1,7-Naphthalenedisulfonic acid, 2-[[4-chloro-6-(cyanoamino)-1,3,5-triazin-2-yl]amino]-5-hydroxy-6-[2-[2-methoxy-5-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, lithium sodium salt (1:?:?)

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:

From Jul. 13, 1999 to Dec. 23, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to EU Method B.7. and OECD Guideline 407 in compliance with GLP

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

440-050-4

EC Name:

-

Cas Number:

243857-97-8

Molecular formula:

Hill formula: C23H(19-x-y)ClLixN8NayO14S4 (x+y=3), x and y > 0

IUPAC Name:

lithium(1+) disodium 2-{[4-chloro-6-(cyanoamino)-1,3,5-triazin-2-yl]amino}-5-hydroxy-6-(2-{2-methoxy-5-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-1,7-disulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Reaktiv Orange DYPR 1466

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HARLAN WINKELMANN Gartenstr. 27, D-33178 Borchen, SPF breeding colony
- Age at study initiation: Approximately 6 wk
- Housing: Macrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet: ssniff R/M (V1534), ad libitum, except for the period in which the animals were kept in diuresis cages
- Water: Tap water in plastic bottles, ad libitum, except for the period in which the animals were kept In diuresis cages
- Acclimation period: At least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 °C
- Humidity (%): 50±20 °C
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

IN-LIFE DATES: From Sep. 19, 2000 to Oct. 318, 2000

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance was suspended in the concentrations of 1.25, 5.0 and 20 % in deionized water. After each measurement of the body weight, the calculation of the application volume was repeated. The final dosing volume was 5 mL/kg bw

VEHICLE
- Concentration in vehicle: 1.25, 5.0 and 20 % w/v
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

29 d, 28 applications

Frequency of treatment:

Once a day for 28 d

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 62.5, 250, 1000
Basis:
other:

No. of animals per sex per dose:

5/sex/dose in main groups
5/sex/dose in recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment (if not random): Randomization using computer-generated algorithm
- Rationale for selecting satellite groups: To check the reversibility of the effects, if any
- Post-exposure recovery period in satellite groups: Yes

Positive control:

Not used in the study

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily in all groups

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before start of the study and once a week during the study

BODY WEIGHT: Yes
- Time schedule for examinations: weekly once

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: Weekly once

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period
- Anaesthetic used for blood collection: Yes (intraperitoneal injection of 67 + 6.7 mg/kg bw Ketamine HCl + Xylazine)
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period
- Anaesthetic used for blood collection: Yes, killed by section of the vena cava cranialis in deep narcosis (intraperitoneal injection of 67 + 6.7 mg/kg bw Ketamine HCl + Xylazine) and exsanguinated

URINALYSIS: Yes
- Time schedule for collection of urine: Few days before termination of the study as well as before the end of the recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before start of the study and once a week during the study
- Dose groups that were examined: All groups
- Battery of functions tested: sensory reactivity, grip strength and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals were necropsied and checked for macroscopically visible abnormalities. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.

HISTOPATHOLOGY: Yes, Following tissues/organs were preserved in a suitable fixative and processed for histopathological investigations: adrenal gland, bone marrow (sternum), brain, heart, colon, jejunum, kidneys, liver, lungs, lymph nodes (mandibular and iliac), ovaries, uterus, thyroid and parathyroid glands, prostate gland, spleen, stomach, testis, epididymides, thymus, trachea, urinary bladder, N. ischiadicus and spinal cord (cervical).

Other examinations:

Following organs were weighed and the organ to body weight ratios calculated:
Heart , liver, kidneys, adrenal glands, spleen, testes, epididymides, thymus and brain

Statistics:

Evaluation was performed by l/S DI&A, Aventis Pharma Deutschland GmbH, with the aid of a program package for the evaluation of toxicological studies.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No deaths occurred throughout the study. Increased salivation during and after substance administration was observed temporarily in some animals of the intermediate and high dose groups from Day 10 on until Day 28 of the study. Behavior and state of health.

BODY WEIGHT AND WEIGHT GAIN: Body weight gains of female animals were noted to be significantly higher in the intermediate and high dose groups during the treatment period. body weight gains in males of the high dose group were noted to be significantly lower compared to the control.

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption remained unaffected by the administration of the test substance throughout the study in all dose groups.

WATER CONSUMPTION AND COMPOUND INTAKE: Water consumption was distinctly increased in all animals of the high dose group and minimally increased in the mid-dose females in the course of the treatment period.

HAEMATOLOGY: The hematological investigations revealed an apparently dose dependent increase of hemoglobin in female animals at the end of the 28-d treatment period. At the end of the recovery period male animals of the high dose group showed an increase in red blood cell count, hemoglobin and hematocrit.

CLINICAL CHEMISTRY: Main group: a statistically significant decrease of sodium in high-dose males and an increase in total bilirubin and total protein in intermediate and high dose males. In females a statistically significant increase in calcium in the intermediate and high doses and of total bilirubin,
total protein and ALAT of animals of the low, intermediate and high dose groups was observed.
Recovery group: An increase in sodium, total bilirubin, total protein and albumin and a decrease in potassium and glucose in males of the high-dose group. In female animals an increase in phosphorus, total bilirubin and glucose and a decrease in sodium and triglycerides was noted in the high dose group.

URINALYSIS: An increase in the volume of the urine in females of the intermediate and high dose group. The urine of most males of the 28-d treatment and the recovery high-dose group was discolored salmon-pink in different intensities. In female high-dose animals the urine was discolored very light to light salmon-pink in one animal of the final and three animals of the recovery group.

NEUROBEHAVIOUR: Neurotoxicological measurements including 'open field' observations, assessment, of sensory function, and forelimb and hindlimb grip strength were not influenced by the administration of the test substance in all groups.

ORGAN WEIGHTS: An increase in the absolute organ weights of the adrenals of the animals of the high dose group and of the heart and kidneys of the high dose females and the liver of the females of the intermediate and high dose groups. In the relative organ weights there was an increase of the spleen weight of the males of the high dose group and of the liver weights of the females of all treated groups observed. Additionally, a decreased mean brain weight in females of the high dose group was noted.
At the end of the recovery period: Increased adrenal weights in female animals of the high dose group. In the relative organ weights an increase of the adrenal weights of male high dose animals was observed.

GROSS PATHOLOGY: The animals of the intermediate and/or high dose group showed light red, red or brown-red discolored kidneys. Additionally, the animals of the high dose group showed light red discolored skin and adipose tissue. The male animals of the high dose group showed light red discolored testes and sporadically light red discolored epididymes. At final value only, the animals of the high dose group showed light red or red stomach and small intestine. Other sporadic findings were pelvic dilatation in the kidney of one female of the intermediate dose group and a constriction in the spleen in one female low-dose animal.

HISTOPATHOLOGY: NON-NEOPLASTIC: Submucosal mixed-cellular infiltrations in the stomach of several animals of terminal sacrifice high dose group and an intratubular deposition of yellowish pigment in the kidneys of nearly all animals of the high dose group (terminal sacrifice and recovery). Sporadic findings were observed in various organs of different animals; type and incidence of these changes were not relevant for the assessment of the test article.

HISTORICAL CONTROL DATA (if applicable): Most changes concerning clinical chemistry were within the historical control data range

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Effect level:

62.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Discoloration of the serum, urine and some organs in the animals of the intermediate and high dose groups, consequently, the NOEL is 62.5 mg/kg bw/kg bw/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the NOAEL and NOEL of the test substance were determined to be 250 and 62.5 mg/kg bw/day respectively in rats.

Executive summary:

A study was conducted to assess the subacute repeated dose toxicity of the test substance in rats according OECD guideline 407 and EU Method B.7.

Groups of male and female rats received test substance by oral gavage at dose levels of 0, 62.5, 250 or 1000 mg/kg bw/day for a period of 28 d. On Day 29, 5 males and five females from each group were killed and necropsied. Five males and five females from the control and high dose group were killed and necropsied after a recovery period of 14 d.

Behavior and state of health were observed daily in all groups. Body weights and food consumption were recorded twice weekly, water consumption once weekly. Once before the first treatment and once a week thereafter detailed clinical observations were performed in all animals outside the home cage in a standard arena ('open field'). Additionally, the animals were examined for opacity of the refracting media of the eyes, damage to the oral mucosa and impairment of dental growth.

Neurotoxicological measurements including assessment of sensory function, motor activity, forelimb and hind limb grip strength were conducted at the end of the treatment period. Hematological examinations, clinical chemistry and urine analysis were carried out at the end of the treatment period and after the recovery period.

During necropsy the animals were examined for macroscopically visible abnormalities, the main organs were weighed and the organ to body weight ratios calculated. Organs and tissues were processed for histopathological examination and checked for microscopically visible changes.

Body weights, hematological and clinical chemistry data, urine data (volume, specific weight), absolute and relative organ weights and neurotoxicological measurements (motor activity, forelimb and hind limb grip strength) were analyzed with the aid of a statistical program.

No deaths occurred throughout the study. Behavior, state of health, neurotoxicological, body weight gains and food consumption remained unaffected by the administration of the test substance in all dose groups.

Water consumption was distinctly increased in all animals of the high dose in the course of the treatment period. Consequently, the urine volume was increased in this group during the treatment period. Polyuria and polydipsia are well-known side effects for lithium. As the content of lithium is rather high in this test substance, these effects were most likely related to the lithium salt content.

The urine of most males of the 28-d treatment and the recovery high-dose groups was discolored salmon-pink in different intensities. Hematological and clinical chemistry investigations and organ weights did not exhibit treatment related adverse effects. The serum of almost all animals of the high-dose recovery and the intermediate and high dose final groups was discolored by the dye.

At necropsy of the final and/or recovery value animals of the intermediate and/or high dose groups showed reddish discolored Kidneys, skin, adipose tissue, testes, epididymes, stomach, and small intestines.

In conclusion, repeated administration of test substance caused reversible slight acute topical irritations in the submucosa of the stomach and pigment depositions in the kidneys of animals of the high dose group without causing any tissue damage. Additionally these animals showed sporadically temporarily increased salivation and distinctly increased water consumption, due to the lithium salt content. The test substance did not cause compound related adverse effects in the intermediate and low dose groups.

Under the test conditions, as a consequence of the combination of the effects in the high dose group the NOAEL is 250 mg/kg bw/day. Test substance caused discoloration of the serum, urine and some organs in the animals of the intermediate and high dose groups, consequently, the NOEL is 62.5 mg/kg bw/kg bw/day (Dr. Seeberger A, 1999).