg/kg bw orally by gavage for aprrox 63 days. Statistically significant decrease were observed in number of rears (250, 500 and 1000 mg/kg bw at pre-treatment and 1000 mg/kg bw at Week 1), number of urine pools (250 mg/kg bw at Week 2) in male as compared to control. In recovery male, statistically significant increase in number of urine pools at week 4 in recovery at 1000 mg/kg bw as compared to recovery control. In female, statistically significant decrease was observed in number of fecal bolus (1000 mg/kg bw recovery at Week 1) as compared to control recovery group. The above changes observed were inconsistent/ biologically insignificant and not dose dependant hence considered as incidental and not attributed to the effect of test item administration. One female was found dead on day 41 at 1000 mg/kg bw due to gavaging error. Statistically significant decrease was observed in percent body weight change of 250, 500 and 1000 mg/kg bw on day 1-20 as compared to control group. Body weight and Percent body weight changes in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. These changes observed were inconsistent, hence not considered as effect of the test item administration. Statistically significant decrease in feed consumption was observed in 250, 500 and 1000 mg/kg bw female on gestation day 14-20 as compared to the control. Feed consumption in animals of the all other test groups of both the sexes was comparable and did not show any significant difference as compared to the respective control group. Changes observed in feed consumption were inconsistent, hence not considered as effect of the test item administration. No effect on Eye was observed in treated rats as compared to control. Similarly, At the end of treatment period revealed statistically significant decreased of aPTT in male rats at 1000 mg/kg, statistically significant increased of aPTT in female rats at 250 mg/kg while statistically significant decreased in Lymphocyte Count in female rats at 500 mg/kg. At the end of recovery period, all of the above altered parameters had returned to normal level similar to control group rats in both sexes. During treatment free recovery period, statistically significant increase was observed in PT of recovery male rats treated at 1000 mg/kg and statistically significant decreased was observed Hematocrit and Hemoglobin in recovery male rats treated at 1000 mg/kg, while statistically significant decreased of Monocyte in female recovery rats at 1000 mg/kg when compared to respective control group. The observed variations in PT, Hematocrit, Hemoglobin and monocyte were considered to be of no toxicological significance, minimal in nature and occurred in the absence of clear dose related response. At the end of treatment period revealed, statistically significant increase in Chloride of male rats treated at 500 mg/kg, Sodium of male rats treated at 1000 mg/kg. Statistically significant decrease in bile acid of male rats treated at 250 mg/kg and 1000 mg/kg respectively while statistically significant decrease in Triglyceride of female rats treated at 500 mg/kg. During treatment-free recovery period revealed statistically significant increase in Potassium in female rats treated at 1000 mg/kg while statistically decrease in Bile acid in female rats treated at 1000 mg/kg was observed when compared to respective control group. The observed variations in Potassium and Bile acid was considered incidental and not test item related as it was observed only in single sex, inconsistent, not dose dependent and further is not evidenced by histopathological observations. The sensory reactivity measurements were comparable and no changes were revealed in any of the animals of all treated groups in both the sexes. Foot splay and fore limb and hind limb grip strength parameters were comparable and no treatment related changes were revealed in any of the animals of all treated groups. Motor activity measurements were comparable and no changes were revealed in any of the animals from all treated groups of both the sexes as compare to control group. The above changes observed were inconsistent, hence considered as incidental and not attributed to the effect of test item administration. At the end of treatment and recovery period, absolute and relative weight of organs of treated group rats of either sex did not differ significantly when compared to respective control group except, statistical significant increase absolute and relative adrenal weight in male rat at 250 mg/kg bw as compared to main control group. Statistical significant decrease relative testes and Epididymis weight in male rat at 1000 mg/kg bw was observed when compared to control rats. Statistical significant increased relative heart weight in male recovery rats was observed when compared to control rats. External examination of all male and female rats of control and all treated groups including recovery groups did not reveal any abnormality of pathological significance. Visceral examination of the rats of control and other treated groups did not reveal any pathological abnormality. Focal mild to multifocal mild mononuclear cell infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration and minimal chronic changes and hydropic degeneration in female liver, Unilateral: focal minimal to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Kidneys, focal mild to multifocal mild mononuclear cells infiltration in male and focal minimal to multifocal minimal mononuclear cells infiltration in female Lungs and focal minimal to multifocal mild GALT hyperplasia in male and focal minimal to multifocal minimal GALT hyperplasia in female intestine at 250 and 1000 mg/kg bw, Vacuolar, degeneration multifocal, mild in male and diffuse vacuolations in female Adrenals at 250 mg/kg bw, minimal cyst in male and mild serosanginous fluid cavity in female Pituitary Gland, focal minimal to multifocal moderate Retention of sperm, focal minimal to focal mild Exfoliation of round spermatid, focal minimal to multifocal mild Seminiferous tubules Vacuolar degeneration, marked diffuse Necrosis of Seminiferous tubules, minimal diffuse Atrophy Seminiferous tubules, minimal to mild Vacuolar degeneration of Leydig cell, marked diffuse Necrosis of Epididymis, marked diffuse decrease in Epididymis sperm and Epididymis of Epididymis in male rat and segmental minimal Vacuolar degeneration of Ovary and minimal Perimetrium and myometrium vacuolations of Uterus in female rats were observed at different doses. Lesions observed in liver, kidneys, lungs, adrenals, intestine, pituitary gland and reproductive organs of high dose treated group rats are well comparable with respective control group rats and exhibited no dose relationship. Further these observed lesions are common in occurrence in rodents during toxicological studies (Boorman et al., 1990, Greaves, 2007, Greaves and Faccini, 1992; Haschek et al., 2002). Hence, occurrence of these lesions could be considered as spontaneous or incidental in nature and not to be attributed because of administration of the Test Item. In addition, no reproductive toxicity were observed in treated rats such as Gestational length, Litter size, No. of live births, Post-implantation loss, Post-natal loss, Pregnancy index, Pups sex ratio, survival, body weight on PND 4 and gross pathology of treated rats were observed as compared to control. One females showed prolonged diestrous i.e more than 3 days with total estrous cycle period of 6 days or more before mating period at 250 and 1000 mg/kg bw. Therefore, No Observed Adverse Effect Level (NOAEL) of the test chemical is considered 1000 mg/kg body weight when Wistar male and female rats orally by gavage for Approx 63 days.

In another study, Combined repeated dose & carcinogenicity study was performed in male and female mice were treated with test chemical at a concentration of 0, 0.1, 0.3 and 1 % (equivalent to 0, 150, 500 and 1500 mg/kg body weight/day) orally. Slightly increased in mortality, significant decrease in body weight of male mice and significant reduction in haemoglobin, haematocrit, red blood cell counts and reticulocyte count, increase in the total number of white blood cells in male and female rat were observed at 1 % dose group as compared to control. Increase in relative heart and caecal weights were observed in female at 1 % dose group. In addition, non-neoplastic lesions of lungs, kidneys and liver were observed. Adrenocortical adenoma and carcinoma, adenoma of mammary gland were noted and were found to be dose independent. The observed changes were mainly isolated findings, of commonly occurring tumours showing no dose-response relationship. Therefore, NOAEL was considered to be 0.3 % (500 mg/kg body weight /day) when male and female mice were treated with test chemical orally for 32 weeks.

Thus based on the above studies, the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg / kg body weight and hence is not likely to classify as per the criteria mentioned in CLP regulation.

Repeated dose toxicity: Inhalation

A short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment.7-Diethylamino-4-methylcoumarin (CAS no 91-44-1) has very low vapor pressure of 0.00257 Pa. (1.92765825e-5 mmHg), so the potential for the generation of inhalable vapours is very low, also the normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be highly unlikely and therefore this end point for repeated dose toxicity by inhalation route of exposure was considered for waiver.

Repeated dose toxicity: Dermal

A short-term toxicity study does not need to be conducted because exposure of humans via dermal in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment. Also, the acute dermal toxicity value for test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. Given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that test chemical shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that test chemical shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.

Justification for classification or non-classification

Based on the data available and applying the weight of evidence approach, the given test chemical does not exhibit toxic nature upon repeated exposure by oral route of exposure. Hence, it is not likely to classify as toxic as per the criteria mentioned in CLP regulation. For repeated inhalation and repeated dermal toxicity wavier was added so, not possible to classify.