1-[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl]pyridinium acetate

Endpoint summary

Description of key information

Repeated dose toxicity: Oral

The No Observed Adverse Effect Level (NOAEL) was considered to be 1250 mg/kg/day, when rodents were treated with the given test chemical during repeated dose toxicity study.

 

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. Also, the given test chemical has very low vapor pressure 2.493128e-10 Pa, so the potential for the generation of inhalable vapors is very low. 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 inhalation toxicity 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:

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:

Weight of evidence approach based on the available information from various test chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

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:

rat

Strain:

other: 2. Fischer 344 3. Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

2. TEST ANIMALS
- Source: Frederick Cancer Research Center (Frederick, MD)
- Age at study initiation: 56 days old
- Housing: rats were housed five per cage. Cages were rotated vertically once every two weeks.
- Diet (e.g. ad libitum): NIH-07 Rat, meal (Zeigler Bros., Inc., Gardners, PA), available ad libitum
- Water (e.g. ad libitum): Tap water (Birmingham Water Works) in glass water bottles with stainless steel sippers (Edstrom Automatic Watering Systems, Waterford, WI), available ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8° C-25.6° C
- Humidity (%): 15%-85%
- Air changes (per hr): minimum 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day
3. TEST ANIMALS
- Source: No data
- Age at study initiation: 9 weeks

Route of administration:

other: 2. oral: feed 3. oral: gavage

Vehicle:

other: 2. NIH-07 rat ration 3. water

Details on oral exposure:

2. PREPARATION OF DOSING SOLUTIONS: No data available

DIET PREPARATION
- Rate of preparation of diet (frequency): No data available
- Mixing appropriate amounts with (Type of food): premix of NIH-07 rat ratioon as meal, with the appropriate amount of C.I. Pigment Red 23.
- Storage temperature of food: 2 weeks for 45 °C
-Other information:The dose formulations were prepared by mixing appropriate quantities of C.1. Pigment Red 23 with feed (NIH-07 Rat Ration) to form a premix, then the remaining feed was added and mixed in a twin-shell blender equipped with an intensifier bar. Studies conducted by the analytical chemistry laboratory to determine stability and homogeneity of the dosed feed formulations indicated that the formulations were homogeneous and stable for at least 2 weeks at temperatures up to 45° C when stored in the dark. The preparations protected from light were stored at 5° C prior to use and at room temperature during use. Storage time was not more than 14 days. Periodic analyses of the dosed-feed formulations were conducted at the study laboratory and at the analytical chemistry laboratory throughout the studies. The original method used the extraction solvent nitrobenzene; the solventwas changed to a solution of 10 g potassium hydroxide in 500 mL methanol diluted to 1,000 mL with tetrahydrofuran because of inconsistent recoveries.


VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: 0, 10,000, 25,000, or 50,000 ppm ((500, 1250 or 2500 mg/kg))
- Amount of vehicle (if gavage): Not applicable.
- Lot/batch no. (if required): Lot UB2158
- Purity: 99.6%.
3. PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with water at dose levels of 0, 40, 200 or 1000 mg/Kg/day (Recovery group: 0, 1000 mg/kg/day)

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0, 40, 200 or 1000 mg/Kg/day (Recovery group: 0, 1000 mg/kg/day)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

2. Dose levels were determined using visible spectroscopy at 478 nm. Homogeneity of the formulations was confirmed by the study laboratory. For the 2-year studies, a total of 142 samples were analyzed and five were remixed in order to be within acceptable limits.Periodically, the dose formulations were sent for referee analyses by MRI. The results from the study laboratory and from the referee analytical chemistry laboratory were generally in good agreement, with all value differences less than 13%
3. not specified

Duration of treatment / exposure:

2. 2years
3. Male: 42 days / - Female: 41 - 47 days

Frequency of treatment:

2. Daily
3. Daily

Remarks:

2. 0, 10,000, 25,000, or 50,000 ppm (0, 500, 1250 or 2500 mg/kg/day)
3. Test group: 0, 40, 200 or 1000 mg/Kg/day
Recovery group: 0, 1000 mg/kg/day

No. of animals per sex per dose:

2. 10 animals/dose
3. No data

Control animals:

yes, concurrent vehicle

Details on study design:

2. Details on study design:
- Dose selection rationale: Because levels of C.I. Pigment Red 23 as high as 2500 or 50,000 mg/kg in the feed did not adversely affect survival and mean body weights in the 17-day and 13-week studies, nor cause any chemical-related lesions, doses of 0, 500, 1250, or 2500 mg/kg were selected for the 2-year studies.
3. No data

Positive control:

Not specified

Observations and examinations performed and frequency:

2. Observations and examinations performed & frequency
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice/day
- Cage side observations checked in table [No.?] were included. No data available

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at each weight check and at terminal sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: once/week for 13 weeks, once/month thereafter and at sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data available
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data available
- 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): No data available
- Time schedule for examinations: No data available

OPHTHALMOSCOPIC EXAMINATION: 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: No data available
- Anaesthetic used for blood collection: No data available
- Animals fasted: No data available
- How many animals: No data available
- Parameters checked in table [No.?] were examined. No data available

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

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: No data available sensory activity / grip strength / motor activity / other: No data available
3. CAGE SIDE OBSERVATIONS: No data
- Time schedule: No data
- Cage side observations checked in table [No.?] were included. No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: No data

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

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: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

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

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

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: No data
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: No data
- Parameters checked in table [No.?] were examined. No data

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

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

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

OTHER: No data

Sacrifice and pathology:

2. GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
3. GROSS PATHOLOGY: Yes. Organ weight was noted
HISTOPATHOLOGY: No data

Statistics:

2. Mean ± standard deviation was observed.
3. No data

Clinical signs:

no effects observed

Description (incidence and severity):

2. no effects observed - There were no clinical findings in rats considered to be chemically related.
3. no effects observed

Mortality:

no mortality observed

Description (incidence):

2. no mortality observed - No mortality were observed at all treated group 500, 1250 or 2500 mg/kg compare to control.
3. not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

2. no effects observed - Male: Mean body weights of male rats were similar to that of the controls throughout the 2-year study
Female: from week 20 to the end of the study, the mean body weights of mid- and high-dose females were consistently lower than that of the controls
3. no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

2. no effects observed - Feed consumption by exposed male and female rats was similar to that of to the controls.
3. no effects observed

Food efficiency:

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. no effects observed - In male rats, there were no biologically significant differences in hematology parameters related to chemical exposure.
In female rats, Hematocrit values, hemoglobin concentration, and erythrocyte counts interim evaluation were significantly less than those of the controls and serum total bilirubin was significantly increased in 50000 ppm female rats at the 15-month
3. no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

2. no effects observed - In male rats, there were no biologically significant differences in clinical chemistry parameters related to chemical exposure.
3. no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

2. not specified
3. no effects observed

Behaviour (functional findings):

not specified

Description (incidence and severity):

2. not specified
3. no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

2. not specified
3. no effects observed

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

2. effects observed, treatment-related - A slightly increased incidence of mitosis, and invasion of adjacent renal tissue by tumor cells and Kidney: nephropathy was observed.
Pituitary Gland: Adenoma or carcinoma (Combined) of the pars distalis occurred with a significant negative trend in female rats. The incidence in the high-dose group was significantly lower than in the control.
Lymphoid Tissue: Red pigment, presumably compound- related, was observed in the lymphoid tissue of the small intestine in females and in the mesenteric lymph nodes in males. There was a dose-related increase in the amount of pigment present. The pigment consisted of distinct red granules or small elongated crystals within the cytoplasm of the macrophages.
3. no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

2. not specified
3. no effects observed

Other effects:

not specified

Dose descriptor:

NOAEL

Remarks:

2

Effect level:

1 250 mg/kg bw/day (actual dose received)

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

haematology

histopathology: non-neoplastic

mortality

Remarks on result:

other: No toxic effect were observed

Dose descriptor:

NOAEL

Remarks:

3

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

other: No adverse effects were noted at the mentioned dose level

Critical effects observed:

not specified

Conclusions:

The No Observed Adverse Effect Level (NOAEL) was considered to be 1250 mg/kg/day, when rats were treated with the given test chemical during repeated dose toxicity study.

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:

 

Two year repeated dose toxicity study was conducted by administering the given test chemical to male and female rats at doses of 0, 500, 1250, and 2500 mg/kg in feed for 103 weeks. All animals were observed twice daily. Clinical findings for rats were noted and recorded during body weight measurements and at sacrifice. Body weights were recorded weekly for the first 13 weeks, and then every four weeks until the end of the study; body weights were also recorded at the end of the study. Feed consumption was recorded daily per cage. Complete necropsies were performed on all animals. During necropsy all organs and tissues were examined for grossly visible lesions. Tissues for microscopic examination were preserved in 10% neutral buffered formalin and routinely processed for microscopic examination (embedded in paraffin, sectioned at 4-5 microgram, and stained with hematoxylin and eosin). Complete histopathologic evaluation was performed on animals from the control and high-dose group, on selected tissues, and on target organs and gross lesions from low- and mid-dose animals. Mean ± standard deviation was observed. No mortality was observed at all treated groups 500, 1250 or 2500 mg/kg compare to control. There were no clinical findings in rats considered to be chemically related. Mean body weights of male rats were similar to that of the controls throughout the 2-year study. From week 20 to the end of the study, the mean body weights of mid- and high-dose females were consistently lower than that of the controls. Feed consumption by exposed male and female rats was similar to that of to the controls. In male rats, there were no biologically significant differences in hematology parameters related to chemical exposure. In female rats, Hematocrit values, hemoglobin concentration, and erythrocyte counts interim evaluation were significantly less than those of the controls and serum total bilirubin was significantly increased in 2500 mg/kg female rats at the 15-month. In male rats, there were no biologically significant differences in clinical chemistry parameters related to chemical exposure. A slightly increased incidence of mitosis and invasion of adjacent renal tissue by tumor cells and Kidney: nephropathy was observed. In Pituitary Gland, adenoma or carcinoma (Combined) of the pars distalis occurred with a significant negative trend in female rats. The incidence in the high-dose group was significantly lower than in the control. In lymphoid Tissue, red pigment, presumably compound- related, was observed in the lymphoid tissue of the small intestine in females and in the mesenteric lymph nodes in males. There was a dose-related increase in the amount of pigment present. The pigment consisted of distinct red granules or small elongated crystals within the cytoplasm of the macrophages. Mononuclear cell leukemia occurred with a decreased incidence in male and female rats. Under the condition of the study, the NOAEL value for repeated dose toxicity of the given test chemical in male and female F344 rats was considered to be 1250 mg/kg/day.

 

In another study, the given test chemical was assessed for its possible toxic potential. For this purpose, Combined repeated dose and reproduction / developmental screening was performed as per OECD Guideline 422. Male and female Crl:CD (SD) rats were used in the study. The test compound was dissolved in water and used at dose levels of 0, 40, 200 or 1000 mg/Kg/day. The male rats were treated for 42 days and female rats were treated for 41-47 days. Recovery group of 0 and 1000 mg/Kg/day was also included in the study. The treated animals were noted for clinical signs, functional battery observations, body weight, food consumption, urinanalysis, hematology, blood chemistry, organ weight changes and histopathology. No adverse effects were noted in the various parameters studied. Based on the observations made, No Observed Adverse Effect Level (NOAEL) for the given test chemical was considered to be 1000 mg/Kg/day.

 

Thus based on the above studies, the No Observed Adverse Effect Level (NOAEL) was considered to be 1250 mg/kg body weight/day and hence is not likely to classify for repeated oral toxicity as per the criteria mentioned in CLP regulation.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 250 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Data is Klimicsh 2 and from handbook or collection of data.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

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

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:

short-term repeated dose toxicity: dermal

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

Endpoint conclusion

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:

 

Two year repeated dose toxicity study was conducted by administering the given test chemical to male and female rats at doses of 0, 500, 1250, and 2500 mg/kg in feed for 103 weeks. All animals were observed twice daily. Clinical findings for rats were noted and recorded during body weight measurements and at sacrifice. Body weights were recorded weekly for the first 13 weeks, and then every four weeks until the end of the study; body weights were also recorded at the end of the study. Feed consumption was recorded daily per cage. Complete necropsies were performed on all animals. During necropsy all organs and tissues were examined for grossly visible lesions. Tissues for microscopic examination were preserved in 10% neutral buffered formalin and routinely processed for microscopic examination (embedded in paraffin, sectioned at 4-5 microgram, and stained with hematoxylin and eosin). Complete histopathologic evaluation was performed on animals from the control and high-dose group, on selected tissues, and on target organs and gross lesions from low- and mid-dose animals. Mean ± standard deviation was observed. No mortality was observed at all treated groups 500, 1250 or 2500 mg/kg compare to control. There were no clinical findings in rats considered to be chemically related. Mean body weights of male rats were similar to that of the controls throughout the 2-year study. From week 20 to the end of the study, the mean body weights of mid- and high-dose females were consistently lower than that of the controls. Feed consumption by exposed male and female rats was similar to that of to the controls. In male rats, there were no biologically significant differences in hematology parameters related to chemical exposure. In female rats, Hematocrit values, hemoglobin concentration, and erythrocyte counts interim evaluation were significantly less than those of the controls and serum total bilirubin was significantly increased in 2500 mg/kg female rats at the 15-month. In male rats, there were no biologically significant differences in clinical chemistry parameters related to chemical exposure. A slightly increased incidence of mitosis and invasion of adjacent renal tissue by tumor cells and Kidney: nephropathy was observed. In Pituitary Gland, adenoma or carcinoma (Combined) of the pars distalis occurred with a significant negative trend in female rats. The incidence in the high-dose group was significantly lower than in the control. In lymphoid Tissue, red pigment, presumably compound- related, was observed in the lymphoid tissue of the small intestine in females and in the mesenteric lymph nodes in males. There was a dose-related increase in the amount of pigment present. The pigment consisted of distinct red granules or small elongated crystals within the cytoplasm of the macrophages. Mononuclear cell leukemia occurred with a decreased incidence in male and female rats. Under the condition of the study, the NOAEL value for repeated dose toxicity of the given test chemical in male and female F344 rats was considered to be 1250 mg/kg/day.

 

In another study, the given test chemical was assessed for its possible toxic potential. For this purpose, Combined repeated dose and reproduction / developmental screening was performed as per OECD Guideline 422. Male and female Crl:CD (SD) rats were used in the study. The test compound was dissolved in water and used at dose levels of 0, 40, 200 or 1000 mg/Kg/day. The male rats were treated for 42 days and female rats were treated for 41-47 days. Recovery group of 0 and 1000 mg/Kg/day was also included in the study. The treated animals were noted for clinical signs, functional battery observations, body weight, food consumption, urinanalysis, hematology, blood chemistry, organ weight changes and histopathology. No adverse effects were noted in the various parameters studied. Based on the observations made, No Observed Adverse Effect Level (NOAEL) for the given test chemical was considered to be 1000 mg/Kg/day.

 

Thus based on the above studies, the No Observed Adverse Effect Level (NOAEL) was considered to be 1250 mg/kg body weight/day and hence is not likely to classify for repeated oral toxicity 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. Also, the given test chemical has very low vapor pressure 2.493128e-10 Pa, so the potential for the generation of inhalable vapors is very low. 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 inhalation toxicity 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.