7-acetamido-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonic acid, sodium salt

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From September 23,1985 to October 23,1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1986

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 (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dilithium 7-acetamido-1-hydroxy-2-(4-((2-sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-3-sulfonate

EC Number:

401-010-1

EC Name:

Dilithium 7-acetamido-1-hydroxy-2-(4-((2-sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-3-sulfonate

Cas Number:

106027-83-2

Molecular formula:

C20H17Li2N3O11S3

IUPAC Name:

dilithium 7-acetamido-1-hydroxy-2-(4-((2-sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-3-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

see below

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is the recommenden species for this study. There exist plenty of historical data for the Wistar rat at the test facility

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: 6 weeks
- Housing: in fully air-conditioned rooms in Makrolon cages (type 4) on softwood granules (lignocellulose) into groups of 5 animals
- Diet: Altromin 1323, ad libitum, no food consumption between 9.30 to 12 (administered test item dose by gavage)
- Water: tap water in plastic water bottles ad libitum, no drink consumption between 9.30 to 12 (administered test item dose by gavage)
- Acclimation period: 5 days
- Health check: The behavior and general health of all animals used during the experiment was 2 x daily, checked on weekends and holidays, 1 x daily. Every week the rats to neurological disorders, clouding of the eye media, adverse effects on oral mucosa and disorders of tooth development were examined.

ENVIRONMENTAL CONDITIONS
- Temperature:22 ± 3° C
- Humidity: 50 ± 20 %
- Air changes (per hr):romm fully air conditioned
- Photoperiod: 12 hours cycle dark/light

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route is a possible route of exposure in humans

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test concentrations were prepared daily by dissolving the test material in deionised water at the following concentrations:

dose mg/kg bw day concentration %(w/v) appl. Volume ml/kg vehicle
0 0 5 water
62.5 1.25 5 water
250 5 5 water
1000 20 5 water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

30 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

62.5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:

The acute oral toxicity test showed an LD50 of 3630 mg/kg body weight in male and female Wistar rats. On the day of application, the following symptoms of toxicity were observed in both males and females: reduced spontaneous activity, drawn-in flanks, squatting position, ruffled coat, irregular breathing, narrowed eyelids, dizziness, prone position, staggering or uncoordinated gait.
In the females, spreading posture of the hind limbs, trembling when moving, tonic-clonic spasms, forward movement in crawling position, hyperthermia and red encrusted eyelid margins were also observed.

In a preliminary test, 5 male and 5 female Wistar rats were each given 1000 mg/kg body weight of the test substance on 12 days within 14 days.
At the beginning of the second week, body weight gain was reduced in both male and female animals.
After 12 applications, the range-finding test was terminated, as it was clear that a limit test with a dosage of 1000 mg/kg body weight per day is not feasible.

- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: no
- Rationale for selecting satellite groups: NA
- Post-exposure recovery period in satellite groups: NA
- Section schedule rationale (if not random): random
- Other:

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
The behavior and general health of all animals used during the experiment was 2 x daily, checked on weekends and holidays, 1 x daily.

DETAILED CLINICAL OBSERVATIONS: Yes
Every week the rats to neurological disorders, clouding of the eye media, adverse effects on oral mucosa and disorders of tooth development were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of all animals was determined at the beginning of the study and twice a week during the study.


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

OPHTHALMOSCOPIC EXAMINATION: No

CLINICAL CHEMISTRY: Yes
sodium
potassium
inorg. phosphorus
uric acid
total bilirubin
creatinine
Glucose in serum
Urea-N (BUN)
calcium
chloride
ASAT (G0T)
ALT (GPT)
alkaline phosphatase (AP)
y-glutamyl
Total protein
Electrophoresis

URINALYSIS: Yes
The urine production was carried out overnight on 29-30. The experiment (16 hours) to non-fed animals and not soaked in metabolic cages (single urine). The urine analyzes were performed on all males and females performed and extended to the following parameters:
Parameters. method
appearance
color
pH hemoglobin
protein
glucose
ketone bodies
bilirubin
urobilinogen
density
sediment

NEUROBEHAVIOURAL EXAMINATION: Yes

HAEMATOLOGY: Yes
The following haematological parameters were determined:
parameter method
hemoglobin
Erythrocyte count
Leucozytenzahl
hematocrit
reticulocyte*
Differential blood count
Platelet count

* Was performed only on the control group and the high-dose group Furthermore, the calculated values ​​for MCV, MCH and MCHC were determined.

Sacrifice and pathology:

Sacrifice:
After the retro-orbital blood sampling for hematological tests, the animals were in nembutal narcosis (injection of approximately 50 mg / kg ip) killed by transection of the vena cava cranialis and exsanguination.

GROSS PATHOLOGY: Yes
skin, body orifices, eyes, teeth, oral mucosa and the inner organs were assessed macroscopically.

HISTOPATHOLOGY: Yes
Of the animals of the main groups 1 - 4 were in accordance with Section I Procedure (Prof. K / G of 04.02.1982) the following bodies or fragments of these fixative in one set and delivered to the histological examination:
heart
lung
liver
kidneys
spleen
stomach
jejunum
Colon
bladder
testicle
epididymis
prostate
seminal vesicle
ovaries
uterus
thyroid
pancreas
adrenal
thymus
pituitary
brain
Eye with N. optic
Bone marrow (femur)

Statistics:

The following measurements were evaluated statistically at a significance level of p = 0.05:
Body weights on the individual time points
Body weight gains
Hematological parameters (except for differential blood count)
Clinical-chemical parameters
Absolute and relative organ weights
Urine analysis (pH and specific gravity)
The evaluation was carried out with the aid of a program package for evaluating toxicological tests, according to the Standard Operating Procedure

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two female and one male animal of the 1000 mg/kg bw/d group showed the following signs from the 22nd day of the study until the end of the study: ruffled fur, squatting, stilted gait, staggering and apathy.
It remained unclear whether these effects were related rather to mis-dosing than substance-related effects. No signs of organ toxicity could be detected during terminal examinations.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female animal of the high-dose group died on Day 29. Due to cannibalism it could not be necropsied, hence, it remained unclear whether the death of this animal was related rather to mis-dosing than substance-related effects.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistical analysis of body weight development showed a statistically significant retardation of body weight in the male animals of the highest dose group (1000 mg/kg bw/d) from the 15th day of the study. All other treated groups showed no statistically significant differences compared to the control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Absolute feed consumption was found to be unchanged by the test substance over the entire test period in the treated groups.

Food efficiency:

no effects observed

Description (incidence and severity):

Relative feed consumption was found to be unchanged by the test substance over the entire test period in the treated groups.

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

effects observed, treatment-related

Description (incidence and severity):

The relative water consumption was significantly increased after administration of 1000 mg/kg bw/d.
In the other dose groups the relative water consumption was unaffected by the administration of the test substance.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes in urinary status were observed. Sediments were inconspicuous. Urine of the 250 and 1000 mg/kg bw/d group was reddish to red in colour.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

In the 62.5 mg/kg bw/d group no statistically significant changes in absolute and relative organ weights occurred.
In the 250 mg/kg bw/d group, the absolute kidney weights of the male animals were statistically significantly increased; however, the deviations are extremely minimal, so that no toxicological relevance can be deduced from this, particularly due to the lack of dose dependence and the absence of histologically detectable changes.
In the absolute organ weights of the highest test concentration (1000 mg/kg bw/d) there was an increase in adrenal gland weight and a decrease in testicular weight in the male animals. The relative organ weights of the 1000 mg/kg bw/d group showed a statistically significant increase in adrenal and brain weights in the males. The increase in relative organ weights can be attributed to the lower terminal body weight in these animals.
In the female animals of this group, an increase in absolute kidney weights and a decrease in ovary weights was observed. The relative organ weights showed an increase in kidney weights and a decrease in ovarian weights in the females.
All these effects were considered to be incidental findings, routinely seen in this strain of rats and to be of no toxicological relevance in the absence of any histopathological correlates.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examinations revealed a pink discoloration of the gastrointestinal tract in the animals of the highest dose (1000 mg/kg bw/d). The reproductive organs of the male animals (seminal vesicle, testes) of the 1000 mg/kg bw. group were in some cases reduced in size. This finding is considered an incidental one, often seen in this strain of rats and has no histopathological correlate.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopy revealed no morphologically detectable substance-related organ damage in the male and female rats.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

ca. 250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

water consumption and compound intake

Remarks on result:

other:

Remarks:

the effects observed were most likely attributed to the lithium salt of the the test material and not related to the chromophore

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

water consumption and compound intake

Remarks on result:

other:

Remarks:

The clinical signs in one male and two female of the high-dose group are most likely related to mis-dosing, as all other animals of this group remained unaffected, and no correlates were seen in clinical and anatomical pathology. The impaired body weight development in males and the increased water consumption in all animals of the high-dose group are related to the high content of lithium, which is well-known to cause polydispsia and not related to the chromophore.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a 30-day test by daily gavage administration of the test substance at a dose-level of 1000 mg/kg body weight of rats, resulted in slight impairment of body weight development in males and water consumption. Two female and one male showed clinical signs of impaired . A female died on 29° day of the experiment. these effects are most-likely rather related to mis-dosing than toxicological effects. The hematology, clinical chemistry, and macroscopic and histological studies revealed no evidence of specific toxicity. A NOEL of 250 mg/kg bw/d was determined. As the impaired body weight development in males and the increased water consumption in all animals of the high-dose group are related to the high content of lithium, which is well-known to cause polydipsia and not related to the chromophore, the NOAEL was considered to be 1000 mg/kg bw/d.

Executive summary:

The test substance was administered orally (gavage) for 30 days at dose levels of 0, 62.5, 250, and 1000 mg/kg body weight per day to male and female SPF-Wistar rats.

Behaviour, mortality and clinical signs were assessed daily; body weight and food consumption twice weekly, and water consumption once a week. Hematological, clinical chemistry and urinalysis tests were performed at the end of the study. At necropsy, the animals were examined macroscopically for gross lesions, the major organs were weighed and relative organ weights calculated. A large number of tissues were examined histolopathologically. The body weights, haematological and clinical chemistry parameters and urinalysis (specific gravity, pH) and the absolute and the relative weights were statistically tested in comparison to the control group.

Neurological disturbances, turbidity of the eye media, disturbances of the tooth growth or changes of the oral mucous membranes which could be related to administration of the test substance, were not observed in any dose group. The behaviour and the general state of health of the animals of the 62.5 and 250 mg/kg bw groups were not affected by administration of the test substance. Two female and one male animals of the 1000 mg/kg bw group showed piloerection, hunched posture, stilted gait, tumbling and ataxia from day 22 onwards. One of the female animals died on day 29. This might be an effect due to a false gavage.

Statistical analysis of the body weight development at 1000 mg/kg bw males showed a statistically significant retardation of the body weight gain from day 15 onwards. All other animals did not show any changes in body weight development. Relative water consumption was increased at 1000 mg/kg bw.

No adverse effects were observed in haematology, clinical chemistry, or urinalysis. The only changes in urine status were reddish discolorations by the dyestuff at 250 and 1000 mg/kg bw.

At necropsy, pink discoloration of the stomach and intestines and partially smaller male sexual organs at 1000 mg/kg bw. These changes were not reflected microscopically.

The observed effects in body weight development in males and increased water consumption is typical for lithium salts an has been observed in many other dyes with lithium as counter cation.

The no observed effect level (NOEL) is 250 mg/kg bw/day, due to the fact that effects on body weight and water consumptiion is a salt effects and not related to the chromophore, the NOAEL is considered to be 1000 mg/kg bw/day.