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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: black powder
- Analytical purity: 99 %
- Storage condition of test material: room temperature

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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)

Results and discussion

Results of examinations

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

Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.