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Description of key information

Nigrosin WLF dissolved in tap water was administered orally by gavage in doses of 0, 100,300 and 1000 mg/kg bw/day to male and female Wistar rats for a period of four weeks according to OECD TG 407 and GLP. Under the conditions described, the NOAEL for oral  administration of Nigrosin WLF to male and female rats was 1000 mg/kg bw(day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guidelinestudy and GLP
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation:
---males: 185g (174-193 g)
---females: 139 g (132-149g)
- Housing: in groups of 2-3 animals per cage
- Diet ad libitum
- Water ad libitum
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 55
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light) 12/12:
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
groups of male and female Wistar rats recieved daily doses of the test item by gavage
(males 30 days and females 31 days) ;the test item was dissolved in water; the administration volume was 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
analyses for stability and homogenicity
Duration of treatment / exposure:
males 30 days and females 31 days)
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
groups of male and female rats received daily doses of the test item which was dissolved ibn water, Treatment time for males included 30 days and for females 31 days. Animals were observed twice daily for mortality and morbidity, once daily for cliinical signs and were weighed, food and water consumption weekly
After termination of treatment determination of clinical chemistry data nd hematology then sacrifice and gross pathologicval examination and histological examination
Positive control:
no
Observations and examinations performed and frequency:
groups of male and female rats received daily doses of the test item which was dissolved ibn water, Treatment time for males included 30 days and for females 31 days. Animals were observed twice daily for mortality and morbidity, once daily for cliinical signs and were weighed, food and water consumption weekly
After termination of treatment determination of clinical chemistry data nd hematology then sacrifice and gross pathologicval examination and histological examination
The following hematological parameters were determined in peripheral blood:
Leucocytes, erythrocytes, hemoglobin, hematocrit, reticulocytes, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), thrombocytes, Hepato Quick, differential blood count
The following clinical chemistry parameters were determined:
Alanine aminotransferase, aspartate aminotransferase, albumin, protein (total), bile acids, cholesterol, creatinine, urea, glucose, sodium, potassium, total bilirubin
Sacrifice and pathology:
sacrifice
--- organ weight determination including rel organ weights
group 1-4
brain, epididymides, heart, kidneys, liver, ovaries/oviducts, prostate, seminal vesicles with coagulation glands
---gross pathologicval examination and histological examination
mainly group 1 and group 4, additional to the weighed organs:
caecum, colon, duodenumeyes, femur with bone marrow, joint, ileum, jejunum, larynx, lungs, lymph nodes mandibular mesemteric iliacale, optic nerves, pancreas, Peyer's patches, rectum, siatic nerve
Other examinations:
Functional observations (FOB) were performed once on all animals in dosing week 430 minutes after treatment
Motor activity assessments were conducted once following the FOB examinations
Statistics:
Dunnett-test, Dunnett exact homogenous test and heterogenous after logarithmic transformation, adjusted Mann-Whitney-U-test, Bonferroni Mann-Whitney U-test
Details on results:
CLINICAL SIGNS AND MORTALITY
---Survival was not affected by treatment
---At clinical observations the only treatment-related finding was that the color of feces was changed (black) in cages of treatment groups. It seems to be obvious that this finding is caused by the color of Nigrosin WLF

BODY WEIGHT AND WEIGHT GAIN
Body weight development of males and females was not retarded by the treatment up to 1000 mg/kg.

FOOD CONSUMPTION
WATER CONSUMPTION
Food intake in treated groups was comparable to the respective control group.
The water intake in treated groups was higher compared to the respective control group

OPHTHALMOSCOPIC EXAMINATION
not done

HAEMATOLOGY
Hematology gave no evidence for treatment-related effects up to 1000 mg/kg.

CLINICAL CHEMISTRY
At clinical chemistry concentration of creatinine was significantly decreased (p<=0.01) at 1000 mg/kg in males:
53.2µmol/l versus 58.8 µmol/l in controls
Furthermore, concentration of sodium in males was significantly decreased starting at 300 mg/kg. :
100, 300, 1000 mg/kg bw/day : 146.3 mmol/l , 144.6 mmol/l (p<=0.05), 143.6 mmol/l (p<=0.01) versus 147.o of controls
However, the differences to control were relatively slight and no corresponding findings were observed.
A toxicological relevant effect is therefore not assumed

URINALYSIS
not done

NEUROBEHAVIOUR
Functional observation battery; motor activity assessment:
the investigations gave no evidence for treatment-related findings

ORGAN WEIGHTS
the diffrences to control were slight and/or statistical significance was absent and
no treatment-related histopathological findings were observed;
therefore a toxicological relevance was not assumed from these data.
---males
slightly increased weights of adrenal glands starting at 300 mg/kg
increased weight of prostate at 1000 mg/kg.
---females
weight of thymus was increased in all dose groups.

GROSS PATHOLOGY and
HISTOPATHOLOGY: NON-NEOPLASTIC
MACROSCOPIC changes at terminal sacrifice where gray or black discolorations were seen in the intestine and/or Peyer's patch in a dose-related manner at 300 and 1000 mg/kg/day in both sexes, and also in one single female at 100 mg/kg/day these discolorations were also seen in the lymph nodes.

HISTOLOGICALLY, a minimal amount of gray-green pigment was noted in small intestinal villi in each one male rat treated at 300 or 1000 mg/kg/day. Minimal gray-green pigment deposition in the Peyer's patch and in sinusoidal macrophages of the mesenteric lymph node occurred in a dose-related manner in all treated groups and often confirmed the mentioned macroscopic changes.
Gray-green pigment in sinusoidal macrophages was also seen in the mandibular lymph node of one female treated at 300 and in the iliac lymph node of another female treated at 1000 mg/kg/day.
EVALUATION
All discolorations were considered to be due to the staining properties of the test item.
In view of the very limited degree of changes, and as any indication of structural change or functional impairment of the organs concerned was lacking, they were considered to be non-adverse.


Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: the treatment related slight discolorations are due to the staining property of the test item
Critical effects observed:
not specified
Executive summary:

100, 300 and 1000 mg/kg bw/day Nigrosin WLF dissolved in tap water, was administered orally by gavage to 5 male and 5 female Wistar rats per dose group for a period of 4 weeks according to OECD TG 407 and GLP. Survival, body weight development as well as food and water intake in treated groups were not affected by treatment, Neither hematology nor clinical chemistry gave evidence for treatment related effects up to 1000 mg/kg bw/day. Test item related macroscopic and microscopic changes at terminal sacrifice were slight gray or black discolorations in a dose-related manner in all treated groups. All these discolorations were considered to be due to the staining property of the test item. In the view of the very limited degree of changes and as any indication of structural change or functional impairment of the organs concerned was lacking, they were considered to be non-adverse.

Therefore, under the conditions described, the NOAEL for administration of Nigrosin WLF to male and female rats was 1000 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
Quality of whole database:
A reliable study which was perforemed according to OECD TG 407 and GLP with Klimisch Score 1; no other study available

Additional information

ORAL APPLICATION

100, 300 and 1000 mg/kg bw/day Nigrosin WLF dissolved in tap water, was administered orally by gavage to 5 male and 5 female Wistar rats per dose group for a period of 4 weeks according to OECD TG 407 and GLP. Survival, body weight development as well as food and water intake in treated groups were not affected by treatment, Neither hematology nor clinical chemistry gave evidence for treatment related effects up to 1000 mg/kg bw/day. Test item related macroscopic and microscopic changes at terminal sacrifice were slight gray or black discolorations in a dose-related manner in all treated groups, predominantly in the lymph nodes and less frequently in the intestine and/or Peyer's patches. Corresponding to these findings at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups. All these discolorations were considered to be due to the staining property of the test item. In the view of the very limited degree of changes and as any indication of structural change or functional impairment of the organs concerned, was lacking, they were considered to be non-adverse.

Therefore, under the conditions described, the NOAEL for administration of Nigrosin WLF to male and female rats was 1000 mg/kg bw/day.

There is no sub-chronic study available as required by regulation (EC) No.1907/2006 (REACH) ANNEX IX, section 8.6. According to ANNEX IX, Section 8.6 Column 2 of Regulation EC (REACH) No 1907 (2006) this sub-chronic study needs not to be conducted if a reliable short-term study (28 days) is available. In the available 28-day study (OECD TG 407 and GLP) male and female rats were given daily doses up to and including 1000 mg/kg bw/day (limit dose). Based on the data generated from this study no significant toxicological effects of the substance were found up to and including 1000 mg/kg bw/day. Therefore the NOEL (No Observed Effect Level) of Nigrosin WLF Is considered to be 1000 mg/kg bw/day. In addition, as required by ANNEX IX section 8.6.2 Column 2 the NOAEL -28days allows the extrapolation towards a NOAEL-90 days for the same route of exposure by application of an appropriate uncertainty factor. Furthermore, as no evidence of toxicological effects up to the limit dose of 1000 mg/kg bw/day are reported, it can be assumed that prolongation of treatment time does not provide additional relevant information on toxic effects. Thus, the requirements REACH Regulation are fulfilled.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A reliable study which was perforemed according to OECD TG 407 and GLP with Klimisch Score 1

Justification for classification or non-classification

Based on the available data no classification and labelling is required.