Registration Dossier

Administrative data

Description of key information

The substance does not cause treatment-related adverse effects up to the limit dose upon subacute oral exposure. This assessment is based on a GLP and OECD 407 compliant study with analogue substance Pigment Red 166 (CAS 3905-19-9) (Vuos 2009b) and a GLP and OECD 422 compliant study with analogue substance Pigment Red 220 (CAS 68259-05-2) (BASF 2012b).

Regarding inhalation route, further information will be provided.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17.9. - 5.11.2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study conducted according to GLP. No analytical verification of test item stability and homogenity. No tables with individual data. No scoring system for severety of histopathology findings. Comparison to historical control data sometimes mentioned in the freetext, but no actual data provided.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
(1995)
Deviations:
yes
Remarks:
No analytical monitoring of test substance. Data for individual animals not reported.
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
(2008)
Deviations:
yes
Remarks:
No analytical monitoring of test substance
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Kolec u Kladna, Czech Republic
- Age at study initiation: 6 -7 weeks
- Mean group weight range at study initiation: males, 182.20 - 183.24 g; females, 136.16 - 136.78 g
- Fasting period before study: the animals starved before blood collection for approximately 18 hours but they received drinking water ad libitum.
- Housing: 2-3 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood.
- Diet (e.g. ad libitum): sterilised complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN) was offered ad libitum.
- Water (e.g. ad libitum): sterilised drinking water with a quality corresponding to Regulation No. 252/2004 Czech Coll. of Law (Health Ministry) was offered ad libitum.
- Acclimation period: at least 5 days
- Diet, water and bedding analysis for contaminants: the standard pelleted laboratory animal diet is analysed for nutrients (once a year) and each batch is microbiologically examined on a regular basis. Certificates of analysis of water (performed twice a year) also are available. Bedding (sterilised clean shavings of soft wood) are examined for bacteriological contaminants once a year. Analysis of diet, water and bedding, did not reveal any findings that could affect study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
olive oil
Remarks:
(Batch No.: L 701 151 and L 803 142; manufactured by Dr. Kulich Pharma s.r.o., Hradec Kralové, Czech Republic)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
The test substance is insoluble in many solvents. Low solubility of the test substance was declared by the Sponsor and it was also checked up during the substance identity verification. In this study the substance was found also insoluble in olive oil. Therefore the test substance was administered as an olive oil suspension. A suitable analytical method was not found for homogeneity and stability testing. Since undissolved particles of the test substance are easily visible in the application form, homogeneity was checked by eye (suspensions were mixed for 15 minutes by magnetic stirrer).The concentrations of suspensions at every dose level were adjusted to ensure the administration of 1 mL/100 g bw. The test substance suspension in olive oil was prepared daily and mixed for 15 minutes by magnetic stirrer just prior administration. The vehicle control group received olive oil in the same volume, without test substance.

TREATMENT
The treated and control groups were administered daily for the period of 28 days. Oral administration was chosen on the request of sponsor. The animals were treated 7 days per week at the specified time (8.00 - 10.00 am).
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No suitable analytical method was available for homogeneity and stability testing. Since undissolved particles of the test substance are easily visible in the application form, homogeneity was checked by eye (suspensions were mixed for 15 minutes by magnetic stirrer).
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
0.16 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Five animals per sex and dose group
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE SELECTION RATIONALE
The doses were selected with respect to the results of a dose-range finding experiment.
SATELLITE GROUPS
A control (vehicle) satellite group as well as a 1000 mg/kg bw/day satellite group, each consisting of five animals per sex were added. These two groups were subjected to a treatment-free post exposure period of 14 days (i.e. day 29 to 42 of experiment).
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS (GENERAL AND DETAILLED) AND MORTALITY
The animals were subjected to general clinical observation daily, during the administration period.
The animals were subjected to detailled clinical observation once prior test initiation and weekly thereafter. At the first part of observation behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerection, stereotypes or bizarre behaviour. The second part was the observation during removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.
Check for mortality was done daily.

BODY WEIGHT
Body weight was recorded weekly.

FOOD CONSUMPTION
Food consumption was recorded weekly.

WATER CONSUMPTION
Water consumption was recorded twice a week

OPHTHALMOSCOPIC EXAMINATION
None

HAEMATOLOGY AND CLINICAL CHEMISTRY
Blood samples were collected on day 29 of study for the main groups and on day 43 for the satellite groups. The blood samples were collected from the orbital plexus under light ether narcosis into PVC test tubes containing anticoagulation systems.
Haematology analyser Coulter and Coagulometer were used for examination and the following parameters were considered: Total erythrocyte count RBC, Mean corpuscular volume MCV, Haematocrit HCT, Haemoglobin concentration HGB, Total leucocyte count WBC, Total platelet count PLT, Partial thromboplastin time APTT, Prothrombin time PT, Fibrinogen FIB, Granulocytes GRAN, Lymphocytes LYM, Monocytes MON.
The biochemical parameters were measured in the serum of the blood samples, which was obtained by centrifugation; the separated serum was collected individually. The following parameters were determined by automatic biochemical analysers: Glucose GLU, Cholesterol total T-Cho, Urea BUN, Bilirubin total T-Bil, Aspartate aminotransferase AST, Alanine aminotransferase ALT, Alkaline phosphatase ALP, Calcium Ca, Phosphorus IP, Protein total T-Pro, Protein albumin ALB, Creatinine Crea, Sodium Na, Potassium K, Chloride Cl.

URINALYSIS
Urine was collected on day 28 of study for the main groups and on day 42 for the satellite groups. For this purpose the rats were kept in metabolic cages and urine was collected for two hours. Immediately before entering metabolic cages the animals were administered with 2 mL/100 g bw of drinking water by gavage. The following parameters were considered: Volume, Colour, Cloud, Odour, Glucose GLU, Protein PRO, Bilirubin BIL, Urobilinogen URO, pH, specific gravity, Blood BLD, Ketones KET, Nitrites NIT, Leucocytes LEU.


NEUROBEHAVIOURAL EXAMINATION
This observation was done at the end of administration period at main groups or at the end of recovery period at satellite groups. During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. Moreover the individual observations of grip strength were performed using dynamometer. Measurements were made on pectoral legs, pelvis legs, and all four legs; grip power was expressed in Newtons.
Sacrifice and pathology:
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. The cranial, thoracic and abdominal cavities were examined and then the organs were collected for weighing, gross pathology and histopathology.

ORGAN WEIGHTS
The absolute and relative weights of liver, kidneys, adrenals, gonads (testes or ovaries), epididymides or uterus, thymus, spleen, brain, pituitary gland and heart were recorded/calculated.

GROSS PATHOLOGY
A revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent pathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde).


HISTOPATHOLOGY
Tissue specimens fixed in 4% buffered formaldehyde were processed by routine paraffin technique and stained by hematoxyline-eosine. Cryotome sections of liver and kidneys were stained by oil red for neutral lipids. Samples of following tissues and organs were considered for histopathology: Adrenal glands, Ovaries, Aorta, Pancreas, Brain (- cerebellum, mid-brain, cortex), Jejunum, Ileum, Caecum, Pituitary gland, Cervix of uterus, Prostate gland, Coagulation gland, Salivary glands (mandibular), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skeletal muscle, Female mammary gland area, Skin, Femur (bone marrow), Spinal cord (midthoracic), Heart, Spleen, Stomach, Testes, Kidneys, Thymus, Liver, Thyroid, Lungs, Trachea, Lymph nodes (mesenteric, paraaortal), Urinary bladder, Oesophagus, Uterus, all gross lesions.
Statistics:
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of haematology, blood chemistry, urinalysis, biometry of organs and body weight. Control group with vehicle was compared with three treated groups and satellite control with vehicle was compared with the satellite treated group. The results statistically significant on probability level 0.05 were indicated.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In 2 of 5 males of the high dose group (1000 mg/kg bw), enlarged paraortal nodes and in one of them enlarged mesenterial nodes with changed colour were recorded.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
reactive hyperplasia of paraaortic lymph nodes (grade not specified) in 2/5 males at 1000 mg/kg bw
Details on results:


FOOD CONSUMPTION
No adverse effect on dietary intake and food efficiency was observed. It was noticed that the application of the test substance caused temporary colouring of the chyme but without marked effect on function of digestive tract. For details, see table below.

WATER CONSUMPTION
Water consumption decreased in both sexes treated with 1000 mg/kg bw/day during the application period. However, urinalysis revealed no changes of concern, thus indicating that the finding was of no toxicological relevance. For details, see table below.

HAEMATOLOGY
All parameters were within the historical control range. Occasionally parameters showed a statisical difference to the control group. There was however no dose-response relationship. Consdering that all parameters were within the historical control range, the changes are considered to be incidental and to be not related to the treatment.

CLINICAL CHEMISTRY
All parameters were within the historical control range. Occasionally parameters showed a statisical difference to the control group. There was however no dose-response relationship. Consdering that all parameters were within the historical control range, the changes are considered to be incidental and to be not related to the treatment.
The measured concentration of urea was below the historic control almost in all females at the middle dose level. Due to absence of changed hepatic parameters or histopathological changes in liver or kidneys, this effect was considered to be of no toxicological importance.

URINALYSIS
Urinalysis showed significantly decreased pH values only in females at the end recovery period. At the end application period there were detected only sporadic presences of protein, cloud or urobilinogen at all dose levels. No microscopic changes were found which could be associated to these sporadic changes; thus they were considered to be of no toxicological importance.

NEUROBEHAVIOUR
Functional observation evidenced no effect of the test substance.

ORGAN WEIGHTS
Sporadic significant changes in weight affecting brain and pituitary gland only were noticed at the end of the recovery period. Differences in absolute or relative weight of liver were sporadically recorded in males and females but were not associated to microscopic changes in this organ. Thus, differences in incidence of findings between control and treatment groups were considered to be of no toxicological significance.

GROSS PATHOLOGY AND HISTOPATHOLOGY
Pathological examination revealed possibly treatment changes in two of the males at the end of application period. Enlarged paraortal nodes (2 of 5 males, none in females) and enlarged mesenterial nodes with changed colour (1 of five males, none in females) were recorded at the highest dose level of 1000 mg/kg bw/day. Histopathology showed reactive hyperplasia of unknown severety of the paraaortic lymph nodes in the two males of the high dose group. No individual data is included in the report, but this information could later be retrieved directly from the CRO. Incidence of reactive hyperplasia in control animals was reported to be zero to two per study.
No neoplastic findings were recorded by histopathological examination.
Dose descriptor:
NOEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
food consumption and compound intake
other: 2 males affected: Enlarged paraortal nodes with reactive hyperplasia (n =2) and enlarged mesenterial nodes with changed colour (n=1) at of 1000 mg/kg bw/day.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Critical effects observed:
not specified

Body weight data:

Mean group body weight (g) for the male rats

Main study (28 days treatment)

Test group

0 mg/kg bw/d

160 mg/kg bw/d

400 mg/kg bw/d

1000 mg/kg bw/d

Day 0

182.54

182.28

183.24

182.22

Week 1

225.46

223.52

217.92

219.02

Week 2

258.36

258.26

249.50

252.76

Week 3

284.16

283.78

272.12

280.18

Week 4

306.16

306.86

289.38

305.74

Recovery groups (28 days treatment + 14 days recovery)

Day 0

182.20

-

-

182.80

Week 1

221.30

-

-

222.02

Week 2

251.34

-

-

255.34

Week 3

275.84

-

-

284.62

Week 4

295.84

-

-

309.72

Week 5

311.14

-

-

325.14

Week 6

320.48

-

-

338.26

Mean group body weight (g) for the female rats

Main study (28 days treatment)

Test group

0 mg/kg bw/d

160 mg/kg bw/d

400 mg/kg bw/d

1000 mg/kg bw/d

Day 0

136.58

136.66

136.60

136.72

Week 1

155.26

156.12

154.42

156.46

Week 2

167.54

170.64

166.90

172.40

Week 3

178.06

179.50

175.14

180.98

Week 4

190.24

190.42

182.74

186.50

Recovery groups (28 days treatment + 14 days recovery)

Day 0

136.16

-

-

136.78

Week 1

152.42

-

-

156.98

Week 2

164.50

-

-

172.04

Week 3

175.98

-

-

181.92

Week 4

186.28

-

-

190.68

Week 5

190.76

-

-

199.04

Week 6

194.86

-

-

206.00

Food consumption data:

Mean food consumption (g/animal/day) for male and females rats

Main study (28 days treatment)

Test group

0 mg/kg bw/d

160 mg/kg bw/d

400 mg/kg bw/d

1000 mg/kg bw/d

Sex

M

F

M

F

M

F

M

F

Week 1

21.09

13.84

19.43

14.77

18.47

12.68

17.76

13.57

Week 2

19.27

12.07

17.91

12.66

17.12

10.44

16.79

12.15

Week 3

18.53

11.46

16.92

11.67

16.15

10.44

16.95

11.30

Week 4

18.60

12.16

15.86

11.00

16.95

9.87

17.22

11.50

Recovery groups (28 days treatment + 14 days recovery)

Week 1

20.63

13.85

-

-

17.53

13.37

Week 2

18.49

12.18

-

-

17.61

11.57

Week 3

17.61

11.31

-

-

18.19

10.65

Week 4

18.19

11.51

-

-

18.32

11.34

Week 5

19.59

13.06

-

-

18.94

14.01

Week 6

19.87

13.09

-

-

20.29

13.74

Water consumption data:

Mean water consumption (mL/animal/day) for male and females rats

Main study (28 days treatment)

Test group

0 mg/kg bw/d

160 mg/kg bw/d

400 mg/kg bw/d

1000 mg/kg bw/d

Sex

M

F

M

F

M

F

M

F

Week 1

40.00

31.71

37.43

33.14

36.86

32.00

36.57

32.29

Week 2

37.14

28.29

35.14

30.57

34.00

28.00

38.57

31.43

Week 3

39.43

32.86

39.14

34.57

38.00

31.71

36.29

33.71

Week 4

40.57

35.71

39.14

36.29

38.57

31.43

36.29

31.43

Recovery groups (28 days treatment + 14 days recovery)

Week 1

39.71

33.14

-

-

35.14

29.14

Week 2

36.57

28.00

-

-

34.57

27.71

Week 3

40.00

34.00

-

-

38.00

30.29

Week 4

40.57

34.29

-

-

36.29

30.57

Week 5

40.57

35.14

-

-

39.71

34.29

Week 6

40.57

32.29

-

-

37.14

30.86

Haematological data for females (means)
dose group 0 140 400 1000 recovery 0 recovery 1000
RBC (10 exp6/uL) 6.57 6.87 6.72 6.82 7.03 6.9
MCV 53.9 54.6 53.4 53.7 53.4 54.1
Haematocrit (%) 35.6 37.5 35.9 36.6 37.6 37.2
Haemogloblin (g/100ml) 12.6 13 12.7 13 13.3 13.2
WBC (1000 /uL) 3.8 4.1 3.7 3.6 4.5 4.7
Platelets (1000 /uL) 900 919 919 820 865 929
APTT (S) 24 26 28 26 26 25
Protrombine Time (s) 25 26 28 26 26 25
Fibrinogen (g/L) 1.66 1.8 1.81 1.82 1.76 2.06
Granulocytes (%) 6.5 5.6 6.8 4.5 5.4 6.4
Lymphocytes (%) 86.9 87.1 81 87.6 86.3 84
Monocytes (%) 6.6 7.3 12.1 7.9 8.2 9.6
Haematological data for males (means)
dose group 0 140 400 1000 recovery 0 recovery 1000
RBC (10 exp6/uL) 6.65 7.01 7.94 6.83 7.08 7.26
MCV 53.9 55.8 53.8 56.2 51.3 53.8
Haematocrit (%) 35.8 38.9 37.7 38.3 37.6 39.2
Haemogloblin (g/100ml) 12.5 13.3 13.2 13 13.3 13.5
WBC (1000 /uL) 7.18 6.84 6.36 7.26 6.22 7.94
Platelets (1000 /uL) 879 893 787 730 826 827
APTT (S) 28.8 25.2 36.2 43.5 40.8 34.9
Protrombine Time (s) 25 32.2 31.8 29.4 33 28.9
Fibrinogen (g/L) 2.23 2.27 2.23 2.43 2.66 2.43
Granulocytes (%) 9.56 7.56 6.5 5.7 7.6 9
Lymphocytes (%) 83.7 86.3 85.1 87 80.7 81.8
Monocytes (%) 6.8 6.1 8.4 7.3 11.5 9.2
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD 422 compliant study with well-characterized test material.
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: RccHanTM: WIST(SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 315 to 364 g, Females: 182 to 212 g
- Fasting period before study: none
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2011-12-15 To: 2012-02-06
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared fresh daily using the test item as supplied by the Sponsor.


VEHICLE
- Concentration in vehicle: as adjusted to dose
- Amount of vehicle (if gavage): 10 mL/kg body weight
:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle
and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of
concentration and homogeneity. During the last week of the treatment, samples were taken from
the middle to confirm concentration.
Duration of treatment / exposure:
Males: Minimum 4 weeks
Females: Approximately 6 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Experimental data on substance of similar structure
Positive control:
Not required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (twic for mortality)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first administration of the test item and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum), detailed clinical observations were performed outside the home cage in a standard arena.
- Cage side observations : changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g.
lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.


BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data



OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males randomly selected from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Anaesthetic used for blood collection: Yes (light isoflurane anesthesia.)
- Animals fasted: Yes
- How many animals: 5
- Parameters checked below were examined:

Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Leukocyte count, total
Differential leukocyte count
Platelet count
Reticulocytes
Prothrombin time (= Thromboplastin time)
Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained on the day before or on the day of the scheduled necropsy from
5 males randomly selected from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Animals fasted: Yes
- How many animals: 5
- Parameters checked below were examined:

Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Gamma-glutamyl-transferase
Bile acids
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females randomly selected from each group. This FOB assessment was conducted following the daily dose administration.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)

At the scheduled sacrifice, the testes and epididymides of 5 parental males were weighed separately.

-Organ weights (5 animals):
Adrenal glands (weighed as pairs)
Brain
Heart
Kidneys (weighed as pairs)
Uterus (including cervix)
Prostate
Liver
Thymus
Spleen
Thyroid (after fixation)
Ovaries (weighed as pairs)
Seminal vesicles (inclusive coagulating gland)


HISTOPATHOLOGY: Yes
Testes, epididymides, prostate, seminal vesicles, ovaries, oviduct, vagina and uterus from all animals of the control and high-dose group were examined. The remaining organs/tissues of 5 randomly selected males and females of the control and high-dose group, respectively, were examined histopathologically:

Brain
Spinal chord (cervical, thoracic, lumbar)
Small and large intestines (duodenum, jejunum, ileum, colon, caecum, rectum, incl. Peyer’s patches)
Stomach (forestomach and glandular stomach)
Liver
Kidneys
Adrenals
Lymph nodes (axillary and mesenteric)
Urinary bladder
Aorta1
Eyes with optic nerve and harderian gland1
Lacrimal gland1
Larynx1
Nasal cavity 1
Esophagus1
Heart
Thymus
Thyroids and parathyroids
Trachea and lungs (preserved by inflation
with fixative and then immersion)
Pituitary gland1
Spleen
Peripheral nerve (sciatic)
Bone marrow (femur)
Femur with knee joint1
Mammary gland (male and female) 1
Pancreas1
Salivary glands – mandibular, sublingual 1
Skeletal muscle 1
Sternum with bone marrow1
Pharynx1
1 = only examined by histopathology in case of macroscopic findings indicative of potential toxicity
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Critical effects observed:
not specified

All males and females had reddish discolored feces one day after treatment start onwards until the end of the treatment period. The severity of this finding was dose-related. This finding is considered to be a typical effect resulting from oral administration of a red dyestuff and is without toxicological relevance.

Conclusions:
The substance does not cause adverse effects upon 28-day gavage application to rats at the limit dose of 1000 mg/kg bw.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).
Reason / purpose for cross-reference:
read-across source
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Critical effects observed:
not specified

All males and females had reddish discolored feces one day after treatment start onwards until the end of the treatment period. The severity of this finding was dose-related. This finding is considered to be a typical effect resulting from oral administration of a red dyestuff and is without toxicological relevance.

Conclusions:
The substance does not cause adverse effects upon 28-day gavage application to rats at the limit dose of 1000 mg/kg bw.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).
Reason / purpose for cross-reference:
read-across source
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
In 2 of 5 males of the high dose group (1000 mg/kg bw), enlarged paraortal nodes and in one of them enlarged mesenterial nodes with changed colour were recorded.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
reactive hyperplasia of paraaortic lymph nodes (grade not specified) in 2/5 males at 1000 mg/kg bw
Details on results:


FOOD CONSUMPTION
No adverse effect on dietary intake and food efficiency was observed. It was noticed that the application of the test substance caused temporary colouring of the chyme but without marked effect on function of digestive tract. For details, see table below.

WATER CONSUMPTION
Water consumption decreased in both sexes treated with 1000 mg/kg bw/day during the application period. However, urinalysis revealed no changes of concern, thus indicating that the finding was of no toxicological relevance. For details, see table below.

HAEMATOLOGY
All parameters were within the historical control range. Occasionally parameters showed a statisical difference to the control group. There was however no dose-response relationship. Consdering that all parameters were within the historical control range, the changes are considered to be incidental and to be not related to the treatment.

CLINICAL CHEMISTRY
All parameters were within the historical control range. Occasionally parameters showed a statisical difference to the control group. There was however no dose-response relationship. Consdering that all parameters were within the historical control range, the changes are considered to be incidental and to be not related to the treatment.
The measured concentration of urea was below the historic control almost in all females at the middle dose level. Due to absence of changed hepatic parameters or histopathological changes in liver or kidneys, this effect was considered to be of no toxicological importance.

URINALYSIS
Urinalysis showed significantly decreased pH values only in females at the end recovery period. At the end application period there were detected only sporadic presences of protein, cloud or urobilinogen at all dose levels. No microscopic changes were found which could be associated to these sporadic changes; thus they were considered to be of no toxicological importance.

NEUROBEHAVIOUR
Functional observation evidenced no effect of the test substance.

ORGAN WEIGHTS
Sporadic significant changes in weight affecting brain and pituitary gland only were noticed at the end of the recovery period. Differences in absolute or relative weight of liver were sporadically recorded in males and females but were not associated to microscopic changes in this organ. Thus, differences in incidence of findings between control and treatment groups were considered to be of no toxicological significance.

GROSS PATHOLOGY AND HISTOPATHOLOGY
Pathological examination revealed possibly treatment changes in two of the males at the end of application period. Enlarged paraortal nodes (2 of 5 males, none in females) and enlarged mesenterial nodes with changed colour (1 of five males, none in females) were recorded at the highest dose level of 1000 mg/kg bw/day. Histopathology showed reactive hyperplasia of unknown severety of the paraaortic lymph nodes in the two males of the high dose group. No individual data is included in the report, but this information could later be retrieved directly from the CRO. Incidence of reactive hyperplasia in control animals was reported to be zero to two per study.
No neoplastic findings were recorded by histopathological examination.
Dose descriptor:
NOEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
food consumption and compound intake
other: 2 males affected: Enlarged paraortal nodes with reactive hyperplasia (n =2) and enlarged mesenterial nodes with changed colour (n=1) at of 1000 mg/kg bw/day.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Critical effects observed:
not specified

Body weight data:

Mean group body weight (g) for the male rats

Main study (28 days treatment)

Test group

0 mg/kg bw/d

160 mg/kg bw/d

400 mg/kg bw/d

1000 mg/kg bw/d

Day 0

182.54

182.28

183.24

182.22

Week 1

225.46

223.52

217.92

219.02

Week 2

258.36

258.26

249.50

252.76

Week 3

284.16

283.78

272.12

280.18

Week 4

306.16

306.86

289.38

305.74

Recovery groups (28 days treatment + 14 days recovery)

Day 0

182.20

-

-

182.80

Week 1

221.30

-

-

222.02

Week 2

251.34

-

-

255.34

Week 3

275.84

-

-

284.62

Week 4

295.84

-

-

309.72

Week 5

311.14

-

-

325.14

Week 6

320.48

-

-

338.26

Mean group body weight (g) for the female rats

Main study (28 days treatment)

Test group

0 mg/kg bw/d

160 mg/kg bw/d

400 mg/kg bw/d

1000 mg/kg bw/d

Day 0

136.58

136.66

136.60

136.72

Week 1

155.26

156.12

154.42

156.46

Week 2

167.54

170.64

166.90

172.40

Week 3

178.06

179.50

175.14

180.98

Week 4

190.24

190.42

182.74

186.50

Recovery groups (28 days treatment + 14 days recovery)

Day 0

136.16

-

-

136.78

Week 1

152.42

-

-

156.98

Week 2

164.50

-

-

172.04

Week 3

175.98

-

-

181.92

Week 4

186.28

-

-

190.68

Week 5

190.76

-

-

199.04

Week 6

194.86

-

-

206.00

Food consumption data:

Mean food consumption (g/animal/day) for male and females rats

Main study (28 days treatment)

Test group

0 mg/kg bw/d

160 mg/kg bw/d

400 mg/kg bw/d

1000 mg/kg bw/d

Sex

M

F

M

F

M

F

M

F

Week 1

21.09

13.84

19.43

14.77

18.47

12.68

17.76

13.57

Week 2

19.27

12.07

17.91

12.66

17.12

10.44

16.79

12.15

Week 3

18.53

11.46

16.92

11.67

16.15

10.44

16.95

11.30

Week 4

18.60

12.16

15.86

11.00

16.95

9.87

17.22

11.50

Recovery groups (28 days treatment + 14 days recovery)

Week 1

20.63

13.85

-

-

17.53

13.37

Week 2

18.49

12.18

-

-

17.61

11.57

Week 3

17.61

11.31

-

-

18.19

10.65

Week 4

18.19

11.51

-

-

18.32

11.34

Week 5

19.59

13.06

-

-

18.94

14.01

Week 6

19.87

13.09

-

-

20.29

13.74

Water consumption data:

Mean water consumption (mL/animal/day) for male and females rats

Main study (28 days treatment)

Test group

0 mg/kg bw/d

160 mg/kg bw/d

400 mg/kg bw/d

1000 mg/kg bw/d

Sex

M

F

M

F

M

F

M

F

Week 1

40.00

31.71

37.43

33.14

36.86

32.00

36.57

32.29

Week 2

37.14

28.29

35.14

30.57

34.00

28.00

38.57

31.43

Week 3

39.43

32.86

39.14

34.57

38.00

31.71

36.29

33.71

Week 4

40.57

35.71

39.14

36.29

38.57

31.43

36.29

31.43

Recovery groups (28 days treatment + 14 days recovery)

Week 1

39.71

33.14

-

-

35.14

29.14

Week 2

36.57

28.00

-

-

34.57

27.71

Week 3

40.00

34.00

-

-

38.00

30.29

Week 4

40.57

34.29

-

-

36.29

30.57

Week 5

40.57

35.14

-

-

39.71

34.29

Week 6

40.57

32.29

-

-

37.14

30.86

Haematological data for females (means)
dose group 0 140 400 1000 recovery 0 recovery 1000
RBC (10 exp6/uL) 6.57 6.87 6.72 6.82 7.03 6.9
MCV 53.9 54.6 53.4 53.7 53.4 54.1
Haematocrit (%) 35.6 37.5 35.9 36.6 37.6 37.2
Haemogloblin (g/100ml) 12.6 13 12.7 13 13.3 13.2
WBC (1000 /uL) 3.8 4.1 3.7 3.6 4.5 4.7
Platelets (1000 /uL) 900 919 919 820 865 929
APTT (S) 24 26 28 26 26 25
Protrombine Time (s) 25 26 28 26 26 25
Fibrinogen (g/L) 1.66 1.8 1.81 1.82 1.76 2.06
Granulocytes (%) 6.5 5.6 6.8 4.5 5.4 6.4
Lymphocytes (%) 86.9 87.1 81 87.6 86.3 84
Monocytes (%) 6.6 7.3 12.1 7.9 8.2 9.6
Haematological data for males (means)
dose group 0 140 400 1000 recovery 0 recovery 1000
RBC (10 exp6/uL) 6.65 7.01 7.94 6.83 7.08 7.26
MCV 53.9 55.8 53.8 56.2 51.3 53.8
Haematocrit (%) 35.8 38.9 37.7 38.3 37.6 39.2
Haemogloblin (g/100ml) 12.5 13.3 13.2 13 13.3 13.5
WBC (1000 /uL) 7.18 6.84 6.36 7.26 6.22 7.94
Platelets (1000 /uL) 879 893 787 730 826 827
APTT (S) 28.8 25.2 36.2 43.5 40.8 34.9
Protrombine Time (s) 25 32.2 31.8 29.4 33 28.9
Fibrinogen (g/L) 2.23 2.27 2.23 2.43 2.66 2.43
Granulocytes (%) 9.56 7.56 6.5 5.7 7.6 9
Lymphocytes (%) 83.7 86.3 85.1 87 80.7 81.8
Monocytes (%) 6.8 6.1 8.4 7.3 11.5 9.2
Endpoint:
sub-chronic toxicity: oral
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
Valid without restriction

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

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

There were no significant toxic effects at doses of less than 300 mg/kg bw upon subacute oral exposure in rats. As a result the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive EC 618/2012.