Registration Dossier

Administrative data

Description of key information

OECD guideline study for subacute (oral) toxicity in Wistar rats, resulting in a NOAEL of 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:
key study
Study period:
1986-Feb-19 to 1986-Apr-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen
- Age at study initiation: 6-8 weeks
- Weight at study initiation: M=108-135g, F=100-119g
- Housing: macrolon cages (type II) on wood granulate, 1 animal/cage; (type III macrocolon cages during acclimation period)
- Diet (e.g. ad libitum): Altromin 1324, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 5d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22 °C
- Humidity (%): ca. 65 %
- Air changes (per hr): 10x/h
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 40. 200, 1000 mg/kg bodyweight
- Amount of vehicle (if gavage): 15 mL/kg body weight
- Lot/batch no. (if required): ÖK-Nr. 2123
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of the application solution was analysed during the application period by TLC. Results showed that the solutions were stable and that even at treatment end the concentration of the test substance did not deviate more than 0,5 % for the 200 mg/kg and 1000 mg/kg dose solutions, but was somewhat (5,4 %) diminished in the 40 mg/kg dose solutions.
Duration of treatment / exposure:
28 d


Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0, 40, 200 or 1000 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on preliminary 17d study (5 rats/sex/dose) using 0, 200, 1000 and 5000 mg/kg body weight of Reactive Blue FC 15353; test substance exposed animals showed no signs of toxicity regarding appearance, behaviour, food/water consumption and body weight development. No functional effects on liver and kidney were observed. Histopathological observation reavealed a dosis-dependant tubular storage in the cortex of the kidney. No changes were found in liver and spleen. Based on this findings, a dosage of 40 and 200 mg/kg body weight plus the limit concentration of 1000 mg/kg body weight was chosen.
- Rationale for selecting satellite groups: Purpose of the satellite group (5 rats/sex/1000 mg/kg body weight) was to investigate the reversibility of tubular storage in the cortex of the kidney.
- Post-exposure recovery period in satellite groups: 4 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 2/day, on week end and public holiday 1/day
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 1/week

BODY WEIGHT: Yes
- Time schedule for examinations: before first exposure, during study: 1/week, before euthanasia

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as mean g food/animal/day: Yes, calculated from difference of supplied food to non-consumed food. Additional cumulative food consumtion per animal was calculated.

FOOD EFFICIENCY:
- Body weight gain data: Yes

WATER CONSUMPTION: Yes, analoug to food consumption

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 0, 200, 400 and 1000 mg/kg bg groups: after 28 d; satellite group: after 56 d
- Anaesthetic used for blood collection: Yes (ether narcotized); except: No (for blood glucose)
- Animals fasted: No data
- How many animals: 5 rats/sex/dose
- Parameters checked: erythrocyte, leucocyte and thromcocyte counts, Hematocrit, Hemoglobin (HB) , Mean corpuscular hemoglobin
(MCH), Mean corpuscular hemoglobin concentration (MCHC), Mean corpuscular volume (MCV), Differential blood count, prothrombin time with quick test

CLINICAL CHEMISTRY: Yes
- How many animals: 5 rats/sex/dose
- Parameters checked: enzymes: alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase; substrates: blood glucose, cholesterine, total bilirubin, creatinine, urea, total protein, triglycerides, albumine, uric acid, sodium, potassium, calcium, anorganic phosphate, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: 16 hours
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: semiquantitative: glucose, blood, protein, pH, ketone bodies, bilirubin, urobilinogen; microscopic analysis of sediments after centrifugation of urine; quantitative: protein (Richterich method); volume, specific weight, phosphate, urea, creatinine, calcium, N-acetyle-glucose aminidase,
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Animals were narcotized with diethyl ether, sacrificed by exsanguation and necropsied. Brain, heart, lungs (pairwise), testes (pairwise), liver, adrenals (pairwise), kidneys (pairwise), spleen and liver were removed for weighing.The following organ material was harvested and fixated in Bouin's solution:
adrenal gland, aorta, brain, epididymis, exorbital lacrimal gland, eyes, eyelids, femur en bloc with skeletal muscle and N. ischiadicus, Harderian gland, heart, intestine (duodenum, jejunum, ileum, colon, caecum and rectum), kidney, larynx, liver, lungs, mesenterial and mandibular lymph nodes, N. opticus, oesophagus and trachea en bloc, ovaries with oviduct, pancreas, pituitary, prostate gland, salivary gland, seminal gland, skin/mamma, spinal cord (cervical, thoracic and lumbar spine), spleen, sternum, stomach, testicles, thymus (if present), thyroid gland, urinary bladder, uterus, vagina, tongue and all macroscopic visible changes. Additionally, 1 lobe of the liver was fixated in formol-calciue.

HISTOPATHOLOGY: Yes
Histopathological examination of liver, lungs, kidney, spleen, adrenal glands and heart from all animals of the control group, 1000 mg/kg bw and satellite group
Other examinations:
No death occured.
Statistics:
Means, standard deviations and lower and upper confidence intervals were determined for α= 0.05 and α=0.01. Testing significance of comparisons was done using the Mann Whitney U test, with P<0.05 or P<0.01 being considered as statistically significant. However, since all treatment groups were compered to the control group (0 mg/kg per day of test substance), some false positive statistical differences may be found.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw: blue dicolored feces, which is reversible within the 28 day recovery group.
Mortality:
mortality observed, treatment-related
Description (incidence):
1000 mg/kg bw: blue dicolored feces, which is reversible within the 28 day recovery group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
≥200 mg/kg bw reduced body weight in females, but not in the 1000 mg/kg bw/d satellite group.
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The finding of blue discolored urine in some animals starting from 200 mg/kg bw /day were without effect regarding to any other parameter of the urine analysis.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Blue discolored stomach and intestine content in some animals, partial blue discolored organs and muscles. The finding has not been considered adverse.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Small light breaking structures in the epithelial cell of the kidney were observed in the 1000 mg/kg bw/day group, which were not accompanied with vital reactions in the tissue.
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical symptoms:
None of the animals died during the observation period and no clinical symptoms were observed, except that for the rats that were treated with the highest dose (1000 mg/kg bodyweight per day) the feces were colored blue. This was seen in both males and females (see table 1) and seemed to be reversible, as in the group in which the observations were continued until week 4 after daily treatment with 1000 mg/kg bodyweight of Reactive Blue FC 15353, the feces of both male and female rats discolored within this 4-week recovery period.

Table 1 Number of animals in which in at least one or more occasions in the indicated time frame blue coloration of the feces occurred.

Dose group (M=males, F= females) Time of treatment/observation (weeks)

1 2 3 4 5 6 7 8
M 0 mg/kg.day - - - - - - - -
M 40 mg/kg.day - - - - - - - -
M 200 mg/kg.day - - - - - - - -
M 1000 mg/kg.day 5 5 5 5 - - - -
M 1000 mg/kg.day - - - - 5 5 - -

F 0 mg/kg.day - - - - - - - -
F 40 mg/kg.day - - - - - - - -
F 200 mg/kg.day - - - - - - - -
F 1000 mg/kg.day 5 5 5 5 - - - -
F 1000 mg/kg.day 5 5 5 5 4 4 2 -



Bodyweights:
Bodyweights of the male rats were not affected by the treatment (see table 2). In female rats the absolute bodyweights were slightly reduced in the 200 and 1000 mg/kg bw/d groups compared to control. This effect is, however, not consistent as it was only observed in the animals that were sacrificed on day 28, but not in those that were followed until day 56 (ie another 4 weeks after the Reactive Blue FC 15353 treatment was stopped). Therfore thefinding was considered to be without toxicological relevance.

Table 2 Bodyweights expressed as mean (s.d.) during the treatment period

Dose group (M=males, F= females) Week
0 1 2 3 4

M 0 mg/kg.day 114 (5.3) 149 (11.9) 190 (10.5) 225 (8.4) 241 (7.0)
M 40 mg/kg.day 123 (5.2) 161 (8.6) 203 (10.9) 241 (12.2) 256 (14.6)
M 200 mg/kg.day 113 (6.2) 148 (5.4) 190 (5.1) 226 (7.2) 242 (4.8)
M 1000 mg/kg.day 111 (2.3) 148 (6.4) 186 (8.7) 219 (9.2) 253 (13.5)
M 1000 mg/kg.day 119 (10) 155 (11.1) 193 (10.9) 230 (11.8) 246 (10.5)

F 0 mg/kg.day 110 (6.1) 130 (6.7) 149 (6.8) 159 (4.9) 164 (5.3)
F 40 mg/kg.day 106 (4.4) 125 (6.3) 140 (6.1) 150 (7.5) 156 (8.7)
F 200 mg/kg.day 107 (7.7) 122 (0.9)* 136 (2.4) # 150 (2.7)* 153 (3.6) #
F 1000 mg/kg.day 106 (5.80 122 (6.7) 137 (3.6)* 147 (4.4)* 160 (6.1) #
F 1000 mg/kg.day 114 (7.4) 129 (7.7) 141 (8.3) 153 (8.9) 153 (12.2)

* indicates significant difference versus control group P<0.05, U-test; # indicates significant difference versus the control group P<0.01, U-test (Mann-Whitney)

Food and water intake:
For both male and female rats, food and water intake were not different between FC 15353 and vehicle treated rats, irrespective of the Reactive Blue FC 15353 dose with which the rats were.

Hematology findings:
Hematology findings are shown in table 3. No differences between Reactive Blue FC 15353 treated rats and the control group were observed regarding erythrocyte counts (Ery), leukocyte counts (Leuco), hematocrit (HCT), mean cellular volume (MCV), mean corpuscular hemoglobin content (MCH) and thrombin (Thro).This was true for both male and female rats for all Reactive Blue FC 15353 doses.
However, male rats, treated with 200 mg FC 15353/kg of bodyweight per day showed a slightly lower clotting time (Hquick) and somewhat higher hemoglobin (Hb) levels than vehicle treated males (P<0.01 and 0.05 respectively). Females showed a somewhat higher mean corpuscular hemoglobin concentration (MCHC) in the 200 mg/kg bodyweight per day group than the vehicle treated female rats (P<0.05).
The authors of the report state that the here observed differences in the hematology parameters are only small and within the range of historic controls and therefore have no toxicological meaning. For example based on an additional table in the report with control values, the value of Hquick in males in historic controls (given in an additional table in the report) ranges between 27.7 and 37.1 seconds in males, Hb ranges between 132 and 169 g/l in historic control male rats, while MCHC in females ranges between 297 and 341 g/L in the historic controls.
Differential counting did not reveal any differences between vehicle treated and FC 15353 treated (all doses) rats both in males and in females.


Table 3 Hematology data from blood harvested at week 4 (treatment end). Data are represented as means of all 5 animals per group.

Dose group (M=males, F= females)

Ery (Tera/L) Leuco (Giga/L) Hb (g/L) HCT (l/L) Hquick (s) MCV(fl) MCH (pg) MCHC (g/L) Thro (Giga/L)
M 0 7.24 7.8 144 0.488 32.5 62 19.7 323 999
M 40 6.95 8.8 143 0.444 30.0 63 20.4 323 979
M 200 7.39 8.8 150* 0.465 28.9# 63 20.2 325 1068
M 1000 7.40 8.8 154 0.470 30.3 63 20.6 329 1020

F 0 7.25 8.1 144 0.455 29.1 63 19.7 319 1103
F 40 7.25 6.8 145 0.460 28.7 63 19.8 318 1069
F 200 7.54 6.9 151 0.465 30.1 61 19.8 325* 1079
F 1000 7.28 6.7 146 0.456 28.3 62 19.9 322 1020

* indicates significant difference versus control group P<0.05, U-test; # indicates significant difference versus the control group P<0.01, U-test (Mann-Whitney)


Clinical chemistry studies:

Table 4 shows the (average of 5 rats) results of the clinical chemistry parameters, including electrolytes.
As can be seen in table 4, after 4 weeks of treatment reactive blue FC 15353 treated male and female rats had similar values as control rats regarding alkaline phosphatase (AP), aspartate amino tranferase (ASAT), alanine aminotransferase (ALAT), total bilirubin (Bili), triglycerides (Trigly), urea, cholesterol (Chol), glucose (Gluc), albumin (Albu), calcium (Ca), chloride (Cl) and uric acid (UA) levels.
However, in male rats potassium (K) levels were somewhat higher in the 40 and 1000 mg/kg bw/day group than in vehicle treated males (P<0.01 and 0.05 respectively) and also phosphate (P) levels were somewhat but higher in the 1000 mg/kg bw/day treated male than in vehicle treated rats (P<0.05). In females no such differences were seen but protein (Prot) levels of 40 mg/kg.day and 1000 mg/kg.day treated females were lower than in vehicle treated female rats (P<0.05 and P<0.01) respectively, while creatinine (Crea) and sodium (Na) levels were lower (P<0.05) in 1000 mg/kg.day treated than in vehicle treated female rats. These differences in turn were not seen in male rats.
Again the authors of the report here state that the observed changes were only small and within the range of historic controls (the results of which they this time did not show) and that therefore the differences that are seen do have no toxicological meaning.


Table 4 Clinical chemical parameters determined in the blood harvested at week 4 (treatment end). Data are represented as means of all 5 animals per group.

Dose group (M=males, F= females)
daily dose in mg/kg

AP (U/L) ASAT (U/L) ALAT (U/L) Bili (µM) Trigly (mM) Prot (g/L) Urea (mM) Crea (µM) Chol (mM) M

M 0 411 41.4 44.0 1.7 1.47 56.1 6.73 62 2.31
M 40 392 38.1 48.4 2.1 1.69 57.2 6.70 54 2.37
M 200 444 40.8 51.0 2.2 1.99 58.1 7.46 83 2.58
M 1000 400 41.5 49.5 1.9 1.66 57.8 7.42 56 2.62

F 0 257 46.4 47.3 2.1 1.30 62.2 6.84 63 2.18
F 40 243 41.2 44.5 2.2 1.28 59.1* 7.02 62 2.36
F 200 234 43.7 43.7 2.4 1.25 58.6 7.61 59 2.10
F 1000 237 38.9 38.8 2.0 1.52 57.5# 7.16 53# 2.31

Dose group (M=males, F= females)

Gluc (mM) K (mM) Ca (mM) Na (mM) Cl (mM) P (mM) Album(g/L) UA (µM)
M 0 5.83 4.7 2.65 143 100 2.33 38.9 62
M 40 5.71 5.4# 2.60 142 100 2.37 38.4 65
M 200 5.68 5.0 2.65 143 101 2.50 38.8 55
M 1000 5.76 5.5# 2.61 143 100 2.51* 40.0 68

F 0 5.47 4.5 2.65 143 102 2.00 40.8 105
F 40 5.76 4.7 2.59 143 103 1.81 40.3 85
F 200 5.33 4.8 2.54 142 103 1.88 41.2 99
F 1000 5.27 5.2 2.55 141* 103 1.77 42.7 77

* indicates significant difference versus control group P<0.05, U-test; # indicates significant difference versus the control group P<0.01, U-test (Mann-Whitney)


Urine investigations:

The urines of the animals that were treated with the 200 mg or 1000 mg/kg bw/day doses of Reactive Blue FC 15353 were partly colored blue. None of the animals had glucose, ketone bodies, bilirubin or blood in their urines, including animals treated with Reactive Blue FC 15353 at the highest (1000 mg/kg bw/day) dose level.

As can be seen from table 5, doses up to 1000 mg/kg bw/day Reactive Blue FC 15353 did not affect urinary parameters in female rats (pH, volume (Vol), protein content (prot), phosphate levels (P), urea levels, creatine levels (CREA), calcium levels (Ca) or N-acetyl glucosamine). In males however, some differences are observed between the group treated with 40 mg/kg bw/day of Reactive Blue Fc 15353 group and the vehicle treated group regarding specific density (Sdens) of the urine and phosphate (P) content that were higher (P<0.05) than in the vehicle treated animals. However, the authors of the report considered none of these differences as toxicologically meaningful, as no dose effect relationships exist.

Table 5 Urinary parameter data from blood harvested at week 4 (treatment end). Data are represented as means of all 5 animals per group.

Dose group (M=males, F= females)
daily dose in mg/kg

pH Vol (mL) Sdens (g/L) Prot (g/L) P (mM) Urea (mM) Crea (mM) Ca (mM) NAG (U/L)
M0 7.2 15 1006 0.44 19.4 254 2.52 0.44 9.39
M 40 6.7 12 1016* 0.67 30.7* 314 3.52 0.55 14.54
M 200 6.9 18 1010 0.61 20.4 243 2.46 0.47 7.49
M 1000 6.8 19 1005 1.03 22.7 202 2.25 0.49 7.42

F 0 6.6 13 1006 0.14 18.6 260 2.32 0.82 7.05
F 40 6.5 11 1011 0.13 18.4 286 2.42 1.02 8.57
F 200 6.4 10 1008 0.14 20.7 336 2.37 0.74 6.59
F 1000 6.4 14 1009 0.15 23.1 250 1.89 0.55 6.16
* indicates significant difference versus control group P<0.05, U-test; # indicates significant difference versus the control group P<0.01, U-test (Mann-Whitney)

Organ weights:
Organ weights and relative organ weights (organ/body weight ratios) are depicted in table 6. Male organ weights were similar between vehicle treated and Reactive blue treated rats for all doses, but relative lung weights are significantly lower in the 40 and 200 mg/kg bw/day dose groups (P<0.05) but not in the higher dose group. For female rats, lung and liver weights were lower in the 40 mg/kg bw/day group and liver weights also in the 200 mg/kg bw/day dose group compared to the vehicle treated female rats, but regarding relative organ weights no differences were observed. The authors of the report conclude that no toxicologically relevant changes in organ weights occurred.

Table 6 Organ weights and relative organ weights (ie organ/body weight ratios) at week 4 (treatment end).

Dose group (M=males, F= females) Organ weights (mg)


heart lungs liver spleen kidneys adre-nals testes brain
M 0 mg/kg.day 903 1166 10368 535 1756 39 3115 1780
M 40 mg/kg.day 964 1098 11068 600 1846 43 3213 1766
M 200 mg/kg.day 950 1024 10421 508 1752 36 3012 1761
M 1000 mg/kg.day 925 1058 10337 519 1695 37 2911 1787

F 0 mg/kg.day 688 832 7184 335 1184 50 1700
F 40 mg/kg.day 641 725* 6082* 323 1062 52 1647
F 200 mg/kg.day 611 763 6104* 330 1089 52 1673
F 1000 mg/kg.day 681 770 6470 320 1095 43 1640

Dose group (M=males, F= females) Relative organ weights (mg/100 g)

heart lungs liver spleen kidneys adre-nals testes brain
M 0 mg/kg.day 375 484 4302 222 729 16 1294 739
M 40 mg/kg.day 375 428# 4313 234 719 17 1257 690
M 200 mg/kg.day 394 424# 4311 210 725 15 1246 728
M 1000 mg/kg.day 396 452 4396 222 722 16 1243 762

F 0 mg/kg.day 418 506 4373 204 721 31 1035
F 40 mg/kg.day 412 467 3916 207 683 33 1061
F 200 mg/kg.day 400 500 4002 216 714 34 1098
F 1000 mg/kg.day 456 515 4329 214 706 29 1097

Data are represented as means of all 5 animals per group. * indicates significant difference versus the control group P<0.05, U-test; # indicates significant difference versus control group P<0.01, U-test


Anatomical/pathological dissectioning:

Starting from 40 mg/kg bw/day, partly blue discoloration of stomach and intestine content, as well as partly discolored organs and muscle were observed but without evidence for any organ damage in all dosage groups.

Histopathological investigations:

Investigation of hearts, lungs, livers, spleens and adrenals did not show any structural changes in the organs. However, in the kidneys of all male and female rats that were treated for 28 days with the highest dose of Reactive Blue FC 15353 (the results of other doses are not explicitly reported) and subsequently sacrificed to harvest the organs, small light breaking structures were found in the epithelial cells of the kidney but were not accompanied by vital reactions in the tissue. The presence of these structures seemed to be reversible since in the animals that received 1000 mg/kg bw/day Reactive Blue FC 15353 for 28 days and were subsequently kept untreated for another 28 days (following sacrificing to harvest the organs), this phenomenon was only observed in 1 out of 5 males while it was not observed in the 5 females.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Slight effects on female body weight at 1000 mg/kg/d where not considered to be of biological relevance.
Critical effects observed:
not specified
Conclusions:
In a 28d GLP guideline study in rat, no substance-induces adverse effects have been reported up to teh limit dose of 1000 mg/kg/d.
Executive summary:

In a subacute toxicity study Reactive Blue FC 15353 was administered to 5 Wistar rats/sex/dose formulated in water by gavage at dose levels of 0, 40, 200 and 1000 mg/kg bw/day).

No relevant clinical symptoms, other than blue colored feces occurred. After treatment, discoloration of blue feces was completed at the end of the 28 day recovery period. Food and drinking water intake were not affected by treatment and assessment of the organs did not reveal pathologies. A slightly reduced absolute body weight of female rats were only observed in the 200 and 1000 mg/kg bw/day dose groups compared to control, but not in the 1000 mg/kg bw/d satellite group. Further, female rats of the control group started with a slightly higher absolute mean body weight. Therefore, the finding was considered to be without toxicological relevance.

Analysis of heamotological parameters revealed a lowered clotting time plus higher hemoglobin in male and higher MCHC in female rats upon 200 mg/kg bw/d. 1000 mg/kg bw/d caused no statistical significant changes. Changes in clinical chemistry parameters related to a lowered protein content upon 40 and 1000 mg/kg/ bw/d and lower creatinine upon 1000 mg/kg bw/d in female rats and in males potassium was higher upon 40 and 1000 mg/kg bw/d and phosphate was higher upon 1000 mg/kg bw/d. As the observed changes regarding heamatological and clinical chemistry parameters were only small and lie within the range of the historical controls, there were considered to be without toxicological relevance.

The blue discolored urine starting from 200 mg/kg bw/d was the sole observed effect in urine analysis. The observed blue discoloration of stomach and intestine content as well as partly blue discolored organs and muscles starting at doses from 40 mg/kg bw/d were without evidence for any organ damage.

Small light breaking structures were histopathologically observed in the epithelial cell of the kidneys, but were not accompanied by vital reactions in the tissue. After end of treatment, these structures were only visible in 1 animal of the 1000 mg/kg bw/d satellite group

Taken together, all observed effects were only minor, mostly laying in the range of historic controls and in all cases where not associated with an adverse effect. Therefore the NOAEL for male and female rats is 1000 mg/kg bw/day.

This subacute toxicity study in the ratis acceptable and satisfies the guideline requirement for a subacute oral study (OPPTS 870.3050; OECD 407) in rat.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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

In a subacute toxicity study Reactive Blue FC 15353 was administered to 5 Wistar rats/sex/dose formulated in water by gavage at dose levels of 0, 40, 200 and 1000 mg/kg bw/day). No relevant clinical symptoms, other than blue colored feces occurred. After treatment, discoloration of blue feces was completed at the end of the 28 day recovery period. Food and drinking water intake were not affected by treatment and assessment of the organs did not reveal pathologies. A slightly reduced absolute body weight of female rats were only observed in the 200 and 1000 mg/kg bw/day dose groups compared to control, but not in the 1000 mg/kg bw/d satellite group. Further, female rats of the control group started with a slightly higher absolute mean body weight. Therefore, the finding was considered to be without toxicological relevance.

Analysis of heamotological parameters revealed a lowered clotting time plus higher hemoglobin in male and higher MCHC in female rats upon 200 mg/kg bw/d. 1000 mg/kg bw/d caused no statistical significant changes. Changes in clinical chemistry parameters related to a lowered protein content upon 40 and 1000 mg/kg/ bw/d and lower creatinine upon 1000 mg/kg bw/d in female rats and in males potassium was higher upon 40 and 1000 mg/kg bw/d and phosphate was higher upon 1000 mg/kg bw/d. As the observed changes regarding heamatological and clinical chemistry parameters were only small and lie within the range of the historical controls, there were considered to be without toxicological relevance.

The blue discolored urine starting from 200 mg/kg bw/d was the sole observed effect in urine analysis. The observed blue discoloration of stomach and intestine content as well as partly blue discolored organs and muscles starting at doses from 40 mg/kg bw/d were without evidence for any organ damage.

Small light breaking structures were histopathologically observed in the epithelial cell of the kidneys, but were not accompanied by vital reactions in the tissue. After end of treatment, these structures were only visible in 1 animal of the 1000 mg/kg bw/d satellite group

Taken together, all observed effects were only minor, mostly laying in the range of historic controls and in all cases where not associated with an adverse effect. Therefore the NOAEL for male and female rats is 1000 mg/kg bw/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
GLP and OECD guideline study examining subacute repeated dose toxicity in male and female rats

Justification for classification or non-classification

No specific target organ toxicity has been reported in an OECD 407 guideline study.