3-amino-2,4,6-triiodobenzoic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 October 1998 to 29 October 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-amino-2,4,6-triiodobenzoic acid

EC Number:

221-493-0

EC Name:

3-amino-2,4,6-triiodobenzoic acid

Cas Number:

3119-15-1

Molecular formula:

C7H4I3NO2

IUPAC Name:

3-amino-2,4,6-triiodobenzoic acid

Test material form:

solid: bulk

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Shoe:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Schering AG, Berlin, Germany
- Females (if applicable) nulliparous and non-pregnant: not reported
- Age at study initiation: not reported
- Weight at study initiation: male: 168-210 g and female: 167-201 g
- Fasting period before study: No
- Housing: conventional (1 animal/cage)
- Diet (e.g. ad libitum): ulverized Altromin® R, ad libitum
- Water (e.g. ad libitum): demineralized acidified water, pH 2-3, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 48-58%
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES:
From: 01 October 1998 To: 29 October 1998 (control, group 1, group 2, and group 3)
From: 01 October 1998 To: 15 October 1998 (group 4 – 1000 mg/kg dose group)
From: 16 October 1998 To: 29 October 1998 (group 4 – 400 mg/kg dose group)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

intragastric by gavage (i.g.)

Vehicle:

other: 0.9% NaCl-solution

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: not reported
- VEHICLE
- Justification for use and choice of vehicle (if other than water): NaCl-solution
- Concentration in vehicle: 0.9%

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28-29 days

Frequency of treatment:

once daily in the morning

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

due to the high rate of mortality at the dose of 1000 mg/kg of the test substanve, the dose was lowered to 400 mg/kg of the test substance

No. of animals per sex per dose:

1 male and 1 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Since oral administration of 2000 mg/kg was tolerated without mortality or signs of intoxication in an acute toxicity study in rats, for the present study a high dose of 1,000 mg/kg was selected as this is the recommended upper limit dose for 4-week studies according to international guidelines. No effects were expected after the low dose of 40 mg/kg; 200 mg/kg was chosen as the mid dose.

- Rationale for animal assignment (if not random): randomization by computer program

Positive control:

N/A

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked twice daily [once in the morning and once in the afternoon]. On weekend the second check was carried out just before the technicians left the laboratory in the late morning. This check consisted of an evaluation of the general condition of each individual animal. All alterations from baseline condition of the animals were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT:
- Time schedule for examinations: Body weight was recorded weekly for the individual animal over a period of 27 days and statistical calculation was performed over this time period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The amount of food consumed by the individual animal was recorded weekly over a period of 27 days and the statistical calculations were performed over this time period.

WATER CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: The quantity of water consumed by the individual animal was recorded weekly over a period of 27 days and the statistical calculation was performed over this time period.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: Ophthalmoscopic examinations were carried out on day 22 or 27 of the test.
- Dose groups that were examined: The pupils of all animals were dilated using a mydriaticum containing tropicamid (Mydriaticum Stulln® 0.5%) and the eyes examined with the naked eye, with an ophthalmoscope (Oculus­ Visuskop, W. Okulus) and a hand-slit-lamp microscope (Kowa SL 5, Kowa Company, Japan).

HAEMATOLOGY:
- Time schedule for collection of blood: Hematological investigations were performed on day 28. The parameters determined included erythrocyte and leucocyte count, hemoglobin, hematocrit (packed cell volume), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), reticulocyte count, platelet count, differential count [neutrophils (myelocytes, immature, band type I and II, segmented), lymphocytes, eosinophils, basophils, monocytes] in 6-4 male and 5-3 female animals per group.

URINALYSIS: Yes
- Time schedule for collection of urine: The following parameters were determined in spontaneous urine samples collected over a period of about 18 hours from 4 to 6 male and 2 to 6 female animals per group on day 23: pH value, specific gravity, urinary volume, protein, glucose, ketones, urobilinogen, bilirubin blood and sediment analysis.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: kidneys, thymus, stomach,

HISTOPATHOLOGY: Yes: kidneys, thymus, spleen, lymph nodes, bone marrow, stomach, and reproductive organs

Statistics:

The Dunnett-test was used for parametric values to assess the statistical significance of differences between the control and the treatment groups.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the animals of control group 1 and of group 2, dosed with 40 mg/kg, no findings were observed over the whole study period.

In group 3, dosed with 200 mg/kg, one out of 6 male animals showed compulsive pushing through the embedding on a single occasion. In the female animals, no findings occurred. Due to the single occurrence of the finding, it is regarded as an incidental finding.

In group 4, slight to moderate apathy, disturbances in gait, slight to moderate emaciation, ruffled fur, complete eyelid closure and compulsive pushing through the embedding were observed in the period, in which the animals received a daily dose of 1,000 mg/kg. After reduction of the daily dose to 400 mg/kg, only compulsive pushing through the embedding occurred.

Mortality:

mortality observed, treatment-related

Description (incidence):

In group 3, dosed with 200 mg/kg, one female rat (no. 32F) died on study day 16. As foamy liquid was found in the bronchi at necropsy, it is suspected, that this animal died due to misadministration.
In group 4, 2 out of 6 male (nos. 39M and 4 1M) and 3 out of 6 female rats (nos. 44F, 46F and 47F) dosed with 1,000 mg/kg died. Death occurred after a treatment period of 3 to 14 days.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was reduced in male and female rats of group 4 (daily dose of 1,000 mg/kg) in the first week of treatment (p < 0.01, p < 0.05).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption was reduced in the first week in the male rats of group 4 (p < 0.01) and in the first and second week in the female rats of group 4 (p < 0.05 to p < 0.01), dosed with 1,000 mg/kg.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Mean water consumption was increased in male rats of group 4 (p < 0.01). Regarding the time course of water consumption, in week 1 (daily dose of 1,000 mg/kg), there was only a slight increase in water consumption (n.s.), in week 2 (daily dose of 1,000 mg/kg) and 3 (daily dose of 400 mg/kg), increase was most expressed (p < 0.01), in week 4, it was no longer observable.

The female rats of group 4 showed a slight increase in water consumption (n.s.) only in week 1 (daily dose of 1,000 mg/kg).

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No compound related findings were observed. In all groups, stripformed inclusions and vesicular inclusions were seen in the cornea. For detailed information on the incidence in the different test groups. In addition, vessels in the anterior chamber and in the vitreous body were seen in the male animal of group 4 showing stripformed inclusions. None of these findings was regarded as being compound-related, since they are all known to be spontaneous findings in the rat eye.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A slight decrease in PCV (p < 0.01) and hemoglobin (p < 0.01) resulting in a slight decrease in calculated MCV (p < 0.01) and MCH (p < 0.05) was noted in male animals of the high dose­ group on day 28.

A slight increase in erythrocyte count (p < 0.05) in female animals of the highest dose group resulting in a slight decrease in the calculated MCV (p < 0.01) and MCV (p < 0.01) were observed on day 28.

Both findings are considered to be of no biological relevance due to the fact that the alterations compared to the control group were rather small.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase (p<0.05 -p<0.01) in sodium and chloride was observed in male animals of all treatment groups on day 28. An increase (p<0.05 -p<0.01) in potassium was noted in males of groups 3 and 4 on day 28. Both alterations are suspected to be compound­related although the effect was not observed in female animals and there is no evidence of a sex specific action of the compound from other parameters. However, since both effects are not pronounced markedly and since all values are well within the historical reference value range, the effect is not regarded as being of toxicological relevance.
None of the other statistically significant differences in biochemical parameters were considered to be compound-related

Coagulation: No compound-related effect was observed.
The slight decreases (p < 0.05) in activated partial thromboplastin time and fibrinogen in male animals at the mid dose of 200 mg/kg were not considered as compound-related , because of lack of dose-dependence.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No compound-related effect was observed.

None of the findings observed in urine, such as protein, blood, ketones and urobilinogen as well as urinary sediment findings were considered to be compound-related, because they were also seen incontrol animals or occurred only sporadically.

The observed presence of bilirubin, mainly in urine of female animals of the high dose group, was not considered as biologically relevant, since this finding did correlate neither with other specific hepatic parameters (serum enzymes), nor with the results of the histological examination of the liver.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No compound-related findings were observed. One female rat in control group 1 showed a delayed reaction in the sensitivity test on day 6 and one female rat in test group 3, dosed with 200 mg/kg, showed a missing response of the hindlimb in the grasping reflex on day 19/20. Both findings were regarded as incidental due to the sporadic occurrence.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute and relative organ weights of the kidneys were increased in the medium and high dose groups while this effect was statistically significant only in females of the high dose group. This correlated to the macroscopic and histologic findings in the kidneys in those dose groups and is therefore considered to be compound-related.

As the significant decrease in relative organ weight of the liver in female animals in the low dose group only had no correlate to any macroscopic or microscopic finding, this effect is considered incidental.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some kidneys in the medium and high dose group showed macroscopic changes, that correlated to histologic findings of degeneration, regeneration and repair.

The diminution of the thymus in two animals of the high dose group correlated to a lymphatic atrophy that was seen in some animals in the high dose group in thymus, spleen, mesenteric lymph node and bone marrow. This macroscopically evident lymphatic atrophy as well as the findings in the stomach, which correlated histologically to stomach ulceration, are considered mainly related to the stress of the animals before death, as they occurred only in animals that died in the course of the study.

All other macroscopical findings are considered as being unrelated to the treatment. They were detected as single events only or in comparable numbers intreated animals and controls and the histopathological correlate was not suspicious for a treatment-related effect.

Blood soaked areas in the subcutaneous tissue of the cervical skin were not collected and examined histopathologically, as these lesions are unequivocal circumscribed paravenous hematomas subsequent to the venous puncture for blood sampling.

The changes in the lungs (not collapsed, reddish discoloration, watery outflow from cut surface and foamy outflow from bronchi) concerned only one animal and were due to a misplaced application.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The male reproductive organs (testes, epididymides, prostate and seminal vesicles) are smaller and immature in those animals that died early in the study. This is due to the younger age of the animals at necropsy and not considered compound-related.

All other findings are common in the used strain of rat. Neither the incidence nor the distribution or morphological appearance give any conclusions as to these being treatment related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity

1

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

kidney

2

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

other: lymphatic system

Organ:

bone marrow

lymph node

spleen

thymus

Applicant's summary and conclusion

Conclusions:

28d repeat dose i.g. with rats according to OECD 407 at dose levels of 40, 200, and 1000 mg/kg resulted in kidney toxicity and lymphoid atrophy (or depletion and apoptosis) at 1,000 mg/kg but concluded as resulting from stress rather than a direct toxic effect.

Executive summary:

28d repeat dose i.g. with rats according to OECD 407 at dose levels of 40, 200, and 1000 mg/kg resulted in kidney toxicity and lymphoid atrophy (or depletion and apoptosis) at 1,000 mg/kg but concluded as resulting from stress rather than a direct toxic effect. The repeated i.g. administration of the test substance to rats over a period of approximately 4 weeks was tolerated without toxicologically relevant findings at the low dose level of 40 mg/kg. Signs of kidney toxicity were observed from the mid dose onwards. However, investigation into biochemical parameters revealed no indication of an impairment of kidney function, which is considered to be due to the great compensatory capabilities of the kidneys. Mortality, preceded by reduction in food consumption and body weight gain occurred after repeated administration of the high dose of 1,000 mg/kg, which made a dose reduction from 1,000 to 400 mg/kg necessary. No mortality occurred after this dose reduction and reduction in food consumption and body weight gain disappeared.