1,4-dimethyl-5-[[4-[methylbenzylamino]phenyl]azo]-1H-1,2,4-triazolium acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Hydrocarbon Nephropathy In Male Rats: Identification Of The Nephrotoxic Components Of Unleaded Gasoline

Author:

Clive A. Halder, Charles E. Holdsworth, Beverly Y. Cockrell And Vincent J. Piccirillo

Year:

1985

Bibliographic source:

Toxicology and Industrial Health, Vol. 1, No. 3, 67-87, 1985

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

28 days repeated dose oral toxicity study was performed to determine the toxic nature of light catalytic reformed naphtha

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Naphtha (petroleum), light catalytic reformed

Molecular formula:

C24H48

IUPAC Name:

Naphtha (petroleum), light catalytic reformed

Details on test material:

- Name of test material: Light catalytic reformed naphtha
- Molecular formula: C24H48
- Molecular weight: Unspecified
- Substance type: Organic
- Physical state: No data
- Impurities (identity and concentrations): No data

Specific details on test material used for the study:

- Name of test material: Light catalytic reformed naphtha
- Molecular formula: C24H48
- Molecular weight: Unspecified
- Substance type: Organic
- Physical state: No data
- Impurities (identity and concentrations): No data

Test animals

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York
- Age at study initiation: 11 weeks approx..
- Weight at study initiation:
- Fasting period before study:
- Housing: The animals were individually housed in suspended polycarbonate cages both during study and for a 2-wk quarantine period. Following the quarantine period, the rats were randomly assigned by weight to groups of 10 each.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 – 23.8 ˚C
- Humidity (%): 40-80%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12-h light/ dark cycle

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

No data

Vehicle:

not specified

Details on oral exposure:

No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

28 days

Frequency of treatment:

5 d/week

Doses / concentrations

Remarks:

0, 500 or 2000 mg/Kg/day

No. of animals per sex per dose:

Total:
0 mg/Kg /day: 10 male rats
500 mg/Kg /day: 10 male rats
2000 mg/Kg /day: No detailed data
Positive control: 10 male rats

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The selection of the dosage levels was based on a single pilot study using unleaded gasoline; pilot studies were not conducted on each test material to assay for toxicity. From the pilot study, a 28-d maximum tolerated dose of 2.0 g/ kg/ d was established. Also, the 0.5 g/ kg/ d dose level was selected because it was effective, yet was not lethal. In each case, the test material was administered neat. Dosages for each animal were calculated based upon body weights taken prior to dosing on day I, and the appropriate dosing volume for each test material was determined using its specific gravity.

- Rationale for animal assignment (if not random): The rats were randomly assigned by weight to groups of 10 each
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random): No data

Positive control:

Yes, Unleaded gasoline blend was used at positive control

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included. Mortality and Morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing on day 1 and at the time of the scheduled sacrifices only

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data
- 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: No data
- 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: No data
- 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: No data
- 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:

GROSS PATHOLOGY: Yes, gross pathology was performed on all animals which were found dead or moribund. The kidneys for these animals were weighed and were
fixed for histopathologic evaluation; however, the weights were not included in the tabulations of the means for each group. On the day immediately following the 20th dose, the remaining study animals were sacrificed, without prior fasting, using an overdose of sodium pentobarbital, and necropsied. At necropsy, only the kidneys
were removed, trimmed free of perirenal fat and connective tissue, and weighed

HISTOPATHOLOGY: Yes, kidneys were sliced longitudinally and fixed by immersion in 10% neutral buffered formalin. All other tissues were discarded after gross examination.

The processed kidneys were embedded in paraffin and sectioned. Two sections were made from each kidney. One section was stained with hematoxylin and eosin and was used to examine for foci of regenerative epithelium in the renal cortex, as well as for foci of intratubular cast formation located between the inner and outer stripe of the renal medulla. The second section was stained with Mallory-Heidenhain stain to assess the degree of hyaline droplet (phagolysosome) accumulation within the epithelial cells of the proximal convoluted tubules.

The severity of the nephrotoxic responses was evaluated using a grading system that was found to be consistent and reproducible.
The changes were graded on a scale of 1 to 4 based on severity or number of pathologic foci, as applicable. The scores for each lesion type were combined and then multiplied by 10. The kidney lesions from animals that died on or before day 14 of exposure were scored, but these scores were not included in the tabulations of the means for each group.

Other examinations:

No data

Statistics:

Mean and standard deviation calculations were made on body weight, kidney weight and kidney pathology data. The body and kidney weight data were compared statistically to the appropriate saline control group using a Student’s t-Test (Snedecor and Cochran, 1980). The means of the kidney lesion scores were compared to that of the appropriate saline control group using the Mann-Whitney U Test (Hays, 1981). Statistical significance was assigned to a probability of <5%.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The test chemical doses were well tolerated but lethargy was the most prominent signs.

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean terminal body weights for all high-dose treatment group was significantly reduced than the respective saline control group

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

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):

Mean kidney weights gave no indication of nephrotoxicity

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The most common pathologic findings were confined to the kidneys and stomachs. The kidney lesions generally consisted of discoloration and mottling, and may have been due to postmortem changes since they were mainly observed in the animals that died prematurely.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The unleaded gasoline, tested as a positive reference material, produced a strong nephrotoxic response

The light catalytic-reformed naphtha induced a very weak nephrotoxic response.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

Dose descriptor:

NOAEL

Effect level:

500 other: mg/Kg/day

Based on:

test mat.

Sex:

male

Basis for effect level:

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Summary of Body weights, Kidney effects and mortality

Test chemical	Dose (mg/Kg/day)	Initial body weight (g)	Terminal body weight (g)	Nephropathy score	Kidney weight (g)	Mortality (%)
Saline (negative control)	2000	163±16	211±23	27±5	1.72±0.15	0
Reference unleaded gasoline	500	162±14	201±21	84±13*	1.77±0.15	0
	2000	163±12	183±25^S-	83±21*	1.73±0.20	0
Light catalytic reformed naphtha	500	162±12	197±17	33±5	1.70±0.14	0
	2000	167±16	176±16^S-	39±9*	1.70±0.18	20

* Significantly elevated kidnet scores relative to saline control. P≤0.05 Mann Whitney U test

S- Significantly lower relative to saline control p≤0.05, Student’s t-test

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for light catalytic reformed naphtha is considered to be 500 mg/Kg/day during 28 days study conducted using male Fischer 344 rats.

Executive summary:

28 days repeated dose oral toxicity study was performed to determine the toxic nature oflight catalytic reformed naphtha. The study was performed using using male Fischer 344 rats at dose levels of 0 (saline), 500 or 2000 mg/Kg/day for 28 days. The animals were doses daily for 5days/week. During the study period, the animals were observed for clinical signs, mortality, morbidity, body weight and organ weight changes, gross and histo-pathology.The test chemical doses were well tolerated but lethargy was the most prominent signs. The mean terminal body weights for all high-dose treatment group (2000 mg/Kg/day) was significantly reduced than the respective saline control group. Mean kidney weights gave no indication of nephrotoxicity. The most common pathologic findings were confined to the kidneys and stomachs. The kidney lesions generally consisted of discoloration and mottling, and may have been due to postmortem changes since they were mainly observed in the animals that died prematurely. The light catalytic-reformed naphtha induced a very weak nephrotoxic response at 2000 mg/Kg/day dose. Based on the observations made, the No Observed Adverse Effect Level (NOAEL) forlight catalytic reformed naphtha is considered to be 500 mg/Kg/day during 28 days study conducted using male Fischer 344 rats.