Hexasodium 4,4'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-morpholino-1,3,5-triazine-4,2-diyl]imino]]bis[5-hydroxy-6-(phenylazo)naphthalene-2,7-disulphonate]

Endpoint summary

Description of key information

The no observed adverse effect level (NOAEL) for general systemic toxicity was 60 mg/kg bw/d (definitive dose: 52.8 mg/kg bw/d) for male and at least 300 mg/kg bw/d (definitve dose: 264.0 mg/kg bw/d) for female Wistar rats.

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

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-27 and 2015-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline conform study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test) 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 weeks (males/females)
- Acclimatization period: 14 days
- Housing: a) individually in Makrolon type M III cages during the study period; b) male and female mating partners were housed together during in polycarbonate cages type III during overnights mating; c) pregnant animals and their litters were housed together until PND 4 (end of lactation); d) for motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2015-03-31 (acclimatization) To: 2015-06-05

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
A appropriate amount of the test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a magnetic stirrer. The test substance preparations were produced daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical investigations of the test substance preparations were carried out as a separate study.

The stability of the test substance in drinking water was demonstrated over a period of 7 days at room temperature. Homogeneity was verified in 3 samples in the highest and lowest concentration (was used as a concentration control at the same time) at the beginning of the study; additional concentration control analyses were done in the mid concentration. Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the test substance was distributed homogeneously in drinking water. The concentrations of the test substance in drinking water were found to be in the range of 103-120 % of the nominal concentration.

Duration of treatment / exposure:

males: 29 days
females: 53 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
12, 60 and 300 mg/kg bw/d
Basis:
other: test material

Remarks:

Doses / Concentrations:
10.6, 52.8, 264.0 mg/kg bw/d
Basis:
other: content of the test substance (88 g/100 g)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a 14-day range finding study in which 0, 300 and 1000 mg/kg bw/day were tested.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Any signs of morbidity, pertinent behavioral changes and signs of overt toxicity were examined. Abnormalities and changes were documented daily for each affected animal. The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except public holidays), the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- The following parameter were examined: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period; on study day 0 (start of administration period) and thereafter once a week at the same time of the day (in the morning)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule: once a week for male and female parental animals
- Exceptions: Food consumption was not determined during the mating period (male and female F0 animals). Food consumption of the F0 females with evidence of sperm was determined on GD 0-7, 7-14 and 14-20. Food consumption of F0 females, which gave birth to a litter, was determined for PND 1-4.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily visual inspection of the water bottles for any changes in volume

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning (on study day 29 for males, on study day 53 for females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- How many animals: in the morning (on study day 29 for males, on study day 53 for females)
- Parameters checked: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET), prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning (on study day 29 for males, on study day 53 for females)
- Animals fasted: Yes
- How many animals: in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group
- Parameters checked: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamytransferase (GGT), sodium (NA), potassium (K), chloride (CL), inorganic phosphate (INP), calcium (CA), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL), bile acids (TBA)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine were collected overnight (on study day 25 for males and on day 50 for females)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, protein (PRO), glucose (GLU), ketones (KET), urobilinogen (UBG), bilirubin (BIL), blood, specific gravity (SP. GR.), sediment, color and turbidity (COL, TURB), volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: in the first five surviving male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups
- Battery of functions tested: sensory activity/ motor activity / other: home cage and open field cage observations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (adrenal glands, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lung, mesenteric and axillary lymph nodes, ovaries, oviducts, peyer's patches, prostate, rectum, sciatic nerve, seminal vesicles, spinal cords, spleen, forestomach and glandular stomach, testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina)

Other examinations:

Organ weight: anesthetized animals, epididymides, testes (all animals); adrenal glands, brain, heart, kidneys, liver, spleen, thymus (5 animals(sex and test group)

Statistics:

- Statistics of the clinical examinations: DUNNETT test (two-sided), FISCHER'S EXACT test (one-sided), WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment, KRUSKALL-WALLIS test (two sided)
- Statistic of clinical pathology: KRUSKAL-WALLIS (bidrectional changes), WILCOXON-test with Bonferroni-Holm adjustment (undirectional changes), WILCOXON-test, KRUSKALL-WALLIS test (one-way analysis), WILCOXON-test (two-sided)
- Statistics of pathology: KRUSKALL-WALLIS (two-sided), WILCOXON-test (two-sided)

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:

not examined

Haematological findings:

effects observed, treatment-related

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:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
- No parental animal died prematurely in the present study.
- Discolored feces from red (test groups 2 [60 mg/kg bw/d] and 3 [300 mg/kg bw/d]) and dark brown (test group 3; females only) to black (test group 3) were observed in both sexes during premating, mating and post mating periods, starting on study day 1. These observations were also made for females during gestation and lactation. The effects were related to the test substance but assessed as being non-adverse. The absence of an open vagina was observed in animal No. 140 during mating. This finding was neither test substance- nor treatment-related but spontaneous in nature.

BODY WEIGHT AND WEIGHT GAIN
- No test substance-related changes in mean body weights were observed for male and female animals of test groups 1-3 (12, 60 and 300 mg/kg bw/d) when compared to control groups. Temporarily, mean body weight gain was significantly slowed down in female animals of test group 3 (300 mg/kg bw/d) during the premating phase, with a body weight loss between study days 0 to 7. As the finding was the limited to the premating phase and as no further findings occurred it was assessed to be non-adverse.

FOOD CONSUMPTION AND COMPOUND INTAKE
- No test substance-related findings were observed.

WATER CONSUMPTION AND COMPOUND INTAKE
- No test substance-related findings were observed.

HAEMATOLOGY
- In males of test group 3 (300 mg/kg bw/d), red blood cell (RBC) counts and mean corpuscular hemoglobin concentration (MCHC) was decreased whereas mean corpuscular volume (MCV) and relative reticulocyte counts were increased. Relative reticulocyte counts were already higher in males of test group 2 (60 mg/kg bw/d) and MCHC was already decreased in males of test groups 1 and 2 (12 and 60 mg/kg bw/d; in test group 2 not statistically significantly decreased). RBC counts in test group 3 was the only measured red blood cell parameter (i.e., RBC counts, hemoglobin and hematocrit values) which was altered. MCHC means in all test groups were within the historical control range (MCHC 20.22-21.48 mmol/L) and, therefore, this change was regarded as incidental and not treatment-related. The relative reticulocyte counts in males of test groups 2 and 3 were above the historical control range (relative reticulocyte counts 1.5-1.9%). At least in test group 2 this alteration without any other change of measured red blood cell parameters was regarded as treatment-related but not adverse because it reflects the capacity of the bone marrow to produce and release red blood cells.

In females of test group 3 (300 mg/kg bw/d) hemoglobin values were lower compared to study controls. The hemoglobin mean in females of test group 3 was 4% below that one of the study controls. The hemoglobin values in females of test group 3 were within, whereas those of the study control were above the historical control range (hemoglobin 8.2-8.9 mmol/L). In these individuals this was the only statistically significantly changed red blood cell parameter. Therefore, this change was regarded as incidental and not treatment-related.

In females of test group 1 (12 mg/kg bw/d) relative neutrophil cell counts were decreased and relative lymphocyte counts were increased. These alterations were not dose-dependent and therefore they were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
- In females of test groups 2 and 3 (60 and 300 mg/kg bw/d) total bilirubin levels were increased, but the values were within the historical control range (total bilirubin 1.29-3.12 µmol/L) and therefore, this change was regarded as incidental and not treatment-related.

URINALYSIS
- No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
1. Functional observation battery
- Home cage observations: No test substance-related effects were observed.
- Open field observations: Each one male animal of test groups 1 (No. 22; 12 mg/kg bw/d) and 2 (No. 31; 60 mg/kg bw/d) showed red-discolored feces. This finding was assessed to be test substance-, but not treatment-related or adverse.
- Sensorimotor tests/reflexes: No test substance-related effects were observed.
2. Motor activity measurement
- There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group.

ORGAN WEIGHTS
- The significantly increased absolute weight of the spleen in males (0.714 g) was within the range of the historical control values (0.502-0.728 g). However, the relative weight (0.216%) was above the historical control range (0.132-0.196%) and correlated with increased extramedullary hematopoiesis. Therefore, it was considered treatment-related. In females of test group 3, although the absolute spleen weight (0.510 g) was marginally above the historical control range (0.382-0.502 g) neither a dose-dependency nor a histopathological correlate were evident. Therefore, the weight increase in females was regarded as incidental.

GROSS PATHOLOGY
- In almost all males and females of test group 3 (300 mg/kg bw/d), the contents of the cecum, colon, glandular stomach and jejunum displayed a red discoloration. In addition, the kidneys of 4 out of 10 males and all females, as well as the mesenteric lymph nodes of all males and females of test group 3 displayed a red discoloration. All of these changes revealed the presence of the test substance and were considered treatment-related, but had no histopathological correlate.

HISTOPATHOLOGY: NON-NEOPLASTIC
- A slightly increased in the severity of the extramedullary hematopoiesis was seen in the spleen of males of test group 3 (300 mg/kg bw/d), only. The extramedullary hematopoiesis was comparable between control animals and treated animals of test groups 1 and 2.

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

52.8 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects

Dose descriptor:

NOAEL

Effect level:

264 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: no adverse effects

Critical effects observed:

not specified

Endpoint conclusion

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is considered acceptable without restrictions as it was conducted according to GLP regulations and OECD guideline.

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

Key study

An OECD 422 study was conducted with the test item (purity: 88 g/100 g). The test item was given daily as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 0, 12, 60 and 300 mg/kg body weight/day (mg/kg bw/d). Control animals were dosed daily with the vehicle only (drinking water). Considering the content of the test substance, i.e. 88.0 g/100 g, the definitive dose levels were 0, 10.6, 52.8 and 264.0 mg/kg bw/d. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals. The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation (days 0-7, 7-14, 14-20) and lactation (days 1-4). In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0 and 4. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 (12, 60 and 300 mg/kg bw/d) during the entire study period.Concerning clinical pathology, in males of test group 3 (300 mg/kg bw/d) a marginal indication of a regenerative, macrocytic anemia was observed because of decreased red blood cell (RBC) counts, increased mean corpuscular volume (MCV) and increased reticulocyte counts.Regarding pathology, target organ was the spleen of male animals. The absolute and relative weight of the spleen in males of test group 3 (300 mg /kg bw/d) were significatly increased (+25% and +30%, respectively). This finding correlated with minimal to moderate extramedullary hematopoiesis. Both, weight increase and extramedullary hematopoiesis were considered treatment-related and adaptive. However, in combination with the marginal anemia observed in the hematology they can be assessed as adverse.Under the conditions of the present Combined Repeated-Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration of the test substance by gavage resulted in a marginal anemia in male Wistar rats at a dose level of 300 mg/kg bw/d (definitive dose: 264.0 mg/kg bw/d). Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 60 mg/kg bw/d (definitive dose: 52.8 mg/kg bw/d) for male and at least 300 mg/kg bw/d (definitive dose: 264.0 mg/kg bw/d) for female Wistar rats.

Supporting study

The test substance was administered by gavage to groups of 4 male and 4 female wistar rats for 14 days at dose levels of 0 (vehicle control, test group 0), 300 (test group 1), and 1000 mg/kg bw/d (test group 2). This study was conducted as dose range finding study for the OECD 422. This study was performed under non-GLP conditions. Body weight was determined on study days 0, 3, 7 and 14. Food consumption and water consumption were determined on study days 3, 7 and 14. All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Clinicochemical and hematological examinations were performed towards the end of the administration period. After the administration period all rats were sacrificed and assessed by gross pathology. Organ weights were determined.

Discolored feces were observed in both sexes from day 1 onwards at 300 and 1000 mg/kg bw/d. Slightly reduced food consumption in females, with a maximum of -6% on day 14 were observed at 1000 mg/kg bw/d.At 300 mg/kg bw/d reduced food consumption were observed in females over the entire study period, significantly on study day 3 (-19%) as well as reduced drinking water consumption in females over the entire study period, significantly on study day 3 (-37%). Body weight gain was decreased in females during the administration period at 1000 mg/kg bw/d, with a maximum of -117% on day 10. At 300 mg/kg bw/d decreased body weight change values were observed in males from day 3 (-53.%) until day 10 and decreased values in females, also from day 3 (-213.3%) until day 14, significantly from day 3 until day 10. Discolored feces in both sexes from day 1 onwards were observed at 300 and 1000 mg/kg bw/d. Hemoglobin and hematocrit were significantly decreased in both sexes and red blood cells in males at 1000 mg/kg bw/d. At 300 mg/kg bw/d significantly decreased values of red blood cells, hemoglobin and hematocrit were determined in both sexes. Significantly increased number of reticulocytes were observed in both sexes at 300 and 1000 mg/kg bw/d. Light red discoloration of several internal organs, of content of glandular stomach and intestine were observed in both sexes as well as discolored content of rectum in males at 1000 mg/kg bw/d. In addtion, significantly increased absolute and relative spleen weights in females were observed. At 300 mg/kg bw/d light red discoloration of several internal organs, of content of glandular stomach and intestine were determined in all male and 1 female animal.Based on these results the dose levels for the subsequent OECD 422 study were set to 0, 12, 60 and 300 mg/kg bw/d.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only one study available

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: spleen

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the test substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.