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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Jun 1990 - 09 Jan 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Lack of test material details and only 5 days/week dosing protocol.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted in 1998
Deviations:
yes
Remarks:
lack of test material details and only 5 days/week dosing protocol, no immunological and reproductive effects addressed
GLP compliance:
yes
Limit test:
yes

Test material

Constituent 1
Reference substance name:
Reference substance 002
Cas Number:
151661-88-0

Test animals

Species:
rat
Strain:
other: Sprague-Dawley CD, SPF quality
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: 4 weeks
- Weight at study initiation: 58 - 80 g (males), 62 - 82 g (females)
- Housing: animals were housed in groups of 2-3 in Makrolon 3 cages with softwood bedding (ARWI-Center, Essen, Germany).
- Diet: Haltungsdiät Altromin 1324 DK Chargen-Nr. 2105901304 (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 40-92
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
18 Jun - 23 Okt 1990

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared daily by dissolving the test substance in peanut oil yielding a final concentration of 20, 60 and 200 mg/mL.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
94/95 days (control and treatment groups)
127 days (recovery groups)
Frequency of treatment:
once daily, 5 days/week
Doses / concentrationsopen allclose all
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:
10 (control)
10 (dose groups)
5 (control and high-dose group as recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: According to the author; yielding of a high security range, observation of a not cumulative toxic dose and identification of toxicological relevant ranges as well as the identification of reversibility of a possibly cumulative toxic effect were aimed.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on weekdays

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD AND WATER CONSUMPTION:
- Food consumption per group in cages: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: one day before section
- Dose groups that were examined: control and high-dose group

HAEMATOLOGY: Yes
- Time schedule: 6 weeks after application and at study termination
- Blood collection: retroorbital
- Parameters checked: erythrocyte count, hematocrit, mean cell volume, hemoglobin, leucocyte count, thrombocyte count, differential blood count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 6 weeks after application and at the end of the study period
- Parameters checked: gamma glutamyl transferase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, sodium, potassium, glucose, urea, protein, calcium, creatinine, cholesterol, chloride, bilirubin
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, whole body, especially eyes, skin, abdomen, chest and skull. Organ weights were examined for brain, testes, heart, liver, spleen, adrenal glands, kidney and thymus.
HISTOPATHOLOGY: Yes, aorta thoracica, eye, colon, proventriculus, small intestine, cerebrum, urinary bladder, skin, heart, testes, hypophysis cerebellum, liver, lung, trachea, axillary lymphnodes, mesentery lymphnodes, spleen, epididymis, adrenal gland, peripheral nerve, kidney, ovary, pancreas, prostate, vesicula seminalis, thyroid gland, salivary gland, esophagus, skeletal muscles, thymus, uterus, tongue, rumen.
Statistics:
t-test according to Dunnett, C.W. (1955)
Stell-test according to Stell, R.G.D. (1959)

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
300 mg/kg bw/day (one male): a lump of the size of a walnut was observed in Week 11 on the right leg which was of the size of a pinhead at the end of the study, non-adverse.
No compound-related symptoms were observed in the study. One male animal in the 300 mg/kg bw/day dose group had a lump of the size of a walnut in Week 11 on the right leg which was of the size of a pinhead at the end of the study and considered to be non-adverse.
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):
300 mg/kg bw/day (males): slight increase in mean body weight until Week 2 due to increased body weights at the start of the study, non-adverse.
The total body weight gain of all groups showed no deviation and was comparable to the control group. The slight increase in mean body weight of the low-dose group up to Week 2 was due to increased body weight at the start of the study and therefore considered to be non-adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
1000 mg/kg bw/day (females): higher food consumption in Week 4, non-adverse.
The mean food consumption in all treated groups was comparable to the control group. The increase in food consumption in the female high-dose group at the beginning of Week 4 up to the end of the study was due to food wasting by the animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The mean water intake of all groups was comparable to the control group.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The examination of the eyes by slit lamp microscope showed no compound-related effects.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
100 and 300 mg/kg bw/day (males): decrease of HCT-value; 1000 mg/kg bw/day (males): decrease of RBC-, HGB-, HCT- value and decrease of lymphocytes ("stabkernige"), increase of monocytes. females: decrease of WBC-values, non-adverse.
The intermediate and the final haematological examinations revealed a decrease of the haematocrit in the male treatment groups. The decrease of the HCT seems to be compound- and partially dose-related (see Table 1 under “Any other information on results incl. tables”). Additionally, the intermediate analysis showed slightly reduced red blood cell count-values for the male high-dose group. The observed deviation of the hematocrit is considered to be incidental, because the diagnosed values are within the +/- 1 SD limits of the historical control. In addition to this, there are no corresponding deviations of the erythrocyte count- or mean cell volume-values to prove the biological relevance of these findings. All other findings were considered to be spontaneous and therefore not related to the treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
1000 mg/kg bw/day (males): increase in protein-, glucose- and cholesterine- content, increase in calcium and creatinine-content; 100 mg/kg bw/day (males): increase in calcium-content, non-adverse.
The biochemical examinations revealed some compound- and dose-independent findings. In the high-dose group (males) a significant increase in protein-, glucose-, cholesterine- -calcium and creatinine-content was observed. In the low-dose group (males) an increase in calcium-content in comparison to the control value was apparent (see Table 2 under "Any other information on results incl. tables").
Urinalysis findings:
not examined
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 absolute and relative organ weights in all groups showed no deviations and were comparable to the control.
Gross pathological findings:
no effects observed
Description (incidence and severity):
single spontaneous findings in all groups including coloured thymus, spleen deformation, hydrometra (see Details on results), non-adverse.
The macroscopical examination of the organs displayed some observations like discolouration of the thymus, deformation of the spleen, cyst of the kidney, hydronephrosis, atrophy of the testes, hydrometra and necrosis of the fatty tissue which were considered to be spontaneous. Compound-related macroscopical effects were not observed. The male and female animals of the high-dose recovery group showed no macroscopical compound-related alterations.
Possible target organs have not been diagnosed. In the male and female animals of all groups (including the recovery groups) the livers, the heart and the mandibular lymph node showed effects which were due to a bacterial infection of unknown aetiology. The observed germinal hyperplasia of the mandibulary lymph node can be interpreted as a consequence of this bacterial infection. The liver and the heart of the recovery groups (high-dose group and control group) showed the same signs of bacterial infection and therefore prove the persistence of the bacterial infection.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
single spontaneous findings in all groups including calcification of the arteria pulmonalis, germinal hyperplasia of the mandibulary lymph node, non-adverse.
The microscopical examination revealed no compound-related effects.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
The microscopical examination revealed no compound-related effects.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Bacterial infection was observed in all test groups.

Table 1. Results of haematology, gorup mean values.

Dose group

[mg/kg bw/day]

Week

HCT

HGB

RBC

WBC

males

control

100

300

1000

6

43.8

42.2*

41.8**

41.5**

16.7

16.3

16.3

16.2*

8.2

8.0

7.9

7.9*

17.0

18.9

17.2

18.9

females

control

100

300

1000

6

40.3

39.8

39.9

39.8

15.8

15.6

15.7

15.7

7.4

7.3

7.3

7.3

14.5

15.1

13.6

13.2

males

control

100

300

1000

13

43.6

41.8**

41.5**

41.2**

16.7

16.1

16.1

16.1*

8.7

8.5

8.5

8.4

16.4

17.8

17.2

17.2

females

control

100

300

1000

13

40.0

39.4

39.6

39.6

15.9

15.7

15.8

15.9

7.5

7.6

7.6

7.5

12.1

11.5

10.6

9.0*

*: Level of significance 95% in comparison with control value.

**: Level of significance 99% in comparison with control value.

HCT: Haematocrit; HGB: haemoglobin; RBC: erythrocyte count; WBC: leucocyte count

Table 2: Results of clinical chemistry, group mean values.

Dose group

[mg/kg bw/day]

Week

glucose

[mmol/L]

calcium

[mmol/L]

creatinin

[µmol/L]

cholesterine

[mmol/L]

proteine

[g/L]

chloride

[mmol/L]

males

control

100

300

1000

6

6.4

6.8

6.6

7.0

2.5

2.5

2.5

2.6**

50

51

53

57**

1.9

2.4

2.2

2.3

64

64

64

67**

103

104

103

103

females

control

100

300

1000

6

5.5

5.9

5.4

5.7

2.6

2.6

2.6

2.6

60

59

57

57

2.2

2.2

2.2

2.1

66

66

67

68

102

102

103

102

males

control

100

300

1000

13

7.4

8.3

7.9

8.7*

2.5

2.6**

2.6

2.6**

56

56

58

60

1.9

2.4

2.1

2.5*

66

66

65

68

103

103

102

102*

females

control

100

300

1000

13

6.8

7.1

6.5

7.1

2.6

2.6

2.6

2.6

70

67

67

71

2.3

2.2

2.2

2.2

69

69

69

71

101

102

102

102

*: Level of significance 95% in comparison with control value.

**: Level of significance 99% in comparison with control value.

Applicant's summary and conclusion