2,2'-[[5-acetamido-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-methoxyphenyl]imino]diethyl diacetate

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

90-Day Oral Toxicity in Rodents

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Completed on February 15, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OTS 798.2650 (90-Day Oral Toxicity in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Common rodent species

Sex:

male/female

Details on test animals or test system and environmental conditions:

From: Charles River
Number: 68 males and 68 females
Age: ca. 7 weeks
Temperature: 20-25°C; Humidity: 40-70%; Light cycle: 12h light/12h dark
Diet: certified rodent chow (no. 5002, Ralston Purina Company) ad libitum; Water: by automatic watering system with demand control valves mounted on each rack

Route of administration:

oral: gavage

Details on route of administration:

Gavage five days/week for 13 weeks at a volume of 10 mL/kg bw/day.

Vehicle:

corn oil

Details on oral exposure:

All animals were dosed based on their individual, most recent body weight.
Animals received the test substance suspensions at concentrations of 250, 1250 or 2500 mg/kg bw/day.
Control animals were administered the vehicle (corn oil) alone.

Analytical verification of doses or concentrations:

yes

Remarks:

Liquid chromatography

Details on analytical verification of doses or concentrations:

Dosing suspensions were prepared weekly at concentrations of 0, 25, 125 and 250 mg/mL of Mazola corn oil.
Homogeneity and stability of the test substance under storage conditions were established prior to the start of the study.
Suspension concentrations were verified for all dose levels for Weeks 1, 2, 3, 4, 8 and 13.
All those parameters were within acceptable values.

Duration of treatment / exposure:

90 days

Frequency of treatment:

five days per week

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Remarks:

maximum feasible dose that can be administered based on the physical properties of the suspension and the considerations of the amount of corn oil administered over the 90-day period

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection: according to a dose range finding study.

Positive control:

no

Observations and examinations performed and frequency:

- Mortality: twice daily
- Clinical signs: daily + detailed observations once each week
- Body weights and body weight gains: weekly
- Food consumption: weekly
- Ophthalmic examinations (indirect ophthalmoscope): prior to the start of dosing and prior to sacrifice
- Hematology following an overnight fasting (retro-orbital sinus): AST, ALT, total protein, gamma glutamyl transferase, calcium, glucose, albumin, globulin (calculated), creatinine, total bilirubin, urea nitrogen, phosphorus, sodium, potassium, chloride
- Clinical chemistry following an overnight fasting (retro-orbital sinus): erythrocyte count and indices, hemoglobin, hematocrit, platelet count, total and differential leukocyte count

Sacrifice and pathology:

- Gross pathology along with complete necropsy (+ gross examinations of lungs, liver, kidneys) for all animals
- Organ weights (after 13 weeks): liver, kidneys, spleen, brain with stem, heart, adrenals, testes and ovaries
- Histopathology (lungs, liver, kidneys, spleen, trachea, esophagus, stomach, intestine, brain with stem, eyes, exorbital lacrymal glands, zymbal glands, pituitary, salivary glands, heart and aorta, adrenals, thymic region, thyroid, nasopharyngeal tissues, vagina, mammary glands, testes and ovaries): saved in 10% neutral buffered formalin

Statistics:

Data for continuous, parametric variables were intercompared for the dose and control groups by use of Levene's test for homogeneity of variances, by analysis of variance, and by pooled variance t-tests. Pathology frequency data were compared using Fisher's exact tests. 0.05 was used as the critical level of significance for all tests.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Blue coloration of the skin and/or tail of some animals
(Females of the control group were not as healthy as the treated ones)

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Dosing accident in the control groups (females)

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the female control group (they were nutritionally affected by the large amount of corn oil)
No dose-response relationship in treated groups

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Increased in females of the treated groups but consistent with the decrase observed in the female control group

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross or microscopic lesions

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

> 2 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed on any of the monitored parameters

Key result

Dose descriptor:

NOEL

Effect level:

> 2 490 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed on any of the monitored parameters

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, the NOEL was greater than the highest dose tested and determined to be >2500 mg/kg bw/day, equivalent to >2490 mg a.i./kg bw/day.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the test substance (99.61% purity) according to EPA OTS Guideline 798.2650. Male and female Sprague Dawley rats (10/sex/dose) were administered by gavage the following concentrations (dissolved in corn oil, at a volume of 10 mL/kg bw): 0, 250, 1250 and 2500 mg/kg bw/day for five days/week during 13 weeks. The homogeneity, stability and concentrations of dosing suspensions were analysed and were within acceptable values. Mortality was checked twice daily. Clinical signs were recorded daily and detailed observations were realised once each week. Body weights and body weight gains and food consumption were measured weekly. Ophthalmic examinations (indirect ophthalmoscope) were made prior to the start of dosing and prior to sacrifice. Haematology following an overnight fasting (retro-orbital sinus) comprised measurements for AST, ALT, total protein, gamma glutamyl transferase, calcium, glucose, albumin, globulin (calculated), creatinine, total bilirubin, urea nitrogen, phosphorus, sodium, potassium and chloride. Clinical chemistry following an overnight fasting (retro-orbital sinus) comprised analyses of erythrocyte count and indices, haemoglobin, haematocrit, platelet count, total and differential leukocyte count. Gross pathology along with complete necropsy (gross examinations of lungs, liver, kidneys) were done for all animals. Organ weights were recorded for the liver, kidneys, spleen, brain with stem, heart, adrenals, testes and ovaries. Histopathology (lungs, liver, kidneys, spleen, trachea, oesophagus, stomach, intestine, brain with stem, eyes, exorbital lachrymal glands, zymbal glands, pituitary, salivary glands, heart and aorta, adrenals, thymic region, thyroid, nasopharyngeal tissues, vagina, mammary glands, testes and ovaries) was carried out on tissues saved in 10% neutral buffered formalin. Except for a blue coloration of the skin and/or tail of some animals, no adverse effects were observed that could be related to the treatment with the test substance. Under the study conditions, the NOEL was greater than the highest dose tested and determined to be >2500 mg/kg bw/day, equivalent to >2490 mg a.i./kg bw/day (Hermansky, 1991).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 490 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good quality

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine the repeated dose toxicity of the test substance (99.61% purity) according to EPA OTS Guideline 798.2650. Male and female Sprague Dawley rats (10/sex/dose) were administered by gavage the following concentrations (dissolved in corn oil, at a volume of 10 mL/kg bw): 0, 250, 1250 and 2500 mg/kg bw/day for five days/week during 13 weeks. The homogeneity, stability and concentrations of dosing suspensions were analysed and were within acceptable values. Mortality was checked twice daily. Clinical signs were recorded daily and detailed observations were realised once each week. Body weights and body weight gains and food consumption were measured weekly. Ophthalmic examinations (indirect ophthalmoscope) were made prior to the start of dosing and prior to sacrifice. Haematology following an overnight fasting (retro-orbital sinus) comprised measurements for AST, ALT, total protein, gamma glutamyl transferase, calcium, glucose, albumin, globulin (calculated), creatinine, total bilirubin, urea nitrogen, phosphorus, sodium, potassium and chloride. Clinical chemistry following an overnight fasting (retro-orbital sinus) comprised analyses of erythrocyte count and indices, haemoglobin, haematocrit, platelet count, total and differential leukocyte count. Gross pathology along with complete necropsy (gross examinations of lungs, liver, kidneys) were done for all animals. Organ weights were recorded for the liver, kidneys, spleen, brain with stem, heart, adrenals, testes and ovaries. Histopathology (lungs, liver, kidneys, spleen, trachea, oesophagus, stomach, intestine, brain with stem, eyes, exorbital lachrymal glands, zymbal glands, pituitary, salivary glands, heart and aorta, adrenals, thymic region, thyroid, nasopharyngeal tissues, vagina, mammary glands, testes and ovaries) was carried out on tissues saved in 10% neutral buffered formalin. Except for a blue coloration of the skin and/or tail of some animals, no adverse effects were observed that could be related to the treatment with the test substance. Under the study conditions, the NOEL was greater than the highest dose tested and determined to be >2500 mg/kg bw/day, equivalent to >2490 mg a.i./kg bw/day (Hermansky, 1991).

Justification for classification or non-classification

Based on the results of a repeated dose oral toxicity study in rat, no classification for this endpoint is required according to CLP (EC 1272/2008) criteria.​