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REACH

Reaction mass of Sodiumbis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodium [1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)][1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-) and Sodiumbis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(1-)

EC number: 915-756-5 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

In a combined repeated dose toxicity study in rats according to OECD Guideline 422 (BASF SE, 2017), a NOAEL of 350 mg/kg bw/d was determined.

In a company internal range finding study in rats, a LOAEL of below 400 mg/kg bw/d was determined (BASF SE, 2016).

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:: 2016-07-12 to 2017-11-17
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:: 2016-07-29
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Limit test:: no
Specific details on test material used for the study:: SOURCE OF TEST MATERIAL
- Batch No: M-R/G
- Purity: Purity: 99.11 area-% (HPLC, 260 nm), 99.71 area-% (HPLC, 375 nm)
- Composition: 78.4 g/100 g of Cr-Complex (C32H18CrN6NaO8) is postulated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Storage stability: The stability of the test substance under storage conditions was guaranteed until 09 Jun 2017 as indicated by the sponsor.
- Stability of the test substance in the vehicle: The stability of the test substance at room temperature in the vehicle DMSO over a period of 4 hours was determined analytically.

OTHER SPECIFICS:
- Date of production: 09 Jun 2014
- Physical state, appearance: Solid, black
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
Species:: rat
Strain:: Wistar
Remarks:: Crl:WI(Han)
Details on species / strain selection:: The test guidelines require the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats. The animals were free from any clinical signs of disease.
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 (F0-generation); All other animals used in this study (F1 generation pups) were derived from the supplier-provided animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males (14 - 15 weeks old); females (about 13 weeks old)
- Weight at study initiation: males ca. 370 g; females ca. 216 g
- Housing: During pretreatment, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp)
- Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
- Diet: Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:
The food used in the study was assayed for chemical and for microbiological contaminants. The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities of Frankenthal and the Environmental Analytics Water/Steam Monitoring of BASF SE.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h.
Route of administration:: oral: gavage
Details on route of administration:: The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.
Vehicle:: water
Details on oral exposure:: PREPARATION OF DOSING SOLUTIONS:
The test substance suspensions in 0.5 % carboxymethylcellulose suspension in drinking water were prepared in intervals, which took into account the analytical results of the stability verification. The test substance preparations were produced weekly, at least. For the preparation of the administration suspensions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5 % carboxymethylcellulose suspension in drinking water and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Concentration control via chrome determination by inductively coupled plasma optical emission spectrometry (ICP-OES) after digestion with mineral acids (5100 ICP-OES, Agilent). For a summary of the results see table 1.
Duration of treatment / exposure:: The duration of treatment covered a 2 weeks premating period and mating period in both sexes (mating pairs were from the same dose group), approximately 2 days post-mating in males, the entire gestation period as well as up to 13 days of the lactation period in females up to one day prior to the day of scheduled sacrifice of the animals.
Frequency of treatment:: Daily
Dose / conc.:: 0 mg/kg bw/day (nominal)
Dose / conc.:: 20 mg/kg bw/day (nominal)
Dose / conc.:: 80 mg/kg bw/day (nominal)
Dose / conc.:: 350 mg/kg bw/day (nominal)
No. of animals per sex per dose:: 10
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale:
Based on the range finding experiment (project no. 01R0317/14R108) showing mortality at 1300 mg/kg bw/d in female after 3 or 4 days of repeated daily administration and at 700 mg/kg bw/d in male rats after 7 days, the following dose levels were selected:
20 mg/kg body weight/day as low dose
80 mg/kg body weight/day as mid dose
350 mg/kg body weight/day as high dose
The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.

- Rationale for animal assignment: The animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 % of the mean weight of each sex. The list of randomization instructions was compiled with a computer.
Observations and examinations performed and frequency:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working days or once daily (Saturday, Sunday or on public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before initial test substance administration and thereafter at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of F0 parents were determined once a week. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20 and on postnatal days (PND) 1, 4, 7, 10 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption of the F0 parents was determined once weekly during premating. In dams, food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7, 7 - 10, 10 - 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at PND 14 (females) and the end of the administration period (males + females). Blood samples were taken by puncturing the retrobulbar venous plexus
- Anaesthetic used for blood collection: Isofluorane
- Animals fasted: No
- How many animals: 5 /sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period.
- Animals fasted: Yes
- How many animals: 5 /sex/dose

URINALYSIS: Yes - Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Following parameters were examined: urine constituents were analysed by test strips (Combur-Test 10 M; Sysmex, Norderstedt, Germany) and evaluated with a reflection photometer (Miditron M; Sysmex, Norderstedt, Germany).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At the end of the administration period
- Dose groups that were examined: 5 parental males and females per group.
- Battery of functions tested: a functional observational battery was performed and motor activity was measured
Sacrifice and pathology:: GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane. On PND 13, one selected male and one female pup per litter were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4 % formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing.

HISTOPATHOLOGY: Yes
Fixation was followed by histotechnical processing, examination by light microscopy according to table 3. Special attention was given on the stages of spermatogenesis in the testes. A correlation between gross lesions and histopathological findings was attempted.
Statistics:: See Table 1.
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: During pre-mating, mating and post-mating discolored feces were seen in all animals of the test group 3 (350 mg/kg bw/d). During mating and postmating discolored feces additionally occured in 9 of 10 males of test group 2 (80 mg/kg bw/d). The sign came up first on premating day 2 in test group 3 (350 mg/kg bw/d) and mating day 11 (study day 24) in test group 2 (80 mg/kg bw/d). Furthermore, discolored feces were observed in all females of test group 3 (350 mg/kg bw/d) during the whole gestation and lactation and in one female of test group 2 (80 mg/kg bw/d) during gestation from gestation day 9 to 15. In test group 3 (350 mg/kg bw/d) additionally discolored skin and eyes were observed in all animals. These signs came up first during pre-mating, discolored eyes from day 8 onwards and discolored skin from day 9 onwards. All these grey-black discolorations were regarded to be caused by the test substance, a black colorant. It was regarded to be test substance related.
Mortality:: no mortality observed
Description (incidence):: There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: Mean body weights and body weight change of all male and female F0 generation parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study period.
For the significantly decreased body weight in males of test group 3 (350 mg/kg bw/d) at mating day 7 (-3.8 %) and significantly decreased body weight changes in males of this group during premating (-58.6 %), it could not be excluded to be treatment related.
The significantly decreased body weight change in males of test group 1 during premating (-40.7 %) was an isolated finding without dose-dependency and assessed as incidental and not related to treatment.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: Food consumption of the F0 males in test group 1 and 2 (20 and 80 mg/kg bw/d) and females in all dose groups (20, 80 and 350 mg/kg bw/d) was not influenced by the treatment throughout the entire study period.
For the significantly decreased food consumption in the high-dose group (350 mg/kg bw/d) of F0 males (-6.4 %) during pre-mating it could not be excluded to be treatment related.
The increased food consumption in females of test group 1 (20 mg/kg bw/d; 15.3 %) during lactation were considered as spontaneous in nature and not treatment-related.
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Description (incidence and severity):: No treatment-related changes among hematological parameters were observed.
Clinical biochemistry findings:: effects observed, non-treatment-related
Description (incidence and severity):: In males of test group 3 (350 mg/kg bw/d) creatinine and calcium levels were significantly decreased. This was also true for calcium in males of test group 1 (20 mg/kg bw/d). In females of test groups 2 and 3 (80 and 350 mg/kg bw/d) aspartate aminotransferase (AST) activities were significantly reduced. However, all means were within historical control ranges (males, creatinine 20.4-29.8 μmol/L; calcium 2.42-2.67 mmol/L; females AST 1.62- 2.73 μkat/L). Therefore, all mentioned alterations were regarded as incidental and not treatment-related.
Urinalysis findings:: effects observed, treatment-related
Description (incidence and severity):: In rats of both sexes of test group 3 (350 mg/kg bw/d) bilirubin levels were significantly higher compared to controls. The urine color in rats of test group 3 compared to controls was not changed. However, this is an isolated finding without any change in liver, kidney or hematologic parameter and without any adverse change in histopathology. Therefore, higher bilirubin levels in the urine of rats of test group 3 (350 mg/kg bw/d) were regarded as treatment-related, but not adverse.
Behaviour (functional findings):: no effects observed
Description (incidence and severity):: Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: no effects observed
Description (incidence and severity):: All mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: A dark grey or black discoloration was generally observed in most organs of all male and female animals of test group 3 (350 mg/kg bw/d), as well as in the contents of stomach and cecum. In test group 2 (80 mg/kg bw/d), only 2 out of 10 males showed the same dark grey discoloration in the cecum contents. All of these changes were due to the black color of the test substance and were considered to be treatment-related. However, since the substance disappeared from the tissues after the histotechnical process, no histopathological correlate was observed.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: A minimal increase in the incidence of basophilic tubules, accompanied by the presence of hyaline casts and / or lympho-histiocytic interstitial infiltrates was noted in the high dose female group when compared with the control group. A treatment-related but not adverse effect cannot be excluded.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:: not examined
: Key result
Dose descriptor:: NOAEL
Effect level:: 350 mg/kg bw/day (nominal)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: no adverse effects were observed up to the highest dose
: Key result
Critical effects observed:: no
Conclusions:: Under the conditions of this Combined Repeated Dose Toxicity Study with the
Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of
Eukesolar Black ER Liquid - dried to Wistar rats revealed no treatment-related adverse
findings in parental animals (F0) and pups (F1).
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity as well as
for reproductive performance and fertility was the nominal dose level 350 mg/kg bw/d in
both sexes of parental animals.
The NOAEL for developmental toxicity in the F1 progeny was the nominal dose level
350 mg/kg bw/d.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 350 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: Klimisch Code 1

Additional information

Repeated dose toxicity: oral

SUMMARY

METHODS

Eukesolar Black ER Liquid – dried was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at nominal doses of 20, 80 and 350 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5% Carboxymethylcellulose suspension in drinking water). The duration of treatment covered a 2 weeks premating period and mating period in both sexes (mating pairs were from the same dose group), approximately 2 days post-mating in males, the entire gestation period as well as up to

13 days of the lactation period in females up to one day prior to the day of schedule sacrifice of the animals. The study was conducted according to OECD 422 guieline and GLP (BASF, 2018).

OBSERVATIONS

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females.

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 once weekly during premating. In dams, food consumption was determined for GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 - 7,

7 - 10, 10 - 13.

Body weights of F0 parents were determined once a week. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20 and on postnatal days (PND) 1, 4,

7, 10 and 13.

Estrous cycle data were evaluated for F0 generation females over a two-week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female

was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. At necropsy on PND 4,

as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. At necropsy on PND 13, one selected male and one female pup per litter

was sacrificed under isoflurane anesthesia by decapitation, all other pups (on PND 13) were sacrificed with CO2, under isoflurane anesthesia. All pups were examined macroscopically

for external and visceral findings.

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized

fashion, using a measuring ocular on all live male and female pups on PND 1.

All surviving male pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted.

Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for

determination of thyroid hormone concentrations.

Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period.

Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone

measurement.

At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group.

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.

RESULTS

Analysis

The various analyses:

- Confirmed the stability of the test substance in 0.5% Carboxymethylcellulose suspension in drinking water over a period of 7 days at room temperature

- Confirmed the homogeneous distribution of the test substance in 0.5% Carboxymethylcellulose suspension in drinking water

- Determined following mean concentrations in the test substance preparations of both timepoints of premating and lactation period: 96% of the nominal concentration in test group 3 (350 mg/kg bw/d), 87% in test group 2 (80 mg/kg bw/d) and 89% in test group 1 (20 mg/kg bw/d) (see Part III, Supplement). Thereby, in the high dose group the strict specifications of the test facility (90-110 %) were met. The determined concentration in the mid and low dose group were slightly below the specification of the test facility, but within generally accepted specification (80-120%). Thereby, the authors assessed the

overall accuracy of the test substance preparations as given. Since the high dose group (350 mg/kg bw/d) in this study represents the no observed adverse effect level (NOAEL) the slight deviation from the strict specification of the test facility in the mid and low dose group does not have any impact on the validity of the study.

Effects

The following test substance-related adverse effects/findings were noted:

Test group 3: nominal 350 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance, clinical pathology, and pathology