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Description of key information

Several studies with either the test substance at a limit dose of 500 mg/kg bw/day administered daily for 14 days or structural analogues of the test substance administered daily for 28 or 30 days up to 1000 mg/kg bw/day resulted in no adverse effects due to the chromophoric structure of the dye. The only effects observed with the structural analogue SA01-Li was due to the adverse effects of the Li-cation. As the test substance is a Na-salt, the NOAEL for Reactive Orange 72/78 is considered to be 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
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
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

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
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
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:
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
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.
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
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.
Critical effects observed:
no
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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From Oct. 01, 1997 to Mar. 03, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: HMR Deutschland Gmbh, Kastengrund, SPF breeding colony
- Age at study initiation: Approximately 5-6 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 Oct. 01, 1997 to Oct. 29, 1997
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:
yes
Details on analytical verification of doses or concentrations:
after each formulation; HPLC
Duration of treatment / exposure:
29 d, 28 applications

Frequency of treatment:
Once a day for 28 d

Remarks:
Doses / Concentrations:
0, 62.5, 250, 1000 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1.25, 5.0 and 20 %
Basis:
nominal in water
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:
- Dose selection rationale: In an acute oral toxicity study, the LD50 in Wistar rats was >2000 mg/kg bw/d. Based on these results test substance was tested in the present study at the dose levels of 0, 62.5, 250 and 1000 mg/kg bw/d.
- 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

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: Before the start of the study and twice weekly throughout the study

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, 2 times/wk

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, measurement of motor activity, rearings, forelimb and hindlimb grip strength and landing foot-spread

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 , 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 IS Research and Preclinical Devolopment; HMR Deutschland GmbH, with the aid of a program package for the evaluation of toxicological studies.

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: No deaths occurred throughout the study. Male and female animals of the high dose group showed orange coloured faeces from Day 7 till the end of the study.

BEHAVIOUR AND STATE OF HEALTH: Remained unaffected by the administration of the test substance

BODY WEIGHT AND WEIGHT GAIN: Remained unaffected by the administration of the test substance in all dose groups.

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 remained unaffected by the administration of the test substance

HAEMATOLOGY: No treatment-related changes observed in any of the dose groups

CLINICAL CHEMISTRY: Increases in urea, cholesterol, triglyceride, and albumin values as well as decreases in inorganic phosphor and serum glucose levels in males of the high dose group. In addition, male animals of the intermediate and high dose groups showed reduced ALAT levels. In female animals of the high dose group decreased uric acid values and in females of the intermediate and high dose groups decreased creatinine values were found. All these changes were within the physiological range of rats and these are not considered as treatment related.

URINALYSIS: No treatment-related changes were detected by urine analysis.

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: No treatment-related changes observed in any of the dose groups

GROSS PATHOLOGY: No treatment-related macroscopically visible changes were observed in all dose groups

HISTOPATHOLOGY: No treatment-related microscopic changes in the organs of the examined animals


Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects (No treatment related effects in any of the treated animals)
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects (No treatment related effects in any of the treated animals). Orange coloured faeces in high dose group was not considered to be of toxicological relevance.
Critical effects observed:
not specified

None

Conclusions:
Under the test conditions, both the NOAEL and NOEL of the test substance were determined to be 1000 mg/kg bw/d in 28-study in rats.
Executive summary:

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

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 5 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, rearings, forelimb and hind limb grip strength, landing foot-spread 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 (pH, 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. Male and female animals of the high dose group showed orange coloured faeces from Day 7 till the end of the study. Behavior, state of health, neurotoxicological, body weight gains, food and water consumption remained unaffected by the administration of the test substance in all dose groups.

Hematological and clinical chemistry investigations and organ weights did not exhibit treatment related adverse effects. At necropsy of the final and/or recovery, no compound-related macroscopically visible changes were observed in all dose groups. Likewise, histopathological examinations revealed no treatment related changes in any dose group.

In conclusion, no treatment-related adverse effects were observed in any of the dose group animals.

Under the test conditions, both the NOAEL and NOEL of the test substance were determined to be 1000 mg/kg bw/day in 28-d study in rats.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17. May 1974 to 30. May 1974
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
duration 14 days, limit dose 500 mg/kh bw
Principles of method if other than guideline:
Internal Guideline Hoechst AG
GLP compliance:
no
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG
- Weight at study initiation: males: mean 113 g (102 - 120 g); females: mean 103 g (96 - 112 g);
- Fasting period before study: none
- Housing: group-housing: 5 rats per sex and cage
- Diet (ad libitum): Altromin 1324 (Altrogge, Lager/Lippe, Germany)
- Water (ad libitum): tap water
- Acclimation period: NA

From: 17. 5. 1974 to 30. 5. 1974
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
5% solution in water


VEHICLE
- Justification for use and choice of vehicle (if other than water): -
- Concentration in vehicle: 5% (50 mg/mL)
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
-
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
NA
Positive control:
NA
Observations and examinations performed and frequency:
Body weight: twice weekly
Clinical signs: daily
Urine: appearence, color, pH, glucose, bilirubin, protein, hemoglobin, sediment
Hematology: hemoglobin, hematocrite, erythrocyte count, leukocyte count, differential blood cell count, heinz bodies
Sacrifice and pathology:
Sacrifice: cervical dislocation and exsanguination
Necropsy: macroscopic evaluation
organ weights: heart, lung, liver, kidney, spleen
microscopic examination: heart, lung, liver, kidney, adrenal, spleen
Other examinations:
behavior
Statistics:
no data
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
urine and feces stained by test article
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
not specified
Conclusions:
No adverse effects were observed at daily administration of the test substance at 500 mg/kg bw/day for 14 days. The NOEL was determined to be 500 mg/kg bw/day in male and female rats
Executive summary:

The test substance was administered daily by gavage at a limit dose of 500 mg/kg bw/day to 10 male and female Wistar rats for 14 days. A control group received the vehicle water unter the same test conditions.

The rats were in good general condition at the start of the experiment; the body weight on the day of the first application was on average 113 g (102 - 120 g) in the males and 103 g (96 - 112 g) in the females. The rats were kept separately by sex in plastic cages on wood shavings in groups of 5 animals each. The behaviour and general state of health of each animal was checked daily, body weights were checked twice a week, and at the beginning and end of the experiment the blood count (haemoglobin content, erythrocytes, leucocytes, haematocrit, differential blood count and the possible presence of Heinz' bodies) and urine (appearance, colour, glucose, bilirubin, protein, pH, haemoglobin and sediment) were examined.

After the end of the study, all animals were killed under nembutal anaesthesia, necropsied and the organs subjected to histological examination.

There were no adverse effects observed in clinical signs, body weight development, urinalysis, hematology, marco- and microscopic evaluation. The test item was excreted via feces and urine.

The No Observed Effect Level is 500 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test substance was administered daily by gavage at a limit dose of 500 mg/kg bw/day to 10 male and female Wistar rats for 14 days. A control group received the vehicle water under the same test conditions.

The rats were in good general condition at the start of the experiment; the body weight on the day of the first application was on average 113 g (102 - 120 g) in the males and 103 g (96 - 112 g) in the females. The rats were kept separately by sex in plastic cages on wood shavings in groups of 5 animals each. The behaviour and general state of health of each animal was checked daily, body weights were checked twice a week, and at the beginning and end of the experiment the blood count (haemoglobin content, erythrocytes, leucocytes, haematocrit, differential blood count and the possible presence of Heinz' bodies) and urine (appearance, colour, glucose, bilirubin, protein, pH, haemoglobin and sediment) were examined.

After the end of the study, all animals were killed under nembutal anaesthesia, necropsied and the organs subjected to histological examination.

There were no adverse effects observed in clinical signs, body weight development, urinalysis, hematology, marco- and microscopic evaluation. The test item was excreted via feces and urine.

The No Observed Effect Level is the limit dose tested of 500 mg/kg bw/day.

 

Structural Analogue 01-Lithium Salt was administered orally (gavage) for 30 days at dose levels of 0, 62.5, 250, and 1000 mg/kg body weight per day to 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 pathology, the major organs were weighed and relative organ weights calculated. A large number of tissues were examined histopathological. 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 animal of the 1000 mg/kg bw group showed piloerection, hunched position, stilted gait, tumbling and ataxia from day 22 onwards. One male animal died on day 28. 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, which are known to cause polydipsia and polyuria, and has been observed in many other dyes with lithium as counter cation.

The no observed effect level (NOEL) is 250 mg/kg bw/day. As Reactive Orange 72/78 has sodium as counter cation and no other adverse effects related to the dye itself could be observed, the NOAEL for Reactive Orange 72/78 is considered to be 1000 mg/kg body weight/day.

 

A study was conducted to assess the subacute repeated dose toxicity of the test Structural Analogue 02 in rats according EU Method B.7. and OECD guideline 407 in compliance with GLP.

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 5 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, rearings, forelimb and hind limb grip strength, landing foot-spread were conducted at the end of the treatment period. Haematological 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 (pH, 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. Male and female animals of the high dose group showed orange coloured faeces from Day 7 till the end of the study. Behavior, state of health, neurotoxicological, body weight gains, food- and water consumption remained unaffected by the administration of the test substance in all dose groups.

Hematological and clinical chemistry investigations and organ weights did not exhibit treatment related adverse effects. At necropsy of the final and/or recovery, no compound-related macroscopically visible changes were observed in all dose groups. Likewise, histopathological examinations revealed no treatment related changes in any dose group.

In conclusion, no treatment-related adverse effects were observed in any of the dose group animals.

Under the test conditions, both the NOAEL and NOEL of the test substance were determined to be 1000 mg/kg bw/day in 28-d study in rats.

Justification for classification or non-classification