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Diss Factsheets

Administrative data

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
Cross-reference
Reason / purpose for cross-reference:
reference to same study
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

Table 1: Summary of the results of the concentration control

Sample No

Date of sample preparation and sampling

Content of Chronium (g/100 g)

Density (g/mL)

Calculated content of test item

(g/100 mL)

Expected content of test item (g/100 mL)

Recovery (%)

1 (Test Item)

-

5.8

 

 

 

 

2 (Vehicle)

-

< 0.003

 

 

 

 

3

02 August 2016

0.010 (102.6 mg/kg)

0.9954

0.1754

0.2

88

4

02 August 2016

0.010 (104.3 mg/kg)

0.9966

0.1785

0.2

89

5

02 August 2016

0.010 (104.15 mg/kg)

0.9972

0.1784

0.2

89

6

02 August 2016

0.041 (407.45 mg/kg)

0.9986

0.6988

0.8

87

7

02 August 2016

0.2 (1952 mg/kg)

1.0012

3.3564

3.5

96

8

02 August 2016

0.19 (1933.5 mg/kg)

1.0077

3.3463

3.5

96

9

02 August 2016

0.19 (1942 mg/kg)

1.0051

3.3525

3.5

96

Table 2: Summary male body weights [g]

Day

 

Group 0
(0 mg/kg bw/d

Group 1
(20 mg/kg bw/d

Group 2
(80 mg/kg bw/d

Group 3
(350 mg/kg bw/d

Male, pre-mating

0

Mean

373.0

372.0

371.4

371.9

 

S.D.

13.5

10.9

12.4

11.7

 

Deviation vs Control

 

-0.2

-0.4

-0.3

7

Mean

380.6

376.3

375.6

373.0

 

S.D.

12.7

13.7

14.1

13.7

 

Deviation vs Control

 

-1.1

-1.3

-2.0

13

Mean

391.8

383.2

384.1

379.7

 

S.D.

14.8

13.5

12.1

14.7

 

Deviation vs Control

 

-2.2

-2.0

-3.1

Male, mating

7

Mean

396.5

389.1

388.8

381.6*

 

S.D.

15.3

12.1

11.0

13.9

 

Deviation vs Control

 

-1.9

-2.0

-3.8

14

Mean

404.9

394.2

394.4

390.4

 

S.D.

16.9

15.0

12.7

16.7

 

Deviation vs Control

 

-2.6

-2.6

-3.6

Table 3: Summary female body weights [g]

Day

 

Group 0
(0 mg/kg bw/d

Group 1
(20 mg/kg bw/d

Group 2
(80 mg/kg bw/d

Group 3
(350 mg/kg bw/d

Female, pre-mating

0

Mean

218.2

214.4

217.3

215.8

 

S.D.

9.2

8.0

8.0

10.1

 

Deviation vs Control

 

-1.7

-0.4

-1.1

7

Mean

219.0

214.8

217.7

216.7

 

S.D.

7.9

9.1

7.8

9.0

 

Deviation vs Control

 

-1.9

-0.6

-1.1

13

Mean

223.2

222.9

224.9

220.2

 

S.D.

9.4

7.3

7.1

10.0

 

Deviation vs Control

 

-0.1

0.8

-1.4

Female, gestation

0

Mean

225.1

221.9

224.4

222.7

 

S.D.

8.0

6.7

8.0

10.6

 

Deviation vs Control

 

-1.74

-0.3

-1.1

7

Mean

248.7

250.2

249.3

243.6

 

S.D.

9.4

9.0

11.9

13.8

 

Deviation vs Control

 

0.6

0.3

-2.1

14

Mean

278.3

276.4

274.9

270.8

 

S.D.

8.8

12.6

12.6

17.2

 

Deviation vs Control

 

-0.7

-1.2

-2.7

20

Mean

333.3

329.0

337.7

330.8

 

S.D.

9.7

24.3

17.3

24.5

 

Deviation vs Control

 

-1.3

1.3

-0.7

Female, lactation

0

Mean

263.2

260.5

255.3

254.3

 

S.D.

14.0

14.3

9.5

20.1

 

Deviation vs Control

 

-1.0

-3.0

-3.4

4

Mean

268.

264.7

266.5

261.3

 

S.D.

11.7

12.7

13.0

22.6

 

Deviation vs Control

 

-1.5

-0.9

-2.8

7

Mean

274.3

276.1

278.0

270.6

 

S.D.

10.7

12.3

10.8

19.3

 

Deviation vs Control

 

0.6

1.3

-1.4

10

Mean

288.4

284.4

288.8

281.6

 

S.D.

8.7

14.5

11.7

16.1

 

Deviation vs Control

 

-1.4

0.1

-2.4

13

Mean

288.3

281.2

283.5

284.6

 

S.D.

10.7

18.1

15.6

19.2

 

Deviation vs Control

 

-2.5

-1.7

-1.3
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
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
Molecular formula:
C32H18NaCrN6O8
IUPAC Name:
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-)
Test material form:
solid
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.

Test animals

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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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,

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

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.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

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.