2,7-NAPHTHALENEDISULFONIC ACID 5-[[4-CHLORO-6-(ETHYLPHENYLAMINO)-1,3,5-TRIAZIN-2-YL]AMINO]-3-[[5-[(2,3-DIBROMO-1-OXOPROPYL)AMINO]-2-SULFOPHENYL]AZO]-4-HYDROXY-,SODIUM SALT

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: JAP (EA 700, MHW 1039, MITI 1014)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40508/A
- Batch No.: Roe TV 1P
- Storage: refrigerator
- Purity: ca. 60%
- Expiration date: December 1997
- Stability in vehicle: at least 4 hours at room temperature

Test animals

Species:

rat

Strain:

other: Tif: RAIf (SPF), hybrids of RII/1 x RII/2 (Sprague-Dawley derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Production, Ciba-Geigy Ltd., 4332 Stein / Switzerland
- Age: approximately 5 weeks at delivery
- Weight at study initiation: 143.9 - 187.7g in males, 126.7 - 158.6 g in females
- Housing: in groups of 5 in macrolon cages type 4 with wire mesh tops and standardized granulated soft wood bedding (Societe Parisienne des Sciures Pantin).
- Diet (e.g. ad libitum): Pelleted, certified standard diet (Nafag No. 8900 FOR GLP) was provided ad libitum.
- Water (e.g. ad libitum): Tap water was given ad libitum.
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±10
- Air changes (per hr): 16-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Volume of solution applied: 10 mL/kg bodyweight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Control analyses of the test article concentration in the vehicle were carried out at all dose levels on samples collected once per experimental week. The samples were collected on completion of dosing, immediately deep frozen and sent to the analytical laboratories of Genetic Toxicology, Ciba-Geigy Ltd., 4002 Basle / Switzerland.

Duration of treatment / exposure:

28 days

Frequency of treatment:

1 dose per day, 7 times per week.

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 200, 1000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

The number of rats assigned to toxicity and recovery testing per group were:
- 5 per sex/dose for the toxicity test
- 5 per sex for 0 and 1000 mg/kg bw for the recovering test

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of the following previously conducted studies: Project no. 945053: Acute oral toxicity in the rat: LD50 in rats of both sexes: greater than 2000 mg/kg bodyweight and Project no. 945058: 5 days range finding study in rats (gavage): FAT 40508/A was administered by daily gavage to 3 male rats per dose group at dose levels of 0, 10, 100 and 1000 mg/kg bodyweight. With reference to appearance, behaviour, bodyweight development and food intake, no signs of toxicological relevance were detected.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

- Mortality: All animals were checked daily (a.m. and p.m. on working days, a.m. on weekends and holidays), in order to record mortalities, and to allow dead or moribund animals to be submitted to necropsy as soon as possible.
- In-life observations: In order to detect changes in state of health or behaviour, or any reaction to treatment, examination was carried out daily, and observations were recorded at least weekly.
- Bodyweight: The weight of all animals was recorded individually at weekly (midweek) weighing sessions. The first weights were recorded during the acclimatation period. Daily bodyweights for accurate dosing were measured but not recorded.
- Food consumption: The food consumption was recorded weekly (cagewise) and was calculated for periods of one week. The calculation was based
on the weight of the offered diet at the beginning of a weighing period and its difference to the re-weighed amount after several days. The individual food consumption values were calculated from the food consumption per cage and the number of animals present.
- Food consumption ratios: The food consumption ratios were calculated as mean of individual ratios.
- Water consumption: The water consumption was recorded weekly (cagewise) and was calculated for periods of one week. The calculation was based
on the weight of the offered water at the beginning of a weighing period and its difference to the re-weighed amount after one day. The individual water consumption was calculated from the water consumption per cage and the number of animals present.
- Laboratory investigations: Laboratory investigations (hematology, blood chemistry and urine analysis) were carried out on all surviving animals of each dose group at the end of the treatment period, and additionally at the end of the recovery period on animals of the control and high dose group kept for reversibility evaluation. To reduce the biological variability due to circadian rhythms, blood sampling was performed in the morning. Ether anesthesia was used to restrain the animals. Blood was withdrawn from the orbital sinus using glass capillary tubes. The following anticoagulants were used: EDTA for performing the complete blood count, 3.8% sodium citrate for coagulation testing and heparin for blood chemistry and methemoglobin investigations. Food was withheld overnight prior to blood removal. Urine for analysis was collected overnight. The individual animals were housed in special metabolism cages. Food and water was withheld during the time of urine collection. The parameters measured: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Leukocyte count, Differential leukocyte count (Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells), Thrombocyte Count, Prothrombin time, Methemoglobin. Blood chemistry: Glucose, Urea, Creatinine, Total bilirubin, Total protein, Albumin, Globulin, A/G Ratio, Cholesterol, Triglycerides, Sodium, Potassium, Calcium, Chloride, Phosphorus inorganic, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Gamma-glutamyl transpeptidase. Urinalysis: Urine volume, Relative density, pH-value, Urine colour, Protein, Glucose, Ketones, Bilirubin, Blood, Urobilinogen.

Sacrifice and pathology:

- Macroscopical examination: At the end of scheduled sacrifices all control and surviving treated animals were bled under ether anesthesia and subjected to detailed necropsy. At necropsy the following weights were recorded from all animals: body (exsanguinated), brain, liver, kidneys, adrenals, ovaries/testes.
- Microscopical examination: The following organs and tissues were preserved in neutral buffered 4% formalin: skin, mammary area, spleen, mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint, skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland (both), liver, pancreas, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), kidney (both), urinary bladder, prostate, seminal vesicle, testis (both), epididymis (both), uterus, vagina, ovary (both), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve (both), orbital gland (both), extraorbital lacrimal gland (both), Zymbal gland (both), muzzle, tongue, any tissue with gross lesions.
After the fixation, organ samples listed below were taken, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin, and subjected to a microscopical examination: spleen, heart, liver, kidney (both), testis (both), epididymis (both), adrenal gland (both), any organ with gross lesions. A complete necropsy with tissue preservation and processing was also performed on animals nos. 18 and 25 which died during the test period. Additionally, tissue samples of the kidneys from male recovery groups were processed and examined microscopically to establish reversibility of changes identified in the main group.

Statistics:

For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods were applied, to allow for non normal as well as normal data distribution. Each treated group was compared to the control group either by Lepage's or by Wilcoxon's two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives. The Lepage test is a combination of Wilcoxon
and Ansari-Bradley statistics, i.e. a combined test for location and dispersion. The Lepage test has a good power against the more general alternative that the distributions differ not only in location but also in dispersion. The Jonckheere test is sensitive to monotone dose-related effects. Statistical significance does not necessarily imply biological relevance. Hence, the responsible scientist may not comment on statistically significant values lying within the physiological
range and on the other hand may comment on values, which differ substantially from the expected normal values although this difference was not statistically significant.

Results and discussion

Results of examinations

Details on results:

- In-life observations: In this study, there was no treatment-related change of the appearance or the behaviour of the animals.
- Mortality: Two animals (no. 18 and 25) died during the study. Microscopical investigation, however, revealed a non test article-related cause of death in both cases.
- Bodyweight: The treatment did not influence the bodyweight development. In the absence of dose-relationship, the slight differences in the males were considered to reflect natural variability.
- Food consumption: The food intake was not influenced by the treatment. Slight differences to the control values were considered to reflect biological variability.
- Food consumption ratios: The mean food consumption ratios were not influenced by the treatment. The minor non dose-dependent differences to the control values noted in male groups 3 and 4 (200 and 1000 mg/kg) were considered to reflect the range of biological variability.
- Water consumption: The overall mean water consumption (week 1-4) was decreased by 18%, 15% and 13% in male groups 2, 3, and 4 (50, 200 and
1000 mg/kg), respectively.
- Hematology: The treatment had no influence on the hematological profile. The minor inter-group differences reaching a level of statistical significance reflect the biological variation of the parameters and were therefore considered not treatment-related.
- Blood chemistry: A dose-related increase of plasma chloride levels was observed at week 5 investigation in all treated groups. Pathophysiological conditions resulting in increased plasma chloride levels are usually associated either with changes in plasma sodium levels or with acid-base disturbances . In the absence of corroborative findings the elevation of plasma chloride concentrations observed in this study is not fully understood. However, regarding the results of histopathological examination of the kidney this finding is considered toxicological relevant. Decreased plasma urea levels and lower activities of alanine aminotransferase such as recorded for high dose males are without toxicological relevance. All other inter-group differences attaining a level of statistical significance were of a small order of magnitude compared with the physiological variation of these parameters and were therefore considered not treatment-related.
- Urine analysis: Reddish discoloured urine was excreted by high dose animals (1000 mg/kg) at treatment end, whereas normal appearing urine was excreted by these animals at the end of the recovery period. Two out of ten male animals of group 4 (1000 mg/kg) excreted a larger amount of urine with decreased relative density. Parameters analysed by means of a spectroscopic method could not be measured in groups with discoloured urins.
- Organ weights and ratios: At treatment end, the absolute and relative mean kidney weights of males of group 4 (1000 mg/kg) were increased by 15% and 16%, respectively. The relative kidney weights of male groups 2 and 3 (50 and 200 mg/kg) were increased by either 11%. Other differences which attained a level of statistical significance were within the expected range and, therefore, not considered of toxicological relevance.
Macroscopical findings: One out of 5 males of group 3 (200 mg/kg) and 3/5 males of group 4 (1000 mg/kg) showed discolouration of various parts of the gastrointestinal tract with one animal of group 4 additionally showing discolouration of the mesenteric lymph node. Incidence of discolouration of various parts of the gastrointestinal tract did not show a consistent pattern but was prevailing in stomach and small intestine. The discolouration was not observed at the microscopical level in H&E stained sections, and therefore is likely to be the result of the physical properties of the test article at higher concentrations. No toxicological relevance is attributed to these findings. Males no. 18 and 25, one each of groups 3 (200 mg/kg) and 4 (1000 mg/kg) died during the study. Apart from discolouration of various parts of the gastrointestinal tract they showed hemorrhagic and mottled lungs, respectively. Microscopical examination established the cause of death as incidental since both animals died from the acute shock lung syndrome.
- Microscopical findings: Three out of 5 males of group 4 (1000 mg/kg) showed cytoplasmic vacuolization of cortical collecting tubules. This lesion was not present after 2 weeks of recovery. Males no. 18 and 25 which died during the study developed acute shock lung syndrome characterized microscopically by acute lung congestion and alveolar edema. This condition was the direct cause of death of these animals. Additionally, there was acute inflammation of the trachea in both cases. Animal no.25 had also renal tubular dilatation and a precipitate in cortical tubules. These lesions are regarded as part of the shock syndrome and were recorded as secondary diagnoses to lung edema. All other recorded microscopical findings in the main group occurred in comparable numbers and were similar in nature to those occurring spontaneously in our colony of laboratory rat. No experimental relevance was attributed to these findings.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Kidney weights and microscopical changes

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

NOAEL was determined to be 200 mg/kg/bw.

Executive summary:

In a GLP compliant repeated toxicity study, performed according to OECD guideline 407, rats were treated with the test substance (50, 200, and 1000 mg/kg bw) by repeated oral gavage, for a period of 28 days. The study was comprised of four groups, each containing five male and five female rats. For the post-exposure recovery period two satellite groups exposed to 0 and 1000 mg/kg bw for 28 days consisting of five male and five female rats each were monitored for an additional 14 days after the end of the 28 days treatment period. No treatment related effects were observed on mortality, clinical signs, food consumption, food consumption ratios, water consumption and hematology. A minimal and reversible elevation of plasma potassium concentrations was recorded for animals of group 4 (1000 mg/kg). A dose-related increase of plasma chloride levels was observed at week 5 investigation in all treated groups (50, 200 and 1000 mg/kg). Reddish discolored urine was excreted by high dose animals (1000 mg/kg) at treatment end, whereas normal appearing urine was excreted by these animals at the end of the recovery period. Two out of ten male animals of group 4 (1000 mg/kg) excreted a larger amount of urine with decreased relative density. At treatment end, the absolute and relative mean kidney weights of males of group 4 (1000 mg/kg) were slightly increased. The relative mean kidney weights of male groups 2 and 3 (50 and 200 mg/kg) were also slightly increased. Microscopical examination revealed in 3/5 males of group 4 (1000 mg/kg) cytoplasmic vacuolization of cortical collecting tubules of the kidneys. This lesion was reversible within 2 weeks of recovery. In conclusion, under the conditions of this test, treatment with the test substance did not provoke overt signs of toxicity. A reversible nephrotropic effect occurred, which was revealed by water consumption data, laboratory investigations, organ weight analysis and microscopical finding. The 200 mg/kg bodyweight dose level is regarded to be a NOAEL, since the effects noted up to this dose level were not accompanied by any overt signs of toxicity or corroborative microscopical finding. Moreover, all effects were reversible within the 2-weeks recovery period at all dose levels.