Reaction mass of lithium sodium 5-amino-3-{[4-(2-{4-[(7-amino-1-hydroxy-3-sulfo-2-naphthyl)diazenyl]-2-sulfophenyl}vinyl)-3-sulfophenyl]diazenyl}-4-hydroxynaphthalene-2,7-disulfonate 2,2'-(methylimino)diethanol (1:1) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, lithium sodium salt, compound with 2,2'-(methylimino)diethanol and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, lithium sodium salt, compound with 2,2'-(methylimino)diethanol

Endpoint summary

Description of key information

Bayscript Blaukomponente (konserviert) was administered orally by gavage to male and female Wistar rats in daily doses of 0, 100, 300, 1000 mg/kg bw/day for a period of four weeks.

The no adverse observed effect level (NOAEL) for administration of Bayscript Blaukomponente (konserviert) to male and female Wistar rats was 300 mg/kg b.w./day mainly due to mild to moderate effects on kidneys and minor changes in nerve roots.

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:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

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
- Age at study initiation: 6-7 weeks
- Weight at study initiation (mean): males 193g , females 154 g
- Housing: in groups of 2 or 3 animals
- Diet ad libitum
- Water . ad libitum:
- Acclimation period: approximately 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:

Bayscript Blaukomponente (konserviert) was adminestered orally by gavage to 5 male and 5 female Wistar rats per dose group using tap water as vehicle in daily doses of 0, 100, 300, 1000 mg/kg bw/day for aperiod of four weeks

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the start of the treatment the suitability of the proposed formulations was confirmed by the analyses of condentration and stability
Stability: the dosage forms prepared were analysed 4 hoursm 1,4,and 7 days after preparation
Concentration: the concentration of samples of control and each test substance dosage form prepared were determined twice during the study

Duration of treatment / exposure:

males: 28 days. females 29 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Bayscript Blaukomponente (konserviert) was adminestered orally by gavage to 5 male and 5 female Wistar rats per dose group using tap water as vehicle in daily doses of 0, 100, 300, 1000 mg/kg bw/day for a period of four weeks.The animals were regularely observed, weighed and food and water intakes were determined. In addition, clinical laboratory investigations of blood samples was performed. Organs and tissues were subjected to gross and histopathological investigation.

Positive control:

no

Observations and examinations performed and frequency:

Inspection of Animals:
for Morbidity and Mortality Twice daily
General Clinical Examinations (in cage) Daily
Detailed Clinical Examinations Daily
Open Field Observation (OFO) Weekly
Functional Observational Battery Once
Motor Actvity Once
Determination of:
Body Weight(s) Daily
Food Consumption Weekly
Water Consumption Weekly
Feeding Period: Approximately 7 days
Clinical Laboratory Investigation:
Hematology Once
Leucocytes, erythrocytes, hemoglobin, hematocrit, reticulocytes, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), thrombocytes, Hepato Quick, differential blood count
Clinical Chemistry Once
Alanine aminotransferase, aspartate aminotransferase, albumin, protein (total), bile acids, cholesterol, creatinine, urea, glucose, sodium, potassium, total bilirubin.

Sacrifice and pathology:

Necropsy : week 5
The organ weights determined before fixation of all animals:
adrenals, brain,epididymidesheart, kidneys,liver, ovaries/oviduct, prostate, seminal vesiceswith coagulation gland, spleen, testes, thymus, uterus with cervix
gross and histological examination:
all weighed organs and additionally
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

Statistics:

Dunnett, U-test, Het-Dunn ( Dunnett exact test heterogeneous test)

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died unsheduled
The only clinical finding noted wss a bluish discoloration of the whole body of all animals at 1000 mg/kg bw/day

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

FOOD CONSUMPTION AND WATER CONSUMPTION
Mean food intake of treated animals over the dosing period was comparable to the respective controls
The water intake was increased starting at 100 mg/kg bw/day. howeverm the difference to control was relatively slight for the low dose group of females nad there was no dose dependencefor the loww and mid dose group of males

HAEMATOLOGY
1000 mg/kg bw/day, males and females:
minor changes in red blood cell parameters were associated with the appearing glolden -brown pigment deposision in the spleen
1000 mg/kg bw/day, males:
thrombocyte count was increased in males at 1000 mg/kg bw/day:
1083.2 G/l (p<=0.01) versus 783.6 G/l of control

CLINICAL CHEMISTRY
Clinical chemistry gave no evidence of treatement.related adverse effects up to 1000 mg/kg bw/day

NEUROBEHAVIOUR
The determination of the motor activita and the Locomotor activity gave no evidence for a relevant treatment realted effect.
Despite the bluishdiscoloration Functional observation battery gave no evidence for treatment related findings

ORGAN WEIGHTS
Determination of organ weights revealed increased absolute and relative weights
The differences given in brackets are the differences of relative organ weights compared to the respective control value.
adrenal glands at 1000 mg/kg b.w./day in females (+13%), of
heart at 1000 mg/kg b.w./day in males (+18%), of
liver at 1000 mg/kg. b.w./day in males (+18%) and females (+19%), of
kidneys starting at 100 mg/kg b.w./day in males (+12%, + 28%, +30%) and at 1000 mg/kg b.w./day in females (+18%),
thymus at 1000 mg/kg b.w./day in females (+13%) and of
uterus starting at 300 mg/kg b.w./day (+ 23%, +21%).

GROSS PATHOLOGY - discolorations of organs
100, 300, 1000 mg/kg bw/day:
At necropsy, dark, blue, gray or black discoloration of the kidneys was noted in most animals treated at 100 mg/kg b.w./day and in all animals treated at 300 or 1000 mg/kg b.w./day, the intensity of the color being approximately dose-related.
at 1000 mg/kg b.w./day.
There was also a discoloration of a variety of other organs, mostly described as gray, and in isolated cases as black, confined to males and females.
Organs concerned were the liver (9/10 rats), skin (9/10 rats), mesenteric lymph node (3/10 rats), stomach (4/10 rats), testis (5/5 rats), uterus (3/5 rats) and generalized lymph nodes (3/10 rats). These discolorations were considered to be related to the test item.

HISTOPATHOLOGY: NON-NEOPLASTIC
---discolorations within organs (deposites) mainly in the 1000 mg/kg bw/day -groups::
kidneys, mesenteric lymph nodes, testes, Peyer's patch, spleen
---histopathological changes
kidneys:
At 1000 mg/kg b.w./day, only, mild or moderate multifocal corticotubular degeneration and a mildly increased incidence and/or severity of cortical basophilic (regenerative) tubules, when compared with the control group. Furthermore, multifocal minimal corticotubular dilation was seen in at 300 (males) and 1000 mg/kg b.w./day (males and females).
nerve roots
In nerve roots at the spinal cord, demyelinated fibers were seen at 1000 mg/kg b.w./day in males and females. The change was minimal in degree, except for one male in which it was mild. There were no other relevant histopathological lesions in the central or peripheral nerve system.
adrenal gland:
in 2/5 females treated at 1000 mg/kg/day, a minimal diffuse cortical hypertrophy of the adrenal gland was seen histologically. However, in view of the individual variability noted in adrenal gland histomorphology and the absence of a statistically significant weight difference in the high dose group, this minor difference was not considered to be sufficient evidence of a test item-related effect.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: due to degenerative effects on kidneys and nerve roots at 1000 mg/kg bw/day

Critical effects observed:

not specified

Conclusions:

Bayscript Blaukomponente (konserviert) was administered orally by gavage to 5 male and 5 female Wistar rats per dose group using tap water as vehicle, in daily doses of 0, 100, 300, 1000 mg/kg bw/day for a period of four weeks. This GLP study was performed according to OECD guideline 407.
Treatment with Bayscript Blaukomponente resulted in several findings which were related to the color of the test substance. The whole body and several organs of animals treated with 1000 mg/kg bw/day appeared discolored. The kidneys were also discolored in the lower dose groups.
In the kidneys, beneath findings associated with the color of the test substance (starting at 300 mg/kg b.w./day), corticotubular degeneration and increased incidence and/or severity of cortical basophilic (regenerative) tubules were noted at 1000 mg/kg b.w./day. Furthermore, demyelinated fibers in nerve roots at the spinal cord were seen in both sexes at 1000 mg/kg b.w./day.
At 1000 mg/kg b.w./day, histopathological findings in the mesenteric lymph node, the interstitium of the testis, and in the Peyer's patch were related to the colored test item and not accompanied by other structural changes of the organs.
Minor changes in red blood cell parameters observed for males and females at 1000 mg/kg b.w./day were associated with appearing of golden-brown pigment deposition in the spleen in females. In males, the thrombocyte count was increased at 1000 mg/kg b.w./day.

Under the conditions described the no adverse observed effect level (NOAEL) for administration of BAYSCRIPT Blaukomponente (konserviert) to male and female Wistar rats was 300 mg/kg b.w./day mainly due to degenerative effects on kidneys and nerve roots. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.

Executive summary:

Bayscript Blaukomponente (konserviert) was administered orally by gavage to 5 male and 5 female Wistar rats per dose group using tap water as vehicle, in daily doses of 0, 100, 300, 1000 mg/kg bw/day for a period of four weeks. This GLP study was performed according to OECD guideline 407.

Treatment with Bayscript Blaukomponente resulted in several findings which were related to the color of the test substance. The whole body and several organs of animals treated with 1000 mg/kg bw/day appeared discolored. The kidneys were also discolored in the lower dose groups.

In the kidneys, beneath findings associated with the color of the test substance (starting at 300 mg/kg b.w./day), corticotubular degeneration and increased incidence and/or severity of cortical basophilic (regenerative) tubules were noted at 1000 mg/kg b.w./day. Furthermore, demyelinated fibers in nerve roots at the spinal cord were seen in both sexes at 1000 mg/kg b.w./day.

At 1000 mg/kg b.w./day, histopathological findings in the mesenteric lymph node, the interstitium of the testis, and in the Peyer's patch were related to the colored test item and not accompanied by other structural changes of the organs.

Minor changes in red blood cell parameters observed for males and females at 1000 mg/kg b.w./day were associated with appearing of golden-brown pigment deposition in the spleen in females. In males, the thrombocyte count was increased at 1000 mg/kg b.w./day.

Under the conditions described the no adverse observed effect level (NOAEL) for administration of BAYSCRIPT Blaukomponente (konserviert) to male and female Wistar rats was 300 mg/kg b.w./day mainly due to degenerative effects on kidneys and nerve roots. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The only available repeated oral dose rat study which is performed according to OECD TG 407 and GLP and evaluated with Klimisch Score = 1

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

ORAL APPLICATION

Bayscript Blaukomponente (konserviert) was administered orally by gavage to 5 male and 5 female Wistar rats per dose group using tap water as vehicle, in daily doses of 0, 100, 300, 1000 mg/kg bw/day for a period of four weeks. This GLP study was performed according to OECD guideline 407 and GLP.

Treatment with Bayscript Blaukomponente resulted in several findings which were related to the color of the test substance. The whole body and several organs of animals treated with 1000 mg/kg bw/day appeared discolored. The kidneys were also discolored in the lower dose groups.

In the kidneys, beneath findings associated with the color of the test substance (starting at 300 mg/kg b.w./day), corticotubular degeneration and increased incidence and/or severity of cortical basophilic (regenerative) tubules were noted at 1000 mg/kg b.w./day. Furthermore, demyelinated fibers in nerve roots at the spinal cord were seen in both sexes at 1000 mg/kg b.w./day.

At 1000 mg/kg b.w./day, histopathological findings in the mesenteric lymph node, the interstitium of the testis, and in the Peyer's patch were related to the colored test item and not accompanied by other structural changes of the organs.

Minor changes in red blood cell parameters observed for males and females at 1000 mg/kg b.w./day were associated with appearing of golden-brown pigment deposition in the spleen in females. In males, the thrombocyte count was increased at 1000 mg/kg b.w./day.

Under the conditions described the no adverse observed effect level (NOAEL) for administration of Bayscript Blaukomponente (konserviert) to male and female Wistar rats was 300 mg/kg b.w./day mainly due to mild to moderate effects on kidneys and minor changes in nerve roots.

There is no sub-chronic study available as required by regulation (EC) No.1907/2006 (REACH) ANNEX IX, section 8.6. According to ANNEX XI further testing does not appear scientifically necessary because there is a reliable short-term study (28 days in rats) available. The available data met the following conditions:1) the data are adequate for the purpose of classification and labeling and/or risk assessment; 2) the provided documentation is sufficient to assess the adequacy of the study and 3) the data are valid for the endpoint being being investigated and the study is performed using an acceptable quality assurance.

Taking the NOAEL of 300 mg/kg bw/day as a starting-point a conservative DNEL was derived because 1) only slight effects were observed at the limit dose 1000 mg/kg/day and no effects are observed at the next lower dose tested, 300 mg/kg/day. 2) Time extrapolation was taken into account and a factor of 6 is applied to extrapolate from sub-acute to chronic exposure. Overall, it is likely that additional sub-chronic testing will not substantially change the conservative risk assessment provided in this dossier, and, consequently, further testing is of low priority and should be omitted also based on animal welfare reasons.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The only available study which is performed according to OECDTG 407 and GLP and evaluated with Klimisch Score = 1

Justification for classification or non-classification

Bayscript Blaukomponente (konserviert) was administered orally by gavage to male and female Wistar rats in daily doses of 0, 100, 300, 1000 mg/kg bw/day for a period of four weeks.

The no adverse observed effect level (NOAEL) for administration of Bayscript Blaukomponente (konserviert) to male and female Wistar rats was 300 mg/kg b.w./day mainly due to mild to moderate effects on kidneys and minor changes in nerve roots. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.