Registration Dossier

Administrative data

Description of key information

NOAEL 28d oral rat = 1500 mg/kg. No substance specific changes were observed. (Safepharm, 1990)

NOAEL 28d oral rat = 1000 mg/kg. (NOTOX, 2006)

NOAEL 90d oral rat = 50,000 ppm (Equivalent to OECD 408; non-GLP; Hoechst, 1967)

These data are based on read across approaches to other category members.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
study is not fully compliant with current guidelines.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Mean weight at study initiation: 130 g (males) and 121 g (females)
- Housing: Makrolon cages on wood shavings, 5 animals per cage
- Diet (e.g. ad libitum): ALTROMIN R, manufactured by Altromin GmbH in Lage/Lippe
- Water (e.g. ad libitum): tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 25
- Humidity (%): 35 - 60
- Air changes (per hr): partially air-conditioned animal rooms
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): pelleted feed was freshly prepared at 7-8-day intervals
- Mixing appropriate amounts with (Type of food): ALTROMIN R, manufactured by Altromin GmbH in Lage/Lippe
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
10 000 ppm
Remarks:
1% in feed
Dose / conc.:
50 000 ppm
Remarks:
5% in feed
No. of animals per sex per dose:
15
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
Each rat was monitored daily to determine its behaviour and general state of health. The body weight of each animal was determined once a week and the food consumption at 7-day intervals.
Before the start and at the end of the trial haematological and urine investigations were carried out in 10 male and 10 female rats from each group. The haematological investigation included determination of the haemoglobin content, number of red and white blood cells as well as the evaluation of the differential blood count and the possible presence of Heinz bodies. The urine investigation covered appearance, colour, protein and sediment.
Sacrifice and pathology:
At the end of the trial all the animals were killed and autopsied and the organs were subjected to histological examination.
Macro- and microscopic examination of the organs heart, lung, liver, kidney, adrenal glands, spleen, cerebrum, cerebellum, testes and ovaries, pancreas, pituitary, thyroid gland, stomach and small intestine.
Clinical signs:
no effects observed
Description (incidence and severity):
All the animals displayed normal behaviour during the trial and no signs of a toxic effect were observed which would have been caused by the pigment. The pigment was excreted via the faeces.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were isolated spontaneous deaths during the trial. One rat (animal no. 304) from group 1 (5%) died after 33 trial days and a further rat (animal no. 337) after 73 trial days. One rat (animal no. 362) from group III (control) died after 23 trial days. The rats died of pneumonia. No connection between the substance administered and the deaths could be detected.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The weight gains of the male and female rats from groups I (5%) and II (1%) were normal and corresponded to those of the control animals.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption of the experimental animals was the same as that of the control rats.
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):
The haematological investigations showed no pathological findings even at the end of the trial.
Clinical biochemistry findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urine investigations showed no pathological findings at the end of the trial. The male rats showed slight protein excretion at the end of the trial which is a physiological feature of adult male rats.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The organ weight gains of the male and female rats from groups I (5%) and II (1%) were normal and corresponded to those of the control animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination of the organs of rats that had received the pigment in their feed in a 90-day trial revealed no pathological organ damage. Inflammatory lung changes were detected at autopsy in experimental animals no. 304 and 337. Animal no. 322 displayed a liver change for which no reason could be found. The controls no. 360 and 362 also had inflammatory lung processes arid the liver of no. 400 was interspersed with yellowish spots the size of a pinhead. The striking feature of no. 413 was very marked liver lobule patterning.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histological examination of the organs of rats that had received the pigment in their feed in a 90-day trial revealed no pathological organ damage. The lymphocytic infiltrates frequently observed in the heart, liver, kidneys and intestine are a manifestation of quiescent infections. The perifollicular hyperplasia in the spleen should also be assessed as a non-specific defense reaction of this type. The histo-lymphocytic granulomas found in the liver may be a manifestation of a pre-existing infection, most probably salmonellosis. The lung changes found are characteristic of rat-specific pneumonia. In many of the animals there was impaired spermiogenesis in the form of varying degrees of desquamation of prespermatid cells. The striking frequency in the control group (11 cases) and in the group that received 5% test material (9 cases) and the only isolated occurrence in the group that was given 1% test item (2 cases) indicates that these changes are not attributable to the test substance. In addition, the changes in the brain in the form of meningoencephalitis occasionally observed developed in some cases with foci of glial proliferation independent of the study. These brain changes are diagnosed in rats more frequently than random findings, without causing clinical symptoms. The thyroid changes in the form of interfollicular solidification, associated with formation of follicles to varying degrees, occurred in both the control and the experimental animals.

In summary, it can be stated that no obvious study-related changes were observed. The test item therefore caused no organ-damaging effects in rats in the dosages given and over the selected period of administration.
Dose descriptor:
NOAEL
Effect level:
50 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects were reported
Critical effects observed:
no
Conclusions:
In summary, it can be stated that no obvious study-related changes were observed. The test substance therefore caused no organ-damaging effects in rats in the dosages given and over the selected period of administration.
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
see attached justification.
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
50 000 ppm
Based on:
other: Read-across
Sex:
male/female
Basis for effect level:
other: no adverse effects assumed
Critical effects observed:
no
Conclusions:
Based on the study performed with the read-across substance, the target substance was not assumed to incude treatment-related adverse effects. Thus, the NOAEL was concluded to be 50,000 ppm.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Manston, Kent
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: males: 130 - 156 g; females: 117 - 145 g
- Housing: 5 per cage
- Diet (e.g. ad libitum): SQC Rat and Mouse Diet No . 1 Expanded, Special Diet Services Limited, Witham, Essex, UK
- Water (e.g. ad libitum): free access to mains water from polycarbonate bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 24 °C
- Humidity (%): 28 - 60 %
- Air changes (per hr): least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h
Route of administration:
oral: feed
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Dietary admixture was prepared for use throughout the study, 2 - 3 days prior to the start of the study and were used throughout the 28-day dosing period.
- Mixing appropriate amounts with (Type of food): The test material was incorporated into the diet at concentrations of 750, 3750 and 15000 ppm as follows: A known amount of test material was mixed with a small amount of basal laboratory diet. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for 30 minutes at a constant speed, setting 1, in a Hobart QE 200 mixer. The diet was stored in labelled black plastic bags and contained in bins with lids when not in use.
- Storage temperature of food: not reported.

VEHICLE
- Concentration in vehicle: incorporated into the diet at concentrations of 50, 250 and 1000 ppm
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
expected concentration: 50 mg/kg;
analytical results:
44/42 mg/kg starting value;
37/36 mg/kg value after 10 days;
43/39 mg/kg value after 32 days;
The test substance is stable in Kliba 343 Mehl for the expected concentration (ca . 50 mg/kg Kliba Mehl) over a period of 32 days within the limits of the errors of the analytical method.

Stability under test conditions: The stability of the test material when mixed with rodent diet was determined by the sponsor. The results showed that the test material is stable under these conditions for at least 32 days. Dietary admixtures were therefore prepared 2 to 3 days prior to the start of the study and were used throughout the 28-day dosing period.
Duration of treatment / exposure:
28 days
Frequency of treatment:
continuously
Dose / conc.:
750 ppm
Remarks:
Mean: 79.25 mg/kg bw/day (actual ingested; males); 78.0 mg/kg bw/day (actual ingested; females)
Dose / conc.:
3 750 ppm
Remarks:
Mean: 404.25 mg/kg bw/day (actual ingested; males); 380.0 mg/kg bw/day (actual ingested; females)
Dose / conc.:
15 000 ppm
Remarks:
Mean: 1572.75 mg/kg bw/day (actual ingested; males); 1501.25 mg/kg bw/day (actual ingested; females)
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Post-exposure recovery period in satellite groups: Animals from satellite groups 5 (additional control group) and 6 (additional high dose group) were maintained for a further 14 days treatment free period following termination of treatment.
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change each day.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on the day before the start of treatment (day 0) and on days seven, fourteen, twenty-one and twenty-eight and in the case of satellite group animals on days thirty-five and forty-two. Bodyweights were also recorded at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was recorded for each cage group at weekly intervals throughout the study.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological and blood chemical investigations were performed on all animals from groups 1 to 4 at the end of the treatment period (day 28). Parameters showing possible treatment-related changes in these animals were examined in satellite group animals at the end of the treatment-free period (day 42).
- Anaesthetic used for blood collection: Yes (identity)
Blood samples were obtained by orbital sinus puncture under Halothane BP anaesthesia.
- Animals fasted: No
- How many animals:
- Parameters examined: measured on blood collected into tubes containing lithium heparin anti-coagulant:
Haematocrit (Hct), Haemoglobin (Hb), Erythrocyte count (RBC), Total leucocyte count (WBC ), Erythrocyte indices - mean corpuscular haemoglobin (MCH), - mean corpuscular volume (MCV), - mean corpuscular haemoglobin concentration (MCHC). Samples were withdrawn into tubes containing potassium EDTA anticoagulant for platelet counts (PLT) and differential leucocyte counts. Clotting time (CT) was assessed by 'Hepato Quick' time using samples collected into sodium citrate solution (0 .11 mol/L).

CLINICAL CHEMISTRY: Yes, see for the schedule, HAEMATOLOGY
- Parameters examined: measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Blood urea, Total protein, Albumin, Albumin/globulin ratio (by calculation), Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Creatinine
Alkaline phosphatase (AP), Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Glucose, Gamma glutamyl transpeptidase (yGT), Triglycerides, Total Cholesterol, Total Bilirubin

URINALYSIS: Yes
- Time schedule for collection of urine: during the final week of dosing (week 4)
- Metabolism cages used for collection of urine: Yes
Urine samples were collected over a period of approximately 16 hours, by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection, but without access to food.
- Animals fasted: Yes
- Parameters examined: Volume, Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Reducing substances, Blood.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weights

HISTOPATHOLOGY: Yes
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights: The following organs from animals that were killed at the end of the study, dissected free from fat, were weighed before fixation:
Adrenals, Brain, Gonads, Heart, Kidneys, Liver, Pituitary, Spleen.
Histopathology: Samples of the following tissues were removed from all animals and preserved in 10% buffered formalin.
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & Bone Marrow (femur), Pituitary, Brain Prostate, Caecum, Rectum, Colon, Salivary glands, Duodenum Sciatic nerve, Eyes, Seminal vesicles, Gross lesions Skin (hind limb), Heart, Spleen, Ileum, Stomach, Jejunum, Testes, Kidneys, Thymus, Liver, Thyroid/parathyroid, Lungs, Trachea, Lymph nodes (cervial and mesenteric), Urinary bladder, Muscle (skeletal), Uterus, Oesophagus.
The following preserved tissues from all high dose and control group animals (groups 1 and 4) were prepared as paraffin blocks, sectioned at nominal thickness of 5 um and stained with haematoxylin and eosin for subsequent microscopic examination: Adrenals, Spleen, Heart, Kidneys, Liver.
Statistics:
Relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating 'F-max' test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal Wallis non-parametric analysis of variance and Mann Whitney U-Test.
Probability values were calculated as: p < 0 .001 ***; P < 0. 01 **; P < 0 .05 *; p >= 0.05 (not significant)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinically observable signs of toxicity were noted in test or control animals throughout the study.
Extensive black/green staining of the external body surface was seen, from day 2 until the end of the treatment period, in animals (males and females) treated with 15000 ppm. Similar, but less extensive, staining was also observed in animals treated with 3750 ppm over the same period. This staining persisted in the satellite treatment groups but diminished with time, with only the animals tails being discoloured by the end of the treatment-free period. Black faeces was common to all treatment groups from day 4 onwards and was also apparent in the satellite treatment animals until day 31, after which the faeces of the satellite high dose groups appeared normal. No external staining was noted in animals treated with 750 ppm.
Mortality:
no mortality observed
Description (incidence):
There were no deaths during the study .
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweight gain in all test animals was comparable with that seen in controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in food consumption during the study.
Food efficiency:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in weekly food efficiency during the study.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No overt intergroup differences were detected.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean values and standard deviations for test and control group animals are given in the tables below (statistically significant differences are indicated).
An anaemia was demonstrated in females treated with 15000 ppm, but not in males from the same dose group, identified by statistically significant reductions in haemoglobin levels, erythrocyte counts and haemotrocrit. Some animals showed values outside the normally expected range for rats of this strain and age but the intergroup difference was exacerbated by some rather high values in the control group. Calculation of mean corpuscular volume showed a slight increase in high dose females compared with controls but all values appeared normal. There is, therefore, no convincing evidence to suggest reticulocytosis and since there were no other changes that could be considered indicative of haemolysis it is possible that the anaemia was megaloblastic in nature. A slight but statistically significant reduction in platelet counts could have been associated with this, but all individual values remained within the normal range. The anaemia was very slight and apparently completely reversible since no such changes were seen in satellite high dose females after fourteen days without treatment. The effect was confined to the high dose group and an apparent increase in mean corpuscular haemoglobin, for females from all treatment groups, was considered to be entirely fortuitous since true hyperchromic cells do not exist.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
A statistically significant reduction in blood glucose was noted in males treated at 750 (111 +/- 10 mg/dL) and 15000 (116 +/- 13 mg/dL) ppm compared with control (133 +/- 16) although all values were within the normal range for animals of this strain and age. In addition, a true dose-related response was not elicited and the difference between test and control groups was considered not to be treatment-related. High dose males also showed a significant reduction in the calculated albumin/globulin ratio but as there were no significant changes in either serum albumin level or total protein and the values were within the normal range, the change is not thought to represent a toxicological effect. Female animals treated at 3750 (353 +/- 134 IU/L) and 15000 (353 +/- 86 IU/L) ppm showed reduced alkaline phosphatase levels compared to controls (500 +/- 77 IU/L), but none of the values could be considered abnormal and, a reduction in this enzyme could not be considered toxicologically significant.
Other minor statistically significant differences were confined to low and intermediate treatment groups only and, as such, were not dose-related .
Urinalysis findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were detected in any of the urine parameters measured.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related differences in organ weight were detected between test and control animals.
A slight reduction in both liver and kidney weights, relative to body weight, was seen in male animals treated at 15000 ppm with only the decrease in relative liver weight being maintained in the satelite group males. However, none of the values could be considered abnormal for rats of this strain and age. In the absence of histopatholigcal abnormalities and any associated relevant changes in either blood chemical or haematological parameters, these reductions are unlikely to be toxicologically significant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Animals treated with 3750 and 15000 ppm had either slight or extensive black/green staining of the external body surface, respectively, at necropsy . Black/green staining of the non-glandular epithelium of the stomach was observed in animals from all the test groups . In addition, the contents of the gastro intestinal tract of all animals fed laboratory diet containing the test material were coloured black/green.
Although satellite treatment group animals still had black/green staining on their tails, there was no residual staining of the stomach epithelium at the end of the fourteen-day treatment-free period.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed. All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
NOAEL
Effect level:
> 1 501.25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse treatment-related effects observed
Dose descriptor:
NOAEL
Effect level:
> 1 572.75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse treatment-related effects observed
Dose descriptor:
NOEL
Effect level:
380 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: slight anaemia which was completely reversible since no such changes were seen in satellite high dose females after fourteen days without treatment
Critical effects observed:
no

GROUP MEAN HAEMATOLOGICAL VALUES AND STANDARD DEVIATIONS (S.D.) - FEMALES

TEST MATERIAL

GROUP

NUMBER

DIETARY

CONCENTRATION

(ppm)

Hb

RBC

Hct

MCH

MCV

MCHC

WBC

DIFFERENTIAL (109/L)

CT

PLT

(g/dL)

(1012/L)

(%)

(pg)

(fL)

(g/dL)

(109/L)

Neut

Lymph

Mono

Eos

Bas

(secs)

(109/L)

1

0 (Control)

MEAN

15.9

7.49

44.3

21

59

36

10.1

0.72

9.31

0.00

0.07

0

26

999

S.D.

0.8

0.62

4.5

1

2

3

1.73

0.33

1.87

0

0.11

0

1

81

2

750

MEAN

15.4

6.86

41.8

23**

61

37

10.9

1.16

9.51

0.11

0.12

0

28

1001

S.D.

0.6

0.38

2.7

1

3

2

1.83

0.79

2.14

0.19

0.08

0

2

74

3

3750

MEAN

15.8

7.02

41.8

23**

60

38

10.73

1.24

9.41

0.02

0.06

0

26

1003

S.D.

0.6

0.63

3

1

1

2

1.44

0.78

0.89

0.05

0.08

0

1

103

4

15000

MEAN

14 .6*

6.35**

39 .3*

23**

62*

37

9.79

0.93

8.76

0.01

0.08

0

27

859*

S.D.

1.5

0.63

4

1

1

1

1.97

0.52

1.75

0.03

0.15

0

2

104

5

0 (Control)

MEAN

15.5

7.6

44.7

20

59

-

-

-

-

-

-

-

-

877

Satelite Group

S.D.

0.4

0.28

2.1

0

2

-

-

-

-

-

-

-

-

55

6

15000

MEAN

15.3

7.43

43.4

21

58

-

-

-

-

-

-

-

-

812

Satelite Group

S.D.

0.5

0.07

0.8

1

1

-

-

-

-

-

-

-

-

117

* = significantly different from corresponding control group value p = 0.05

** = significantly different from corresponding control group value p = 0 .01

- = not applicable

Conclusions:
Administration of the test material to rats for a period of twenty-eight consecutive days by dietary admixture resulted in toxicologically significant changes at a dietary concentration of 15,000 ppm only. However, the effect was not regarded as adverse and therefore, the NOAEL was set for both male and female to 15,000 ppm.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
see attached justification.
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
> 1 501.25 mg/kg bw/day (actual dose received)
Based on:
other: Read-across
Sex:
female
Basis for effect level:
other: no adverse effects assumed
Dose descriptor:
NOAEL
Effect level:
> 1 572.75 mg/kg bw/day (actual dose received)
Based on:
other: Read-across
Sex:
male
Basis for effect level:
other: no adverse effects assumed
Critical effects observed:
no
Conclusions:
Based on the study performed with the read-across substance, the target substance was assumed no to cause treatment-related adverse effects. Thus, the NOAEL was concluded to be greater than 1500 mg/kg bw/day.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 November 2005 - 04 March 2006 (experimental)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
(1995)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar Crl : (WI) BR (outbred, SPF-Quality)
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation: no data
- Housing: 5 animals per sex in Macrolon plastic cages
- Diet (ad libitum): standard pelleted laboratory animal diet (from Altromin (code VRF- 1, Lage, Germany).
- Water (ad libitum): tap water
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0°C (actual range : 19.2 - 22.6°C)
- Humidity (%): 30-70% (actual range : 23 - 94%)
- Air changes (per hr): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Milli-U) (Millipore Corporation, Bedford, USA)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose volume: 5 mL/kg bw/day

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at the testing laboratory and on information from the sponsor.
- Purity: (Milli-U) (Millipore Corporation , Bedford, USA).
- Method of formulation: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level.
- Storage conditions: At ambient temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance formulations in water (Milli-U) were noted as stable for at least 5 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 88% to 99% of nominal. The accuracy level of 88% obtained for one group 3 sample was slightly outside the acceptable range of 90 - 110% of nominal. However, since all other accuracy values were within this range and the accuracy results were in line with the procedural recovery results, the overall accuracy for formulations was considered to be acceptable.
Duration of treatment / exposure:
28 days followed by a 14 day recovery period (control and high dose only)
Frequency of treatment:
daily for at least 28 days, 7 days per week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (low and intermediate dose groups)
10 (vehicle control and high dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on the results of a 5-day range finding study and in consultation with the sponsor, the dose levels for the 28-day toxicity study were selected to be 0, 100, 300 and 1000 mg/kg bw/day.
- Rationale for selecting satellite groups: There were two satellite (recovery) groups: 0 and 1000 mg/kg bw/day
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for mortality was made at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, detailed clinical observations were made in all animals. Detailed clinical observations were also performed outside the home cage in a standard arena on a weekly basis.

BODY WEIGHT: Yes
- Time schedule for examinations: Treatment period: on days 1, 8, 15, 22 and 28. Recovery period: on days 1, 8 and 14.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes (Weekly)

WATER CONSUMPTION: Yes (but no quantification)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination at the end of the treatment period.
- Anaesthetic used for blood collection: Yes: iso-flurane (Abbott Laboratories Ltd., Zwolle, The Netherlands)
- Animals fasted: Yes: overnight
- How many animals: all
- Parameters examined: White blood cells (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration, platelets. Clotting Potential: Prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination at the end of the treatment period.
- Animals fasted: Yes: overnight
- How many animals: all
- Parameters examined: Alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate (Inorg. Phos.).

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (approximately 16 hrs) from all animals at the end of the treatment period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume, colour score, Clarity, specific gravity, pH, protein, glucose, ketone, bilirubin, occult blood, leucocytes, nitrite, urobilinogen, sodium, potassium, calcium, sediment (white blood cells (WBC-SED), red blood cells (RBC), casts, epithelial cells, crystals, bacteria.

NEUROBEHAVIOURAL EXAMINATION: Yes (Functional Observations)
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity:
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period : 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution:
Adrenal glands, Aorta, Brain [cerebellum, mid-brain, cortex], Caecum, Cervix, (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve [if detectable] and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid [if detectable], (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

Tissues mentioned within brackets were not examined mlcroscopically as there were no signs of toxicity or target organ involvement. Histological examinations were performed on organs and tissues from all Main Group 1 and 4 animals (0 and 1000 mg/kg bw/d), and all gross lesions. All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands, Heart, Epididymides, Liver, Kidneys, Spleen, Testes, Thymus, Brain.

In addition, a histopathological investigation of liver, kidneys and adrenal glands of the animals of test groups 2 and 3 (100 and 300 mg/kg bw/day) was performed to meet the additional requirements by the Japanese authority (MHLW) (BASF SE, 99P0617/04P001, 30 Apr 2010).
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. No statistical analysis was performed on histopathology findings.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant clinical signs were observed.
Black faeces observed at 1000 mg/kg bw/day from week 3 of treatment onwards and incidental cases of black staining of parts of the fur were considered to be due to staining properties of the test substance (a black powder). These findings had resolved during the recovery phase. Incidental findings that were noted in single animals during the treatment or recovery phase included alopecia, scabs and a broken tail apex. These findings are occasionally noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were noted in control animals and in males at 300 mg/kg bw/day and females at 100 and 300 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain were considered to have been unaffected by treatment with the test substance.
The statistically significant lower body weight gain of females at 100 mg/kg bw/day (day 15, 24% compared to 30% in the control) was absent at higher dosages, and was therefore considered to be of no toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was considered to have been unaffected by treatment with the test substance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant lower prothrombin time (PT) of females at 1000 mg/kg bw/day at the end of the treatment phase was well within the historical control range. Considering the direction (i.e. a decrease) and slight degree of change, this was considered to be of no toxicological significance (17.4 s compared to 18.2 s in the control). The lower mean corpuscular haemoglobin (MCH) level of males at 300 mg/kg bw/day achieving a level of statistical significance occurred in the absence of a treatment-related distribution and was therefore also considered to be of no toxicological significance (1.15 fmol compared to 1.19 fmol in the control).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant deviations in clinical biochemistry parameters were within the historical control range and occurred in the absence of a (clear) dose-related response. Therefore these changes were considered to be without toxicological significance. These changes comprised higher albumin (33.8 and 33.7 g/L as compared to 32.5 g/L in the control) and chloride levels (102, 103, and 102 mmol/L as compared to 101 mmol/L in the control) in females at 100, 300 and/or 1000 mg/kg bw/day, lower aspartate aminotransferase activity levels (ASAT) in males at 100 mg/kg bw/day (71.2 U/L as compared to 85.6 U/L in the control) and higher sodium levels in females at 300 mg/kg bw/day (138.3 mmol/L as compared to 135.9 mmol/L in the control).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in urinary parameters of treated rats.
Any statistically significant changes in urinary parameters of females at 300 mg/kg bw/day were absent at 1000 mg/kg bw/day and were therefore considered to be of no toxicological relevance. These changes comprised a lower urinary volume, a higher specific gravity, and a higher sodium and potassium concentration (absent when corrected for urinary volume).
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in organ weights of treated rats.
The lower heart to body weight ratio of males at 100 mg/kg bw/day occurred in the absence of a dose-related distribution (0.319 % as compared to 0.347% in the control) and the mean was well within the historical control range. This change was therefore considered to be of no toxicological relevance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant necropsy findings were observed.
Black contents of the gastro-intestinal tract (or parts thereof, i.e. stomach, caecum and/or colon) in most animals at 1000 mg/kg bw/day and in one male at 300 mg/kg bw/day at the end of the treatment phase were considered to represent test substance (a black powder). These findings had resolved at the end of the recovery phase and occurred in the absence of any correlating histopathological tissue reaction. Therefore, these observations were considered to be of no toxicological relevance. Incidental findings among control and/or treated animals included pelvic dilation of the kidneys, red foci on the kidneys, red discolouration of the thymus, enlarged mandibular lymph node, fluid in the uterus, and tail apex fracture. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment. Variations noted in individual motor activity data between treated and control animals occurred in the absence of a dose-related response, similar changes of the low or high sensors, and/or supportive clinical signs. Therefore, they were considered to be of no toxicological relevance.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No histopathological abnormalities were observed that were considered to be of toxicological significance.
Black pigment deposits in the lumen or on the mucosal surface of the gastrointestinal tract in 5/5 males and 4/5 females at 1000 mg/kg bw/day occurred in the absence of any histopathological tissue reaction and were absent at the end of the recovery period. Therefore, these observations were considered to be of no toxicological relevance. The range of other microscopic observations recorded in this study was within the normal range of physiological changes and background alterations that may be seen in untreated animals of this age and strain.

In addition, liver, kidney and adrenal glands of all male and female animals of test group 2 and 3 (100 and 300 mg/kg bw/day) were investigated.
Livers of all animals revealed minimal to slight multifocal lymphoid infiltrates, characterized by a randomly scattered distribution of aggregates of lymphoid cells and minimal Kupffer cell granuloma. In addition, two males (1 of test group 2, 1 of test group 3) revealed in the area of lymphoid infiltration/Kupffer cell granuloma minimal single cell necrosis (3-5 hepatocytes) as accompanying finding. In two females of test group 3 an extramedullary hematopoiesis was seen.
One male of test group 2 revealed in the kidney a focal basophilic tubule. Additionally, two females of test group 3 showed a minimal unilateral pyelitis.
Accessory cortical tissue (accessory nodule) was found in the adrenal gland of 2 males and 1 female of test group 3.
All these findings noted were single observations and/or were biologically equally distributed in both test groups. They were all considered to be incidental and spontaneous in nature and not related to treatment.
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:
other: Up to the highest dose tested, no toxicologically relevant changes were observed in any of the parameters determined in this study.
Critical effects observed:
no
Conclusions:
From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for the test article of 1000 mg/kg/day was established.
Executive summary:

The repeated dose toxicity of the test article was investigated in guideline and GLP compliant 28-day oral toxicity study by daily gavage in the rat, followed by a 14-day recovery period. Based on the resutls of a 5-day range finding study and in consultation with the sponsor, the dose levels for the 28-day toxicity study were selected to be 0, 100, 300 and 1000 mg/kg/day. Each group consisted of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery. Formulation analyses were conducted once during treatment to assess accuracy, homogeneity and stability of formulations. The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology at the end of treatment; macroscopy at termination; organ weights and histopathology on a selection of tissues. Accuracy, homogeneity and stability over 5 hours of formulations of test substance in Milli-U water were demonstrated by analyses. No toxicologically relevant changes were observed in any of the parameters determined in this study. No evidence for neurotoxicity or immunotoxicity was obtained in this study. NOAEL was therefore determined to be 1000 mg/kg.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
see attached justification
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
other: Read-across
Sex:
male/female
Basis for effect level:
other: no toxicologically relevant effects assumed
Critical effects observed:
no
Conclusions:
Based on a study performed with the read-across substance, it was suggested that the NOAEL of the target substance is equal to the NOAEL established for the read-across substance, hence, 1000 mg/kg bw/day.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No studies with repeated administration are available for the test substance. However, one 90-day and three 28-day studies are available for other category members (see attached category justification).

In the available 90-day oral repeated dose study performed with a read across substance from the same category (comparable to OECD guideline 408, but not fully compliant), Sprague-Dawley rats (15/sex/dose) were fed with the test substance at 10000 and 50000 ppm in the diet (Hoechst AG, 1967). A control group received normal diet without test compound. The following parameters were evaluated: clinical signs, body weight, food consumption, haematology, urinalysis, macroscopy and microscopy. The following organs were examined histologically: heart, lung, liver, kidney, adrenal glands, spleen, cerebrum, cerebellum, testes and ovaries, pancreas, pituitary, thyroid gland, stomach and small intestine. The general behavior in all animals of all dose groups was normal throughout the entire trial period and did not differ from that of the control animals. None of the experimental animals showed signs of a toxic effect. The pigment was excreted via the feces. The food consumption and the weight gains of the treated rats were normal and did not differ from that of the control rats. The haematological investigations revealed no pathological findings. Investigation of the urine showed no pathological findings which could be attributed to the pigment administered. Macroscopic and microscopic examination revealed no pathological organ damage. A NOAEL of 50000 ppm for male and female rats was observed for the test article.

In a 28-day oral toxicity study with a second structural analogue according to OECD guideline 407 and under GLP, Sprague-Dawley rats (5/sex/dose) were administered the test substance at 750, 3750 and 15000 ppm in the diet (Safepharm Laboratories Ltd, 1990). These concentrations in the diet are equal to 79, 404 and 1573 mg/kg bw/day for males and 78, 380 and 1501 mg/kg bw/day for females. The following parameters were evaluated: clinical signs, body weight, food efficiency, water consumption and compound intake, haematology, blood chemistry, urinalysis, organ weights and histopathology. None of the animals died during the study. No clinically observable signs of toxicity were noted in test or control animals throughout the study. Bodyweight gain in all test animals was comparable with that seen in controls. There were no treatment-related changes in food consumption and food efficiency during the study. Anaemia was demonstrated in females treated with 15000 ppm, but not in males from the same dose group, identified by statistically significant reductions in haemoglobin levels, erythrocyte counts and haemotrocrit. The anaemia was very slight and apparently completely reversible since no such changes were seen in satellite high dose females after fourteen days without treatment. No toxicologically relevant changes were detected in any of the urine parameters measured. No treatment-related differences in organ weight were detected between test and control animals. All morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance. In conclusion, a NOEL of 3750 ppm / 380 mg/kg bw/day for females was established based on slight anaemia which was completely reversible. The NOAEL was therefore set at the highest dose level, i.e., at 15000 ppm / 1573 and 15000 ppm / 1501 mg/kg bw/day for male and female rats, respectively.

Two more 28-day studies are available for two additional substances from the same category (Notox, 2006 and RTC, 2006). Both studies were compliant with GLP and OECD guideline 407. In both studies, the test article was administered to groups of 5 rats per sex and group at dose levels of 100, 300 and 1000 mg/kg bw. Control animals were treated with the vehicle alone. Five additional animals for each sex were included in the high and control groups for recovery assessment over a period of two consecutive weeks. The following parameters were evaluated: clinical signs daily, functional observation tests, body weight and food consumption weekly, clinical pathology at the end of treatment, macroscopy at termination, organ weights and histopathology on a selection of tissues. No toxicologically relevant changes were observed in any of the parameters determined in these studies. Consequently, a NOAEL of 1000 mg/kg body weight was established for both tested chemicals.

Conclusion: No data from repeated dose studies with the test article are available. But according to the category approach, the data available for other category members is used to assess the toxicity of the test article. Based on the available and reliable data obtained in long term application studies with four category members, no classification for repeated dose toxicity is warranted. The established NOAELs for the tested substances were all above 1000 mg/kg. Therefore, a NOAEL of 1000 mg/kg bod weight was also established for the test article based on this read across/category approach.

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 the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for repeated dose toxicity is not warranted under Regulation (EC) No.1272/2008.