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-)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In an oral combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats according to OECD Guideline 422 (BASF SE, 2017), a NOAEL of 350.0 mg/kg bw/d was determined.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

Reason / purpose for cross-reference:

reference to same 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)
- During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
- 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

Vehicle:

water

Details on 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.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

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 schedule 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.

Parental animals: Observations and examinations:

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 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.
- Anaesthetic used for blood collection: Isoflourane
- 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

Oestrous cyclicity (parental animals):

For all females of the pool estrous cycle normality was evaluated before the beginning of the administration period. Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 2 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); surplus pups or 2 preferably female pups per litter were sacrificed. Standardization of litters was not performed in litters with less than or equal to 8 pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
total number and sex of pups, stillbirths, live births, stillborn pups postnatal mortality, presence of gross anomalies, sex ratio, weight gain, physical or behavioural abnormalities, anogenital distance (AGD) and anaogenital index, presence of nipples/areolae in male pups,

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-bycase basis, depending on the type of finding noted.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed on study day 29.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.

GROSS NECROPSY
- All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 2 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected by randomization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation on PND 4. All stillborn pups and all pups that died before PND 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination)

GROSS NECROPSY
- After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.

Statistics:

See Table 4.

Reproductive indices:

Male: Mating Index, Fertility Index
Female: Mating Index, Fertility Index, Gestation Index, Live Birth Index, Postimplatation Loss,

Offspring viability indices:

Viability Index, Survival Index, Sex Ratio, Anogenital Index,

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, non-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

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

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data revealed regular cycles in the parental females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) was between 3.82 and 4.10 days. The mean number of cycles in the different test groups ranged from 2.4 to 2.8.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stages of spermatogenesis in the testes of males of the high dose (350 mg/kg bw/d) were comparable to those of the controls.

Reproductive performance:

no effects observed

Description (incidence and severity):

For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100 % in all test groups. Fertility was also proven for all of the F0 parental males within the scheduled mating interval for F1 litter. Thus, the male fertility index was also 100 % for all test groups.
The female mating index calculated after the mating period for F1 litter was 100 % in all test groups. The mean duration until sperm was detected (GD 0) varied between 2.2 and 2.5 (test groups 0 – 3). All female rats delivered pups. The fertility index was 100 % in all test groups. The mean duration of gestation was similar in all test groups (i.e. between 22.0 and 22.5 days). The gestation index was 100 % in all test groups 0 - 3. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. Implantation sites were not affected by the treatment since the mean number of implantation sites was comparable between all test groups (11.1 / 9.8 / 13.4 and 12.9 implants/dam in test groups 0, 1, 2, and 3, respectively). These values reflect the normal biological variation of the used strain. The postimplantation loss did not show any significant differences between test groups 0 and 1 or 2,. Whereas postimplantation loss was significantly increased in test group 3 (350 mg/kg bw/d; 14.0 %). But this finding is not considered to be adverse because a postimplantation loss of 14 % is in the range of historical control data (0% – 18.1 %).
For this reason, there were no indications for test substance-induced intrauterine embryo-/fetolethality. The mean number of F1 pups delivered per dam remained unaffected (10.8 / 9.5 / 13.2 and 11.2 pups/dam in test groups 0, 1, 2, and 3, respectively). The gestation index was 100 % in all test groups.
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 99.1 % / 98.9 % / 99.2 % and 100% in test groups 0, 1, 2, and 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.

Key result

Dose descriptor:

NOAEL

Effect level:

350 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For all pups of test group 3 (350 mg/kg bw/d) a black discoloration of the skin was observed. This discoloration was regarded to be caused by the test substance, a black colorant. It was regarded to be test substance related.
In test group 0 one female pup has had a short tail which was narrowed at the root. This is considered to be spontaneous in nature. There were no further test substance-related, adverse clinical signs observed in any of the other F1 generation pups of the different test groups.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 98.6 % / 99.3 % / 98.1 % and 99.1 % in test groups 0, 1, 2, and 3, respectively, showing no association to the treatment. The survival index indicating pup mortality on PND 4 – 13 varied between 100% / 100% / 98.8 % and 100% in test groups 0, 1, 2, and 3, respectively without showing any relation to the treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and body weight change of all male and female pups of all test groups (20, 80 and 350 mg/kg bw/d) were comparable to the concurrent control values throughout the entire lactation period. Two female runts were seen in test group 1 (20 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Urinalysis findings:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A few F1 pups showed spontaneous findings at gross necropsy, such as discolored liver lobe, diaphragm hernia, misshapen or small spleen, dark discolored liver lobe, reddish discolored stomach and intestine content, abdomen fluid-filled, small testis, distended stomach and short tail. These findings occurred without any relation to dosing and are considered to be spontaneous in nature.
Furthermore, 94.2 % of the male and 85 % of the female F1 pups of test group 3 (350 mg/kg bw/d) showed grey discolored skin at their entire body. This discoloration was regarded to be caused by the test substance, a black colorant. It was regarded to be test substance related.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Neither in anogenital distance nor in anogenital index treatment-related effects were noted in all F1 male and female pups of all test groups. The significantly decrease of anogenital distance in male pups of test group 2 (80 mg/kg bw/d; -4.23 %) is considered to be caused by incidentally grouped smaller F1 pups, since the anogenital index is comparable to the concurrent control. It was assessed as not treatment related.
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
In male and female pups at PND13 (test groups 11, 12 and 13; 20, 80, 350 mg/kg bw/d), no treatment-related alterations of T4 levels were observed.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

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

Key result

Reproductive 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.

Effect on fertility: via oral route

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

Toxicity to reproduction

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

- No test substance-related adverse findings

F1 PUPS

Clinical examinations/ gross findings

- No test substance-related adverse findings

Test group 2: nominal 80 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

F1 PUPS

Clinical examinations/ gross findings

- No test substance-related adverse findings

Test group 1: 20 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance, clinical pathology, and pathology

- No test substance-related adverse findings

F1 PUPS

CLINICAL EXAMINATIONS/ GROSS FINDINGS

- no test substance-related adverse findings

CONCLUSION

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.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

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

Justification for classification or non-classification

Classification, Labeling, 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 substance is not considered to be classified for reproductive or developmental toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.