Registration Dossier

Administrative data

Description of key information

In a sub-acute oral toxicity study in rats, performed according to OECD guideline 407, a NOEL of 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 1573 and 1501 mg/kg bw/day for male and female rats, respectively.

Read-across:

Equivalent to OECD 408; non-GLP; oral fed rats, continuous treatment; 90 days; 10,000 and 50,000 ppm; No mortality, no clinical signs, no pathological findings; NOAEL=50,000 ppm.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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 conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 501 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

In a 28-day oral repeated dose study (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 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. 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. A statistically significant reduction in blood glucose was noted in males treated at 750 and 15000 ppm compared with control, 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 and 15000 ppm showed reduced alkaline phosphatase levels compared to controls, but none of the values could be considered abnormal and, a reduction in this enzyme could not be considered toxicologically significant. 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 since no such changes were seen in satellite high dose females after fourteen days without treatment. 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.

This result is supported by three additional studies (one subchronic and two subacute oral toxicity studies) performed with other members of the "Perylene based pigments" category (see attached category jutification). All of these studies resulted in NOAELs set at the highest administered dose level, demonstrating the lack of toxicity for the compounds of this category:

A subchronic toxicity study was performed with the read-across substance using crossbred albino rats in a 90-day feeding trial. Three groups Sprague-Dawley rats (15/sex/dose) were administered the test article at 0 (group III), 10,000 (group II) and 50,000 ppm (group I) in the diet (Hoechst AG, 1967). Control animals received plain diet. 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 behaviour in groups I and II 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 test article 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 test article administered. Macroscopic and microscopic examination revealed no pathological organ damage. Based on the results of this study, a NOAEL of 50,000 ppm for male and female rats was observed for the test article.

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: Based on the available and reliable data obtained in long term application studies with the test article and three category members, no classification for repeated dose toxicity is warranted. The established NOAELs for the various tested substances were all above 1000 mg/kg. The NOAEL for the test substance was determined at 1501 mg/kg and based on the available subacute toxicity study.

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.