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Toxicological information

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:
16 February 1987 to 17 June 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
hamster
Strain:
other: Bio F1 Alexander
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 55-75 %
- Photoperiod: 12 hours light/12 hours dark

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Spratt's LAD 2 Standard Rodent Diet
- Storage temperature of food: -20 °C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC analysis on samples taken from all dose groups from residues left in the foodhopper at the end of weeks 3, 6, 9 and 12 and in the fresh untouched diets of week 12 (used as reference).
Duration of treatment / exposure:
90 days (up to 4pm on the day before terminal kill in week 14 for main group)
Frequency of treatment:
Feed was available ad libitum and fresh food was given once a week
Doses / concentrationsopen allclose all
Dose / conc.:
41 mg/kg diet
Dose / conc.:
209 mg/kg diet
Dose / conc.:
1 289 mg/kg diet
Dose / conc.:
4 648 mg/kg diet
Remarks:
reduced after two weeks from 7500 mg/kg diet
No. of animals per sex per dose:
Ten (main group and recovery group for controls and 4648 mg/kg diet). Twenty for main group controls.
Control animals:
yes, plain diet

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
- At autopsy animals were weighed after overnight fasting to determine autopsy body weights to be used for calculating organ weights relative to body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: individual food intake calculated for periods of seven days

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before treatment started and in 11th week of treatment
- Dose groups that were examined: control and highest dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment and from the recovery animals, after 4 weeks recovery
- Anesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: Yes (18 hours)
- How many animals: all
- Parameters checked: RBC, Hb, PCV, MCV, MCH, MCHC, platelet count, WBC and differential WBC, reticulocytes, PTT and APTT. At autopsy 1.8 mL of blood was taken for clotting observations.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment and from the recovery animals, after 4 weeks recovery
- Animals fasted: Yes (18 hours)
- How many animals: all
- Parameters checked: AST, ALT, ALP, GCT, glucose, total protein, albumin, urea, creatinine, cholesterol, phospholipids, calcium, sodium and potassium
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- a full macroscopic examination was done on all animals and any abnormalities found were recorded.
- the following organs were weighed: brain, heart, liver, spleen, kidneys, thymus, prostate, seminal vesicles, testes, uterus and ovaries.
- for the recovery animals, the liver and kidney weights were recorded.

HISTOPATHOLOGY: Yes
- the following tissues were taken, fixed, sectioned and stained where appropriate: aorta, heart, lungs, trachea, salivary glands, liver, gall-bladder, pancreas, oesphagus, stomach, duodenum, ileum, jejunum, caecum, colon, rectum, urinary bladder, kidneys, testes, prostate, epididymides, seminal vesicle, ovaries, uterus, vagina, mesenteric lymph node, spleen, thymus, adrenals, pituitary gland, thyroid with parathyroid, skin with pelvia mammary gland, muscle (quadriceps femoris), sciatic never, brain, spinal cord, eyes and optic nerve
Statistics:
The basic analysis consists of linear regression analysis with treatment and block effects as parameters assuming constant error variance. The regression analysis was followed by Williams's test.

With some variables, one or two animals showed outlying responses. In such cases the Williams's test was replaced by its nonparametric version and the data were summarised by medians instead of means.

The incidence of macroscopic findings was subjected to a test for trend against dose level in a 5 x 2 contingency table. The statistical significance level for the trend statistic was obtained by enumeration of all possible permutations (permutation test).

Microscopic findings obtained on an ordinal scale were subjected to Shirley's test (as modified by Williams for comparing several dose levels with a zero-dose control).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed.
Mortality:
no mortality observed
Description (incidence):
No treatment-related mortality was observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The initial diet concentration of 7,500 mg/kg diet was not tolerated, after two weeks of treatment the animals had lost weight and were only 75 % and 80 % of the weight of concurrent controls (males and females respectively). Reducing the diet concentration to 4,648 mg/ kg resulted in an immediate return to weight gain although at a slower rate than that of the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption of the males was reduced at the 1,289 and 4,648 mg/kg diet level during the entire treatment period and up to week 6 in the females of the 4,648 mg/kg diet group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related ophthalmoscopic abnormalities were observed.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Haematology effects indicated a very slight, reversible, macrocytic tendency in the highest dose group. Withdrawal of treatment for 4 weeks resulted in an increased number of platelets, reticulocytes and neutrophils, in the males at this dose level, indicating an active bone marrow.
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The prostate showed a dose-related weight reduction. Uterus and spleen weights, absolute and relative, were reduced at the 4,648 mg/kg diet level of the females; however, histopathology examination did not show any abnormality.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were indications of reversible morphological and functional effects on the liver. Following the withdrawal of treatment it was evident that the liver had recovered normal function and morphology
An increase in the incidence of mineralisation in the prostate at 209 - 4,648 mg/kg diet levels was noted. Tubular degeneration in the testes and decreased numbers of spermatocytes in the epididymis were seen in the 4,648 mg/kg diet group. Gall-bladder calculi occurred in both sexes at the highest diet level, this effect remained after the recovery period.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No histopathological abnormalities were seen in uterus and spleen in the female group at the 4,648 mg/kg diet level .
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
41 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Body weight gain (g)

   Dose rate (mg/kg diet, actual)
 Time period (weeks)  Sex  0  41  209  1289  7500/4648  7500 /4648 (R)
 -2 - 0  M  19.4  19.2  20.9  21.6  19.3  
   F  20.2  21.7  21.1  20.0  17.8  
 1 - 3  M  16.4  18.0  17.7  15.4  -12.5**  
   F  15.2  14.2  16.9  15.2  -10.3**  
 4 - 6  M  15.8  17.6  14.5  13.8  7.4**  
   F  14.5  14.6  15.6  15.6  18.0*  
 7 - 9  M  5.8  6.7  4.7  5.0  2.9**  
   F  8.5  8.7  10.6  9.3  12.6**  
 10 - 13  M  1.0  0.4  0.0  -1.1  5.2**  
   F  -1.6  0.3  2.7  -1.3  8.8**  
 14 - 17  M  2.0 (R)          15.6**
   F  -0.7 (R)          6.5*

*statistical significant P≤0.05; **statistical significant P≤0.01

Table 2: Food consumption (g)

     Dose rate (mg/kg diet, actual)
 Time period (weeks)  Sex  0  41  209  1289  7500/4648  7500/4648 (R)
 -2 - 0  M  116  114  118  113  117  
   F  130  128  130  123  125  
 1 - 3  M  175  171  176  163*  130**  
   F  189  184  190  183  155**  
 4 - 6  M  183  177  177  171**  150**  
   F  193  189  198  197 173**  
 7 - 9  M  161  159  152  151*  133**  
   F  187  177  182  176  169  
 10 - 13  M  193  185  178*  179*  171**  
   F  214  212  222  207  212  
 14 - 17  M  194 (R)          199
   F  226 (R)          216

Table 3: Selected clinical chemistry values

     Dose rate (mg/kg diet, actual)
     After treatment period (13 weeks)  After recovery period (4 weeks)
 Parameter  Sex  0  41  209  1289  4648  0  4648
 AST (U/L)  M  34  39  34  33  37  40  31**
   F  43  47  41  36  33*  42  35
 ALT (U/L)  M  53  56  54  65**  64**  62  56
   F  73  84*  101*  85*  100**  94  84
 ALP (U/L)  M  161  149  161  163  168  138  180**
   F  213  202  200  194  200  228  223
 Glucose (mmol/L)  M  4.64  4.05  4.62  4.08  4.66  4.07  4.73
   F  5.16  5.21  5.05  5.74  4.25*  4.71  4.98
 T. prot (g/L)  M  70.1  69.6  69.1  69.3  71.3  70.9  72.6*
   F  72.4  72.2  73.1  72.4  75.3*  75.7  73.2
 Albumin (g/L)  M  55.4  55.6  55.7  57.2  59.8**  45.3  46.8*
   F  53.4  53.3  54.2  55.2  60.1*  43.2  41.7*
 Urea (mmol/L)  M  7.00  6.76  7.30  7.31  8.33**  7.98  7.73
   F  8.10  7.88  8.26  8.71  9.39*  9.02  8.46
 Creat (µmol/L)  M  28.5  27.0  27.0  26.0*  25.0**  27.2  26.8
   F  31.0  29.0  29.4  28.6  26.0**  29.5  27.3**
 Chol. (mmol/L)  M  3.45  2.99+  2.78 ++  2.94+  4.27 ++  3.64  3.59
   F  4.08  4.18  4.56  4.88**  6.31**  4.76  4.75
 Ph. Lip. (mmol/L)  M  2.91  2.70  2.61  2.74  3.65**  2.95  3.06
   F  3.10  3.24  3.62**  3.67**  4.82**  3.19  3.40
 Sodium (mmol/L)  M  147.2  147.7  145.4  146.9  147.1  147.0  145.0
   F  145.8  146.3  145.5  146.4  145.3  146.3  143.8*

*statistical significant, P≤0.05; **statistical significant, P≤0.01; results of Student's test since the mean response is not a monotonic function of dose: + P≤0.05, ++ P≤0.01

Table 4: Selected absolute and relative organ weights

       Dose rate (mg/kg diet, actual)
       After treatment period (13 weeks)  After recovery period (4 weeks)
 Organ    Sex  0  41  209  1289  4648  0  4648
 Body weight (g)     M  138  140  137  133  101**  139  119**
      F  136  139  147 ++  138  128+  136  131
 Brain (g)  abs1  M  0.980  0.985  0.990  0.995  0.990  -  -
   rel1  0.709  0.690  0.730  0.752*  0.955*  -  -
 Heart (g)  abs  M  0.536  0.529  0.528  0.525  0.437**  -  -
   rel1  M  0.390  0.381  0.385  0.397  0.435**  -  -
   abs  F  0.591  0.569  0.574  0.578  0.547*  -  -
   rel  F  0.436  0.411  0.391  0.419  0.428  -  -
 Liver (g)  abs  M  4.39  4.34  4.29  4.97**  6.66**  4.42  4.44
   rel  M  3.18  3.09  3.12  3.73**  6.59**  3.17  3.72**
   abs  F  4.74  4.93  5.52**  6.36**  8.78**  4.93  5.11
   rel  F  3.47  3.54  3.75*  4.59**  6.84**  3.60  3.88
 Spleen (g)  abs  M  0.112  0.118  0.107  0.107  0.074**  -  -
   rel M  0.081  0.085  0.077  0.081  0.074  -  -
   abs  F  0.166  0.151  0.164  0.157  0.126**  -  -
   rel  F  0.122  0.110  0.112  0.114  0.100**  -  -
 Kidneys (g)  abs1  M  1.005  0.975  0.990  0.990  0.900**  0.949  0.903
   rel1  M  0.735  0.690  0.725  0.755  0.880**  0.683  0.760**
   abs1  F  1.160  1.190  1.255  1.230  1.205  1.151  1.019*
   rel1  F  0.840  0.835  0.830  0.925  0.905**  0.848  0.779**
 Thymus (g)  abs  M  0.040  0.048  0.043  0.042  0.034*  -  -
   rel  M  0.029  0.034  0.030  0.030  0.031  -  -
 Prostate (mg)  abs  M  388  342  313*  297**  182**  -  -
   rel  M  283  247  228*  224**  182**  -  -
 Sem. ves. (g)  abs  M  1.41  1.35  1.19  1.19  0.72*  -  -
   rel  M  1.01  0.97  0.84  0.90  0.70**  -  -
 Testes (g)  abs  M  3.27  3.26  3.07  3.15  2.20**  -  -
   rel  M  2.38  2.35  2.25  2.39  2.19  -  -
 Uterus (g)  abs  F  0.40  0.41  0.37  0.35  0.30**  -  -
   rel F  0.29  0.29  0.25  0.26  0.23*  -  -

*statistical significant, P≤0.05; **statistical significant, P≤0.01; 1 medians are presented and nonparameteric test was applied because of an outlying observation; results of Student's t-test since the mean response is not a monotonic function of dose: + P≤0.05, ++P≤0.01

Table 5: Selected microscopic observations

      Dose rate (mg/kg diet, actual)
      Sex  After treatment period (13 weeks)  After recovery period (4 weeks)
        0  41  209  1289  4648  0  4648
 Numbers examined  M  20  10  10  10  9  10  10
      F  20  10  10  10  10  10  10
 Organ  Observation                
 Epididymis No abnormalities  M  19      10  4    
  Spermatocytes decreased  M  1      0  5*    
 Gallbladder No abnormalities  M  20  10  9  9  2**  8  2
   No abnormalities  F  20  10  10  9  0**  9  0**
 Prostate No abnormalities  M  16  9  6  5  5    
  Mineralisation (marginal/slight/moderate)  M  0/0/1  0/0/1  0/1/2  1/3/0  0/2/1    
  Prostatis (marginal/slight/moderate)  M  0/2/2  0/0/1  0/0/2  1/2/0  0/1/1    
 Testes No abnormalities  M  18        2    
  Tubules degenerated (marginal/slight/moderate/marked)  M  0/1/0/1        ** 2/0/4/1    

* incidences yielding statistical significance (P≤0.1) in a two-sided test against trend with dose level; **Shirley's test P≤0.1

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, the NOEL of the test material in a 90 day repeat dose study in the hamster was determined to be 41 mg/kg diet.
Executive summary:

In a GLP compliant repeat dose (oral) study conducted in line with EPA OPP 82-1, the effects of the repeat dose of the test material over 90 days was determined. Hamsters were fed a diet containing 0, 41, 209, 1289 and 4648 mg/kg diet (7500 mg/kg diet for first two weeks) test material.

Under the conditions of the test, the NOEL of the test material in a 90 day repeat dose study in the hamster was determined to be 41 mg/kg diet.