Registration Dossier

Administrative data

Description of key information

ORAL

The NOEL was determined to be 1 mg/kg bw/day according to a 4 week dog study performed in line with OECD Guideline 410 and EPA Guideline 82-1.

The NOEL was determined to be 41 mg/kg bw/day according to a 13 week hamster study performed in line with OECD Guideline 410 and EPA Guideline 82-1.

The NOEL was determined to be 25 mg/kg bw/day according to a 13 week mice study performed in line with OECD Guideline 410 and EPA Guideline 82-1.

The NOEL was determined to be 1 mg/kg bw/day according to a 52 week dog study performed in line with OECD Guideline 410 and EPA Guideline 83-1.

DERMAL

The NOAEL was determined to be 1000 mg/kg bw/day according to a study performed in line with OECD Guideline 410 and EPA Guideline 82-2.

INHALATION

The NOAEC was determined to be 12 mg/m^3 according to a study performed in line with EU Method B.8.

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
Study period:
16 February 1987 to 17 June 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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
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
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
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).
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
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
Critical effects observed:
not specified

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

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.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 January 1987 to 23 October 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55-75 %
- Photoperiod: 12 hours light/12 hours dark
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
Frequency of treatment:
Food was available ad libitum and fresh food was given once a week.
Dose / conc.:
25 mg/kg diet
Dose / conc.:
125 mg/kg diet
Dose / conc.:
625 mg/kg diet
Dose / conc.:
3 125 mg/kg diet
No. of animals per sex per dose:
20 animals per sex per dose in the control, 10 animals per sex per dose in the treatment groups.
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (three times daily in the first two weeks)

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: calculated for periods of seven days

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before treatment started and in 13th week of treatment
- Dose groups that were examined: control and 625 and 3125 mg/kg dose groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment and from the recovery animals
- 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

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment and from the recovery animals
- Animals fasted: Yes (18 hours)
- How many animals: all
- Parameters checked: AST, ALT, ALP, GCT, glucose, total protein, albumin, urea, cholesterol, phospholipids, 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 and testes.

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, kidney, testes, prostate, epididymides, seminal vesicle, mesenteric lymph node, spleen, thymus, adrenals, pituitary gland, thyroid with parathyroid, skin with pelvic mammary gland, muscle (quadriceps femoris), sciatic nerve, brain, spinal cord, eyes, optic nerve and harderian gland.
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' test.

In the clinical chemistry and in the testes weights there are some outlying values. In these cases, the assumption of normally distributed errors with constant variance, underlying the regression analysis, does not hold. Williams' test was replaced by its nonparamateric equivalent and the data were summarised by medians instead of means.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs in animals treated at 3125 mg/kg diet included hunched posture and piloerection in week one which were transient by week 2.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two deaths were recorded, one control animal in week 10 and one of the 25 mg/kg treatment in week 12. These deaths were not considered to be treatment related.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the highest dose level a temporary decrease in body weight gain was noted. Onset of recovery for males and females, in this group, was seen in week 3 and week 2 of treatment, respectively. This was associated with reduced food intake, reduction was 8.2 % (males) and 15.4 % (females) in comparison with their controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Increased water consumption was noted in the females of the highest dose group during the first two weeks of treatment.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
During the pre-treatment period one male and one female with eye lesions were replaced. Ophthalmoscopic findings are normal for the mouse and are not considered related to the treatment with the test material.
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Red blood cell variables and white blood cell variables were unaffected by treatment. Changes in the plasma levels of cholesterol, phospholipids, glucose and an increase in glycogen retention indicated that treatment with the test material at 625 and 3,125 mg/kg diet for 13 weeks caused adverse effects on the livers of males and females.
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 following changes were observed with organ weights: Absolute and relative liver weights were increased in the highest dose level; testis weights were increased at 125 mg/kg and above, but histopathological examination revealed no abnormalities. Absolute and relative thymus weights were increased at 3,125 mg/kg diet group of the males, but also here no histopathological abnormalities were found.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the stomach of all groups changes were observed, consisting of white areas in the cardiac regions in both sexes and fundic ulceration mainly in the females. Twenty out of the 59 females examined at the end of the study had cysts, haemocytes or haemorrhages on the ovaries. The appearance of the uteri indicated similar distributions in the stages of oestrus between the treatment groups. All these changes found were not related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the liver two apparently treatment-related effects were observed, an increase in the severity of parenchyma cells at 625 and 3,125 mg/kg in both sexes; and an increase in glycogen storage in the males at 3,125 mg/kg and in the females at 625 and 3,125 mg/kg.
Examination of the kidneys showed slight inflammatory changes at 625 and 3,125 mg/kg and possibly an increase of the incidence of basophilic cortical tubules at 3,125 mg/kg in males. This was not seen in the females of the same level and therefore kidneys from the intermediate dose levels were not examined.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
25 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
not specified

Table 1: Mean body weight gain (g)

   Group mg/kg diet
 Time period (weeks)  Sex  0  25  125  625 3125
 -2 - 0  M  5.2  4.8  5.6  5.5  5.3
   F  4.0  4.1  4.5  4.1  4.3
 1 - 4  M  4.8  4.8  4.8  4.4  3.4*
   F  3.5  3.3  3.5  3.3  3.2
 5 - 8  M  1.3  1.5  1.5  0.9  2.6*
   F  1.4  1.2  1.6  1.0  1.8
9 -13  M  2.1  2.2  2.1  3.0  2.5
   F  2.1  1.5  1.0  1.8  1.0*

*difference from control significant P<0.05

Table 2: Selected clinical chemistry values

     Group mg/kg diet
 Parameter  Sex  0  25  125  625  3125
 Glucose (mmol/L)1 M  8.66  7.83  8.03  7.30*  7.83*
   F  7.19  7.04  7.50  6.16  7.22
 T. prot (g/L)  M  45.4  47.0  48.1  46.0  47.9*
   F  47.9  46.4  47.3  46.5  47.5
 Chol. (mmol/L)1  M  1.84  2.20  2.12  1.86  2.73**
   F  1.22  1.03  1.36  1.43  1.74**
 Ph. Lip. (mmol/L)  M  1.78  2.18  2.06  1.81  2.60**
   F  1.28  0.98  1.42  1.48  1.79**

1medians are given and non-parametric test was applied due to one or two outlying values

*difference from control significant, P < 0.05; **difference from control significant P < 0.01

Table 3: Selected absolute and relative organ weights

       Group mg/kg diet
 Organ    Sex  0  25  125  625  3125
 Body weight (g)     M  30.6 30.3  31.3  31.1  30.0
      F  24.7  23.6  24.9  24.2  23.7
 Heart (mg)  abs  M  154  152  163  151  154
   rel  M  50.6  50.5  52.1  48.7  51.3
   abs  F  130  125  130  127  120*
   rel  F  53.1  53.3 52.3  52.4  50.7
 Liver (mg)  abs  M  1229  1248  1286  1256  1441**
   rel  M  402  412  411  404  481**
   abs  F  1036  987  1049  1056  1227**
   rel  F  420  417  421  436  518**
 Thymus (mg)  abs  M  20.5  21.9  21.6  21.8  26.2**
   rel  M  6.7  7.2  7.0  7.0  8.8**
   abs  F  28.4  24.7  32.8  31.4  26.2**
   rel  F  11.6  10.5  13.1  13.0  12.3
 Testes (mg)  abs1  M  228  217  258*  258*  244*
   rel1  M  73  75  81*  81*  78* 

1medians are given and non-parametric test was applied due to one or two outlying values

*difference from control significant, P < 0.05; **difference from control significant P < 0.01

Table 4: Treatment-related liver effects

   Males              Females            
 Dose in mg/kg diet  0  25  125  625  3125  0  25  125  625  3125
 Number of mice  19  10  10  10  10  20  10  10  10  10
 Swelling of centrilobular cells
 Marginal  4  2  1  0  0  0  1  0  0  0
 Slight  0  1  1  3  2  0  0  2  0  3
 Moderate  0  0  0  3  2  0  0  0  2  2
 Marked  0  0  0  0  0  0  0  0  0  2
 Mean score  0.21  0.40  0.30  1.50  1.00  0.00  0.11  0.40  0.60  2.00
 Diffuse increases in glycogen
 Marginal  0  -  -  0  2  1  -  -  1  0
 Slight  0  -  -  0  0  1  -  -  1  0
 Moderate  0  -  -  0  1  0  -  -  0  1
 Marked  0  -  -  0  1  0  -  -  0  1
 Severe  0  -  -  0  0  0  -  -  0  1
 Mean score  0.00  -  -  0.00  0.60  0.15  -  -  0.30  1.20
 Scattered parenchyma cells with increased glycogen
 A few  0  -  -  1  2  7  -  -  0  2
 Several  0  -  -  0  0  1  -  -  2  1
 Many  0  -  -  0  0  0  -  -  0  3
 Mean score  0.00  -  -  0.10  0.20  0.45  -  -  0.40  1.30

Scoring: marginal/a few -1; slight/several -2; moderate/many -3; marked -4; severe -5

Table 5: Treatment-related kidney effects

 Males              Females            
 Dose in mg/kg diet  0  25  125  625  3125  0  25  125  625  3125
 Number of mice  19  10  10  10  10  20  10  10  10  10
 Pyelitis
 Marginal  0  3  0  1  1  0  -  -  -  2
 Slight  0  0  1  1  2  1  -  -  -  0
 Moderate  1  0  0  0  0  0  -  -  -  0
 Marked  0  0  0  1  1  0  -  -  -  0
 Mean score  0.16  0.30  0.20  0.70  0.90  0.10  -  -  -  0.20
 Interstital nephritis
 Marginal  0  0  0  0  2  0  -  -  -  0
 Slight  0  0  1  0  1  0  -  -  -  0
 Moderate  0  0  0  0  0  1  -  -  -  0
 Mean score  0.00  0.00  0.20  0.00  0.40  0.15  -  -  -  0.00
 Basophilic cortical tubulus
 A few  2  1  0  0  3  0  -  -  -  0
 Mean score  0.10  0.10  0.00  0.00  0.30  0.00  -  -  -  0.00

Scoring: marginal/a few -1; slight/several -2; moderate/many -3; marked -4; severe -5

Conclusions:
Treatment with the test material at 625 and 3125 mg/kg diet for 13 weeks resulted in adverse effects on the liver of male and female mice. Under the conditions of the test, the NOEL of the test material in a 90 day repeat dose study in the mouse was determined to be 25 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.

Mice were fed a diet containing 0, 25, 125, 625 and 3125 mg/kg diet test material.

Under the conditions of the test, treatment with 625 and 3125 mg/kg diet resulted in adverse effects on the liver of male and female mice. The NOEL of the test material in a 90 day repeat dose study in the mouse was determined to be 25 mg/kg diet.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 August 1994 to 25 August 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
EPA OPP 82-1 (90-Day Oral Toxicity)
Deviations:
yes
Remarks:
(see below)
Principles of method if other than guideline:
The following deviations had no impact on the outcome of the study:
- the diet was pelleted and not powdered
- on day 2 the diet was given after the adminsitration of the test material and not before
- on weighing days and during the first week, the animals were treated using body weights from the previous week insred of the latest recorded body weight
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 5 months
- Weight at study initiation: 6.7-8.2 kg (males); 6.2-8.0 kg (females)
- Housing: individually in pens
- Diet: 400 g/animal/day pelleted diet
- Water: mains drinking water ad libitum (1200 g per day up to day 6 and then 1500 g per day)
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: >50 %
- Air changes: minimum 8 per hour
- Photoperiod: 12 hours light / 12 hours dark
Route of administration:
oral: capsule
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days (males); 29 days (females)
Frequency of treatment:
Once daily during the morning
Dose / conc.:
1 mg/kg diet
Dose / conc.:
6 mg/kg diet
Dose / conc.:
36 mg/kg diet
Dose / conc.:
72 mg/kg diet
No. of animals per sex per dose:
Two
Control animals:
yes
Details on study design:
- Dose selection rationale: based on interpretation of previous toxicity studies
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at least once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated: Yes - daily

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once before study initiation and once during week 4
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- Parameters checked: haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, packed cell volume, red blood cell count, mean corpuscular volume, platelet count, prothrombin time, activated partial thromboplastin time, total white blood cell count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once before study initiation and once during week 4
- Animals fasted: Yes
- How many animals:
- Parameters checked: sodium, potassium glucose, blood urea nitrogen, total cholesterol, triglycerides, phospholipids, total protein, albumin, creatinine, alkaline phosphatase, alanine aminotransferase
Sacrifice and pathology:
GROSS PATHOLOGY: Yes - examination of the external surface, all orifices, cranial cavity, external surface of the brain and samples of the spinal cord, thoracic and abnormal cavities and organs, cervical tissues and organs, carcass.

The following organ weights were weighed for all animals: adrenals, brain, kidneys, liver, ovaries, prostate, spleen, testes, thymus, thyroids and uterus.

HISTOPATHOLOGY: Yes - adrenals, aorta, bone (sternum) with bone marrow, bone (rib) with cartilage, bone marrow smears, bronchi (mainstream), brain, caecum, colon, duodenum, epididymides, eyes, gall bladder, heart, ileum, jejunum, kidneys, lacrimal gland, liver, lungs, lymph nodes, mammary gland, nasal cavities, oesophagus, optic nerves, pancreas, parathyroids, pituitary, prostate, rectum, salivary gland, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroids, trachea, urinary bladder, uterus and all gross lesions.
Statistics:
No statistical analysis was performed due to the small number of animals in each group.
Clinical signs:
no effects observed
Description (incidence and severity):
- Occasional vomiting occurred at all dose levels except the highest but the fact that it did not occur at the highest dose and the very low occurrence, it is not considered to be related to the test material. One high dose female produced a large quality of orange-coloured liquid diarrhoea on day 3 and orange-coloured faeces on day 6; this is not considered to be significant.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- Both high dose males gained slightly less weight than the controls over the treatment period. One high dose female lost 200 g in weight and the other female gained no weight. The body weight of other animals was similar to, or slightly less than, the controls
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- The highest dose had some effect on normal food consumption profile although this was limited to the first two weeks of treatment in males.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
- At the end of the treatment period, the activated partial thromboplastin times of males in the highest two doses were slightly shorter than those of the controls and their pre-dose values. The individual values were within the standard deviations of the normal background range of the laboratory and are considered to be of doubtful toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- Males in the highest two dose levels tended to have slightly lower serum potassium concentrations than controls although the levels were within the normal range. Triglyceride concentrations of both sexes in the three highest dose-groups were higher than controls at the end of the treatment. Cholesterol concentration was increased in the group treated with 36 mg/kg (males only) and the group given 72 mg/kg (both sexes). Phospholipid concentrations were higher than controls in males in the two highest dose-groups, and in females in the three highest dose-groups. Females administered 36 and 72 mg/kg bw and one male given 72 mg/kg bw had alkaline phosphatase activities above the control range. The mean glucose concentrations of females in the highest dose-group were lower than the respective control mean.
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 mean absolute and relative liver weights of the animals in the two highest dose-groups were greater than those of the controls. Other small differences in the weights of the organs were considered to be unrelated to treatment
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- There were no treatment-related macroscopic findings. Microscopic changes, considered to be related to treatment, were confined to the liver. These were the homogeneous appearance of the cytoplasm of the centrilobular hepatocytes in both males and one female treated at the high dose level and in one male and one female administered 36 mg/kg bw. Cytoplasmic aggregations in the hepatocytes were seen in all animals of the highest dose-group and in one male given 36 mg/kg bw.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
other: Increased cholesterol, phospholipid and triglyceride concentrations and elevated alkaline phosphatase activity. Increased liver weight and associated microscopic lesions.
Critical effects observed:
not specified

Table 1: Body weights (kg)

     Dose group (mg/kg bw)
 Time (day)  Sex  0  1  6  36  72
 -14  M  6.6  6.9  7.2  7.4  6.9
 -7  M  6.9  7.1  7.1  7.3  7.1
 -1  M  7.2  7.6  7.3  7.7  7.6
 1  M  7.1  7.5  7.5  7.6  7.5
 8  M  7.5  7.9  7.7  7.8  7.7
 15  M  7.8  8.2  7.8  8.0  7.8
 22  M  8.1  8.5  8.1  8.3  8.1
 28  M  8.3  8.7  8.4  8.5  8.6
 -14  F  7.1  7.0  6.6  6.3  6.5
 -7  F  6.8  7.0  6.7  6.5  6.5
 -1  F  7.2  7.4  7.2  6.9  6.9
 8  F  7.5  7.6  7.4  7.1  7.0
 15  F  8.0  8.1  7.8  7.5  7.1
 22  F  8.0  8.1  7.8  7.5  7.1
 28  F  8.2  8.2  8.0  7.6  6.9

Table 2: Food and water consumption (g/animal/day)

   Dose group (mg/kg bw/day)
 Time (day)  Sex  0  1  6  36  72
 -14  M  276/725  288/843  260/584  232/530  275/747
 -7  M  318/861  344/1011  308/747  301/737  330/856
 1 M  355/965  386/1139  386/863  343/907  330/925
 8  M  358/987  375/1186  375/867  341/916  314/939
 15  M  383/1050  391/1193  391/891  394/1069  362/1054
 22  M  377/981  392/1234  392/899  387/1075  370/1131
-14  F  221/580  247/565  233/611  315/755  234/667
 -7  F  340/835  287/668  291/788  325/850  299/798
 1  F  382/957  310/698  326/913  379/974  289/849
 8  F  370/947  324/740  317/853  341/953  285/880
 15  F  382/994  365/809  329/874  368/1017  317/917
 22  F  379/991  336/743  346/886  358/941  273/836

Table 3: Blood clinical chemistry values (pre-dose and after 4 weeks)

     Dose group (mg/kg bw)
 Parameter  Sex  0  1  6  36  72
Na (mEq/L)  M  148/150  148/148  146/147  147/147  148/149
   F  147/149  148/146  147/149  148/151  148/148
 K (mEq/L)  M  4.9/5.3  4.9/5.2  4.9/4.8  4.9/4.5  5.3/4.4
   F  4.6/4.6  4.9/4.5  4.9/4.5  4.7/4.4  4.6/4.4
 GLUC (g/L)  M  1.21/1.17  1.13/1.16  1.09/1.10  1.32/1.03  1.15/1.12
   F  1.21/1.19  1.14/1.15  1.22/1.15  1.20/1.07  1.13/0.83
 BUN (g/L)  M  0.21/0.27  0.24/0.30  0.23/0.26  0.18/0.24  0.25/0.27
   F  0.34/0.30  0.22/0.25  0.21/0.25  0.22/0.25  0.26/0.40
 CHOL (g/L)  M  1.19/1.10  1.26/1.17  1.48/1.56  1.45/2.28  1.69/2.54
   F  2.14/1.22  1.55/1.38  1.39/1.61  1.06/1.71  1.53/2.40
 TRIGS (g/L)  M  0.26/0.36  0.30/0.38  0.27/0.62  0.27/1.57  0.31/1.85
   F  0.36/0.46  0.32/0.33  0.25/0.64  0.22/0.80  0.34/2.24
 P.LIP (g/L)  M  2.40/2.44  2.50/2.64  2.84/3.15  3.12/4.23  3.37/4.64
   F  3.55/2.63  3.13/3.03  2.73/3.49  2.35/4.03  3.00/4.64
 PROT (g/L)  M  54/58  56/61  56/60  56/63  57/62
   F  57/59  56/56  51/57  56/61  57/62
 ALB (g/L)  M  33/34  32/34  33/33  34/33  33/32
   F  32/35  33/32  33/33  34/34  34/34
 CREAT (mg/L)  M  5.8/7.2  6.8/8.2  6.0/6.8  5.6/6.3  6.7/7.4
   F  6.3/7.7  5.8/7.1  5.7/6.4  6.1/6.7  7.1/7.9
 A.P. (IU/L)  M  369/386  261/255  286/349  325/440  387/581
   F  282/294  223/217  277/283  309/401  202/1145
 ALAT (IU/L)  M  31/52  24/29  27/28  32/24  32/81
   F  33/36  35/35  32/33  31/20  33/28

Table 4: Body and organ weights at termination

     Dose group (mg/kg bw)     
 Parameter  Sex  0  1  6  36  72
 Body weight (g)  M  8250  8600  8150  8300  8200
   F  8150  8000  8000  7500  6800
 Organ                  
 Adrenals (% bw)  M  0.0093  0.0096  0.0092  0.103  0.0141
   F  0.0083  0.0095  0.0107  0.0127  0.0106
 Testes (% bw)  M  0.060  0.031  0.049  0.067  0.052
 Ovaries (% bw)  F  0.0086  0.0105  0.0088  0.0081  0.0073
 Prostate (% bw)  M  0.0130  0.0135  0.0213  0.0104  0.0097
 Thyroid (% bw)  M  0.0065  0.0064  0.0081  0.0089  0.0097
   F  0.0084  0.0074  0.0072  0.0088  0.0100
 Uterus (% bw)  F  0.0207  0.0122  0.0208  0.0173  0.0124
 Spleen (% bw)  M  0.619  0.242  0.565  0.444  0.224
   F  0.263  0.563  0.627  0.392  0.600
 Kidneys (% bw)  M  0.547  0.470  0.500  0.667  0.558
   F  0.454  0.532  0.526  0.541  0.579
 Liver (% bw)  M  3.47  3.20  3.84  5.10  5.20
   F  3.16  3.33  3.34  4.62  5.89
 Thymus (% bw)  M  0.1327  0.0933  0.1398  0.1035  0.1107
   F  0.1601  0.1564  0.2000  0.1120  0.1112
 Brain (% bw)  M  1.036  0.868  0.921  0.941  0.872
   F  0.985  0.983  1.004  0.877  1.110
Conclusions:
In a GLP compliant 28-day repeat-dose study conducted in line with standardised guideline EPA OPP 82-1, the repeat-dose effects of the test material was determined. Under the conditions of the test, the NOEL of the test material in the dog was determined to be 1 mg/kg/day.
Executive summary:

In a GLP compliant 28-day repeat-dose study conducted in line with standardised guideline EPA OPP 82-1, the repeat-dose effects of the test substance was determined.

Under the conditions of the test, oral administration of the test material to the dog at 36 and 72 mg/kg bw, brought about altered clinical chemistry parameters, notably increased cholesterol, phospholipid and triglyceride concentrations and elevated alkaline phosphatase activities, increased liver weight and associated microscopic lesions in this organ. In addition,there was an effect on body weight observed at the dose level of 72 mg/kg bw/day. The changes in the liver, described microscopically as a homogeneous appearance of the centrilobular hepatocyte cytoplasm with occasional cytoplasmic aggregations, was considered to represent hypertrophy and enzyme induction and is probably an adaptive response to test material elimination. Animals treated at a dose level of 6 mg/kg/day had only slightly increased serum phospholipid and triglyceride concentrations. A NOEL of 1 mg/kg/day was determined.  The most suitable high dose level for a subsequent 52-week study is 36 mg/kg/day, based on the body weight effect noted at the high dose level of 72 mg/kg/day.

Under the conditions of the test, the NOEL of the test material in the dog was determined to be 1 mg/kg/day.

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 February 1994 to 22 December 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 83-1 (Chronic Toxicity)
Deviations:
yes
Remarks:
(see below)
Principles of method if other than guideline:
- On weighing days, during the first week of treatment, on one day between day 44 and day 50 and on day 56, animals were treated based on body weights from the previous week instead of the latest recorded body weight.
- Clinical pathology investigations were performed during week 26/27 and during week 52/53 instead of weeks 26 and 52 only.
- During week 27, urine was collected by catherisation of the urinary bladder from female no. 188 (group 4) and not in metabolism cages.
GLP compliance:
yes
Limit test:
yes
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 5 to 6 months
- Weight at study initiation: 5-8 kg (males); 4-8 kg (females)
- Housing: singly in pens
- Diet: 400 g diet/animal/day
- Water: mains drinking water; 1200 g water/animal/day until day 98 and 1500 g thereafter
- Acclimation period: 25 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: >50 %
- Air changes: minimum 8 changes per hour
- Photoperiod: 12 hours light/12 hours dark

IN-LIFE DATES: From: 8th March 1994 To: 10th March 1995
Route of administration:
oral: capsule
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The test material was administered in capsules which were stored at rom temperature in the dark and prepared at least once a week
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
364 to 367 days
Frequency of treatment:
Daily - 400 g diet/animal/day
Dose / conc.:
1 mg/kg bw/day (nominal)
Dose / conc.:
6 mg/kg bw/day (nominal)
Dose / conc.:
26 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Five
Control animals:
yes
Details on study design:
- Dose selection rationale: range-finding study 65/509. Dose levels were 1, 6, 36 and 72 mg/kg bw/day with effects observed at 36 and 72 mg/kg bw/day and a NOEL of 1 mg/kg bw/day.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily with a full clinical examination before treatment

BODY WEIGHT: Yes
- Time schedule for examinations: at least weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes
- Time schedule for examinations: daily up to week 16 and then daily for weeks 20, 24, 28, 32, 36, 40, 44, 48 and 52

FOOD EFFICIENCY:
- Body weight gain: Yes for each week food consumption was measured

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily up to week 16 and then daily for weeks 20, 24, 28, 32, 36, 40, 44, 48 and 52

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data
- Time schedule for examinations: before the start of treatment and during week 52
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before the start of treatment, during week 26/27 and during week 52/53.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes - at least 16 hours
- How many animals: all
- Parameters checked: haemoglobin, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, packed cell volume, red blood cell count, mean corpuscular volume, reticulocyte count, platelet count, prothrombin time, activated partial thromboplastin time, total and differential white blood cell counts.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before tea start of treatment, during week 26/27 and during week 52/53.
- Animals fasted: Yes - at least 16 hours
- How many animals: all
- Parameters checked: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, total cholesterol, triglycerides, phospholipids, total bilirubin, total protein, albumin, globulin (calculated), creatinine, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase and creatine phosphokinase.

URINALYSIS: Yes
- Time schedule for collection of urine: before tea start of treatment, during week 26/27 and during week 52/53.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes - at least 16 hours
- Parameters checked: volume, specific gravity, appearance, microscopic examination of spun deposit and semi-quantitative estimation of pH, protein, glucose, ketones, urobilinogen, bilirubin, blood and reducing substances.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were submitted to full necropsy procedures including examination of: the external surface, all orifices, the cranial cavity, the external surface of the brain and samples of the spinal cord, the thoracic and abdominal cavities and organs, the cervical tissues and organs and the carcass.
- The following organs were weighed: adrenals, brain, kidneys, liver, ovaries, prostate, spleen, testes, thymus, thyroids and uterus.

HISTOPATHOLOGY: Yes
- The following organs/tissues were sampled: adrenals, aorta, sternum with bone marrow, rib, bone marrow smears, brain, caecum, colon, duodenum, epididymides, eyes, gall bladder, heart, ileum, jejunum, kidneys, liver, lungs, submaxillary and mesenteric lymph nodes, mammary gland (females only), nasal cavities, oesophagus, optic nerves, ovaries, pancreas, parathyroids, pituitary, prostate, rectum, salivary gland, sciatic nerves, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus (where identified), thyroids, trachea, urinary bladder, uterus and all gross lesions.
Statistics:
Statistical analysis of parameters was performed using Levene's test.

Where Levene's test showed no significant difference in the group variances, data were analysed using parametric procedures. Such analysis consisted of a one-way analysis of variance (ANOVA) followed by the protected Student's t-test for pre-treatment data or of the Williams test for treatment data.

Where Levene's test showed a significant difference in variance across groups, the data were analysed using non-parametric methods. Such analysis consisted of the Kruskal-Wallis ANOVA followed by the protected Wilcoxon's rank sum test for pre-treatment data and of the Shirley's test for treatment data.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical symptoms.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
High dose males gained less weight than controls over the first 26 weeks of the treatment period. This resulted in a lower mean body weight in this group compared with the controls at the end of the treatment period. Although high dose females had a slightly lower mean body weight at the end of the treatment period than controls (9 %), the difference was small and there had been a small difference before the start of treatment. The effect in high dose females is therefore considered equivocal. There were no other differences in body weight that were of sufficient magnitude to indicate an effect of treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
High dose males ate slightly less food than controls during the first 26 weeks of treatment although the differences did not attain statistical significance. The mean average food efficiency was calculated from the body weight gain and food consumption, using the following equation: food efficiency = body weight gain (g) / food consumption (g/animal/day) x 100.
There were no other biologically significant differences in food consumption between the groups.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no treatment-related differences in water consumption.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related eye lesions.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Intermediate and high dose males had lower haemoglobin concentrations, packed cell volumes and red blood cell counts than concurrent controls. These changes were dose-related and present at both sampling occasions during the treatment period. Other small differences in the haematology parameters were considered not to represent an effect of treatment with the test material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
High dose animals (particularly males) and to some extent intermediate dose males had lower calcium concentrations than controls at some stage in the study. High dose females also had lower potassium concentration compared with controls in week 52. Intermediate and high dose animals had higher serum cholesterol, triglyceride and phospholipid concentrations than controls in weeks 26 and 52 and these differences were dose-related. There was some evidence for a similar, slight effect in low dose animals but only the mean triglyceride and phospholipid concentrations of low dose males in week 52 were statistically different from the control mean. High and intermediate dose males tended to have lower albumin concentrations throughout treatment and high dose females higher globulin concentrations in week 26 when compared with concurrent controls. High dose animals and intermediate dose males had elevated alkaline phosphatase activities compared with controls in weeks 26 and 52.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Treated females tended to pass greater quantities of urine with a correspondingly lower specific gravity when compared with controls although the pattern of change was not dose-related. At both sampling occasions during treatment a small number of high dose animals had reducing substances in the urine although the number at termination was lower than in week 26.
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):
High and intermediate dose animals had greater absolute and relative liver weights than concurrent controls and the mean relative liver weight of low dose females was also statistically significantly higher than the corresponding control mean. Treated females and high dose males had greater absolute thyroid weights when compared with controls and the relative weight of this organ was also higher than the corresponding control mean in high dose animals. The mean absolute and relative kidney weight of high dose males were higher than in controls. High dose females had lower mean absolute and/or relative ovary and uterus weights than controls and this was considered related to the degree of sexual maturity in these animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings that were considered related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examination revealed that all high dose animals and one intermediate dose male had a hepatocytic hypertrophy in the periportal region of the liver. Two high dose animals and another intermediate dose male had a more generalised hypertrophy. In a number of high dose animals the hypertrophy was accompanied by the formation of myeloid bodies representing condensed membranes. The majority of high dose animals had increased cortical vacuolation in the zona glomerulosa of the adrenals. The majority of treated females (including all high dose females) showed only early maturity at microscopic examination. Haemosiderin was present at increased levels in the spleen of occasional animals from all treated groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
other: Changes in the liver. Increased liver weight and hepatocytic hypertrophy. Reduced body weight gain, reduced food consumption Elevated phosphatase activities
Critical effects observed:
not specified

Table 1: Mean body weights (kg)

     Dose group (mg/kg/day)
 Time (days)  Sex  0  1  6  36
 -14  M  5.9  6.1  5.9  5.8
 -7  M  6.3  6.3  6.1  6.2
 -1  M  6.3  6.6  6.3  6.3
 1  M  6.4  7.0  6.7  6.7
 29  M  7.5  7.6  7.3  7.2
 92  M  9.3  9.1  8.9  8.2*
 183  M  10.0  9.9  9.6  8.4*
 274  M  10.2  10.2  9.7  8.8*
 364  M  10.3  10.1  9.6  8.9*
 -14  F  5.6  5.7  5.9  5.1
 -7  F  5.8  5.9  6.2  5.3
 -1  F  5.9  6.2  6.3  5.5
 1  F  6.0  6.2  6.4  5.6
 29  F  6.9  7.0  7.3  6.5
 92  F  8.0  8.3  8.8  7.5
 183  F  8.4  8.5  9.5  7.8
 274  F  8.8  8.8  9.3  8.0
 364  F  8.8  8.8  9.5  8.0

* P≤0.05 (Williams test)

Table 2: Mean average food efficiency (%)

     Dose group (mg/kg/day)
 Time (days)  Sex  0  1  6  36
 1  M  17.3  17.0  11.3  14.9
 29  M  9.1  10.6  7.9  7.2
 92  M  4.6  7.8  5.6  3.4
 190  M  3.0  4.2  -3.0  6.1
 274  M  3.1  0.0  0.7  3.5
 358  M  -9.5  -15.7  -5.4  -9.0
 1  F  14.1  13.8  16.2  21.1
 29  F  11.0  7.5  7.8  3.2
 92  F  3.0  2.4  5.1  3.1
 190  F  5.3  4.4  -0.5  2.5
 274  F  2.4  1.3  5.4  -1.2
 358  F  -20.6  -9.3  -10.1  -14.3

Table 3: Clinical haematology

       Dose group (mg/kg/day)
 Parameter  Time (weeks)  Sex  0  1  6  36
 Hb, g/dL  0  M 13.2  14.6  13.5  13.4
   0  F  14.5  13.9  13.6  13.8
   26  M  15.8  15.7  14.6*  14.2**
   26  F  15.9  15.3  14.9  15.2
   52  M  16.2  16.3  15.3*  14.8*
   52  F  16.0  15.8  14.9  15.1
 MCH, pg  0  M  20.7  22.0  21.7  21.4
   0  F  21.6  22.2  21.2  21.3
   26  M  21.9  22.7  22.7  22.4
   26  F  22.7  22.6  22.1  21.7
   52  M  22.6  23.3  23.2  22.7
   52  F  23.1  23.3  22.6  21.9*
 MCHC, g/dL  0  M  34.6  35.3  34.5  34.8
   0  F  34.9  35.2  34.8  34.6

 

 26

 M

 35.1

 35.1

 35.3

 35.3

   26  F  35.4  35.0  34.7  34.7

 

 52

 M

 35.9

 36.1

 36.1

 35.5

 

 52

 F

 36.0

 36.3

 35.8

 35.4

 PVC, %  0  M  38.3  41.4  39.1  38.6
     F  41.5  39.5  39.1  39.9
   26  M  45.1  44.6  41.5*  40.2*
     F  44.7  43.6  43.0  43.9
   52  M  45.1  45.1  42.2*  41.6*
     F  44.4  43.5  41.5  42.7
 RBC M/mm3  0  M  6.42  6.63  6.21  6.28
     F  6.70  6.25  6.40  6.48
   26  M  7.21  6.90  6.45**  6.33**
     F  6.98  6.74  6.75  7.01
   52  M  7.17  6.98  6.59*  6.52*
     F  6.92  6.78  6.58  6.90
 MCV, fl  0  M  60  62  63  62
     F  62  63  61  62
   26  M  63  65  64  64
     F  64  65  64  63
   52  M  63  65  64  64
     F  64  64  63  62
 RETI/1000  0  M  4  3  3  3
     F  3  4  3  3
   26  M  4  3  4  1#
     F  3  5  4  3
   52  M  1  0  0  1
     F  1  2  1  1
 PLAT k/mm3  0  M  330  375  344  343
     F  372  422  404  417
   26  M  291  291  343  338
     F  310  412  402  511**
   52  M  286  282  360  336
     F  378  441  387  478
 PT, sec  0  M  6.3  6.1  6.1#  6.5
     F  6.1  6.2  6.3  6.3
   26  M  6.4  6.2  6.1  6.5
     F  6.2  6.2  6.2  6.4
   52  M  6.6  6.3  6.2  6.8#
     F  6.2  6.3  6.2  6.5
 APPT, sec  0  M  12.1  11.8  11.8  11.7
     F  11.7  11.9  11.8  11.6
   26  M  10.4  10.2  10.1  10.0
     F  10.3  10.3  10.1  10.1
   52  M  11.5  11.2  10.9  10.6#
     F  10.6  11.1  10.7  10.5
 WBC, k/mm3  0  M  11.8  13.5  15.0  13.5
     F  13.9  16.3  13.8  13.0
   26  M  12.2  11.8  10.0  10.3
     F  12.3  14.2  14.8  12.2
   52  M  11.7  10.5  9.0  11.1
     F  10.9  11.1  13.9  10.8

*P≤0.05

**P≤0.01

#P≤0.05 (non-parametric)

Table 4: Blood clinical chemistry

       Dose group (mg/kg/day)
 Parameter  Time (weeks)  Sex  0  1  6  36
 Na, mEq/L  0  M  145  147  147  147
     F  146  147  146  146
   26  M  146  146  145  145
     F  147  147  146  147
   52  M  148  146  145  145
     F  145  146  146  145
 K, mEq/L  0  M  4.7  5.2  4.7  5.1
     F  4.4  4.7  4.8  4.8
   26  M  4.6  4.6  4.6  4.6
     F  4.5  4.4  4.4  4.5
   52  M  4.4  4.6  4.5  4.5
     F  4.5  4.4  4.5  4.2
 Cl, mEq/L  0  M  108  109  108  110
     F  109  110  109  109
   26  M  114  115  114  115
     F  115  116  115  116
   52  M  112  114  113  114
     F  113  113  113  114
 Ca, mg/L  0  M  115  115  112  114
     F  112  113  113  114
   26  M  110  108  106  103**
     F  108  113  110  108
   52  M  107  108  103  104
     F  109  108  107  104
 P, mg/L  0  M  79  73  79  82
     F  73  76  77  77
   26  M  46  46  46  46
     F  48  43  45  47
   52  M  40  39  36  40
     F  43  41  39  40
 GLUC, g/L  0  M  0.99  0.96  0.97  0.95
     F  1.04  1.01  0.96  0.97
   26  M  0.88  0.92  0.95  0.91
     F  0.99  1.05  0.99  0.97
   52  M  0.95  0.91  0.95  0.91
     F  1.00  0.97  0.98  0.91
 UREA, g/L  0  M  0.21  0.24  0.23  0.26
     F  0.25  0.26  0.26  0.26
   26  M  0.25  0.29  0.29  0.27
     F  0.27  0.34*  0.33*  0.32*
   52  M  0.28  0.32  0.28  0.28
     F  0.33  0.35  0.32  0.36
 CHOL, g/L  0  M  1.71  1.72  1.58  1.56
     F  1.36  1.42  1.27  1.40
   26  M  1.37  1.65  2.05##  3.00##
     F  1.55  1.85  2.27**  3.06**
   52  M  1.23  1.54  1.90**  2.56**
     F  1.72  1.74  2.14  2.99*
 TRIGS, g/L  0  M  0.29  0.40  0.38  0.34
     F  0.33  0.32  0.34  0.34
   26  M  0.33  0.38  0.48#  1.85##
     F  0.45  0.43  0.61  1.13**
   52  M  0.32  0.43#  0.46#  1.55##
     F  0.51  0.52  0.66#  1.34##
 P.LIP, g/L  0  M  3.22  3.25  2.96  3.05
     F  2.77  2.85  2.65  2.84
   26  M  3.01  3.58  3.90##  5.33##
     F  3.43  3.80  4.27*  5.47**
   52  M  2.78  3.45*  3.86**  4.94**
     F  3.69  3.53  4.36  5.39**
 T.BIL, mg/L  0  M  0.4  0.8  0.6  0.9
     F  0.8  0.4  0.7  0.5
   26  M  1.3  1.0  1.3  1.0
     F  1.3  1.1  1.1  0.8
   52  M  1.4  1.1  0.5  0.7
     F  1.3  1.1  0.9  0.8
 PROT, g/L  0  M  59  59  62  58
     F  57  57  56  57
   26  M  68  68  69  64
     F  66  69  67  68
   52  M  65  67  69  63
     F  65  67  67  65
 ALB, g/L  0  M  32  32  33  32
     F  34  32  33  34
   26  M  36  35  33##  31##
     F  36  36  34  34
   52  M  33  34  32*  31*
     F  34  35  34  33
 GLOB., g/L  0  M  27  26  29  26
     F  24  25  23  23
   26  M  33  33  36  33
     F  30  34  32  34
   52  M  32  33  37  32
     F  30  32  33  32
 A/G  0  M  1.2  1.2  1.1  1.2
     F  1.5  1.3  1.5  1.5
   26  M  1.1  1.1  0.9  1.0
     F  1.2  1.1  1.1*  1.0*
   52  M  1.0  1.1  0.9  1.0
     F  1.1  1.1  1.1  1.0
 CREAT, mg/L  0  M  6.0  6.5  6.2  6.2
     F  5.9  6.7  6.5  6.5
   26  M  8.1  7.9  7.9  6.5**
     F  7.5  8.5  7.8  8.0
   52  M  8.4  8.9  8.4  7.0**
     F  7.8  8.7  7.9  7.8
 A.P., IU/L  0  M  278 283  269  268
     F  253  281  298  293
   26  M  133  156  191#  575##
     F  135  177  174  404##
   52  M  110  129  191#  525##
     F  142  174  178  462 **
 ASAT, IU/L  0  M  39  51  45  39
     F  40  48  39  48
   26  M  43  42  41  37
     F  39  51  40  37
   52  M  44  46  39  39
     F  41  52  39  40
 ALAT, IU/L  0  M  24  30  27  25
     F  34  33  29  31
   26  M  31  31  24**  22**
     F  36  36  29  26
   52  M  35  37  27  28
     F  36  44  27  41
 CPK, IU/L  0  M  339  860  455  332
     F  373  492  304  404
   26  M  250  268  235  253
     F  211  628  246  199
 GGT, IU/L  0  M  3  4  4  4
     F  5  3**  4*  5
   26  M  3  4  4  5*
     F  4  4  3  5
   52  M  0  2  2#  3#
     F  2  2  1  3

*P≤0.05

**P≤0.01

#P≤0.05 (non-parametric)

##P≤0.01 (non-parametric)

Table 5: Urine analysis (mean values)

        Dose group (mg/kg/day)
 Parameter  Time (weeks)  Sex  0  1  6  36
 Volume  0  M  89.0  115.0  117.0  107.0
     F  117.0  132.0  119.0  98.0
   26  M  146.0  130.0  150.0  131.0
     F  88.0  164.0  136.0  157.5
   52  M  127.6  112.4  113.6  127.6
     F  62.0  233.6*  135.6*  169.6*
 Specific gravity  0  M  1.049  1.048  1.044  1.044
     F  1.039  1.039  1.042  1.043
   26  M  1.048  1.047  1.045  1.039
     F  1.053  1.037*  1.046*  1.042*
   52  M  1.051  1.046  1.053  1.044
     F  1.050  1.035  1.046  1.040

*P≤0.05

Conclusions:
Under the conditions of the test, the NOAEL of the test material was determined to be 1 mg/kg bw/day on this basis that this dose did not result in any histopathological lesions and there was no evidence for functional impairment in any organ system.
Executive summary:

In a GLP compliant repeat dose (oral) study conducted in line with EPA OPP 83-1, the effects of the repeat dose of the test material over 52 weeks was determined in the dog.

Dogs were administered the test material by oral (capsule) at 0, 1, 6 and 36 mg/kg day. The highest dose was associated with slightly reduced body weight gain and reduced food consumption in males and elevated alkaline phosphatase activities, increased liver weight and hepatocytic hypertrophy in both sexes. Increased thyroid weight in the follicles is considered unlikely to be related to the change in the liver or reflect any functional impairment in the gland. There were increased concentrations of serum cholesterol, triglyceride and phospholipid and to a lesser extent, serum protein concentration was altered. Other changes at this dose level included vacuolation in the adrenals, considered to represent changes in mineralocorticoid production, which could also explain altered serum electrolyte concentrations. The minor delay in sexual maturity in females treated at the highest dose level was probably related to retarded growth and probably an indirect effect of treatment. Some clinical chemistry changes implied an effect on the kidney (increased urine output, reducing substances in the urine) and the weight of this organ was increased, but there was no microscopic evidence of change in this organ. Males treated at the highest dose had slightly lowered red cell parameters and there was some evidence of haemosiderin deposits in the spleen of animals of both sex. Animals treated at the intermediate dose had more or less similar changes to those described above but at a less severe degree. Vacuolation of the adrenals and reducing substances in the urine were not seen. At the lowest dose, the only change was slightly higher triglyceride concentrations in males and increased urine volume and increased liver thyroid weight in females.

Under the conditions of the test, the NOAEL of the test material was determined to be 1 mg/kg bw/day on this basis that this dose did not result in any histopathological lesions and there was no evidence for functional impairment in any organ system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1 mg/kg bw/day
Study duration:
chronic
Species:
dog

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 March 2002 to 30 October 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation: 175-198 g (males); 165-184 g (females)
- Housing: individually in suspended, stainless steel, wire-mesh type cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 66-73 °F
- Humidity: 30-58 %
- Photoperiod: approximately 12 hours fluorescent light per day
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Mean MMAD
2.3 mg/M3 - 1.64 ± 0.169 µ/M; 5.1 mg/M3 - 2.11 ± 0.165 µ/M; 12.0 mg/M3 - 1.62 ± 0.113 µ/M

Mean GSD
2.3 mg/M3 - 1.653 ± 0.223; 5.1 mg/M3 - 1.753 ± 0.332 µ/M; 12.0 mg/M3 - 1.620 ± 0.136 µ/M
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 63 L stainless steel and acrylic nose-only exposure chamber with stainless steel baffle (see Figure below).
- Method of holding animals in test chamber: nose-only restraint tubes
- System of generating particulates/aerosols: test substance was packed into a cylindrical holder with high pressure to provide a compact, uniform surface. A scraping blade in a Wright Dust Feeder (WDF) rotated over the surface removing small amounts of the test substance. The dislodged test substance entered the air stream passing through the WDF and was transported out of the WDF into the jet mill. The jet mill pulverized the test substance by directing air streams containing the test substance particles into each other from opposing directions. Smaller pulverized particles exited the jet mill whereas larger particles went through the pulverization process again. The test substance then passed through a cyclone which further separated out the larger particles from the aerosol prior to entering the exposure chamber.
- Temperature, humidity, pressure in air chamber: 20-24 °C; relative humidity 40-60 %
- Air flow rate: 83, 82, 70 and 68 L/min for controls, low dose, mid dose and high dose groups respectively.
- Air change rate: at least 64 per hour
- Method of particle size determination: particles were accelerated through a nozzle in a TSI Aerodynamic Particle Sizer and measuring the time taken for each particle to pass between two closely spaced laser beams
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber atmosphere samples for the gravimetric determination of the test material exposure levels were collected. The samples were withdrawn from the breathing zone of the animals in the exposure chambers through glass-fibre filters.

The gravimetric test material exposure levels were measured at least twice during the exposure. The aerosol concentration was calculated as the filter weight gain in mg divided by the volume of air sampled. The volume of air sampled was calculated as the sample flow rate multiplied by the sample duration.

The samples collected were transferred to polypropylene jars, diluted with HCl solution and analysed with HPLC.
Duration of treatment / exposure:
Six hours
Frequency of treatment:
Once per day, 5 days/week for 20 exposure days
Dose / conc.:
2.3 mg/m³ air (analytical)
Dose / conc.:
5.1 mg/m³ air (analytical)
Dose / conc.:
12 mg/m³ air (analytical)
No. of animals per sex per dose:
Ten
Control animals:
yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes - evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, reactivity to handling, bizarre behaviour and palpatation of tissue masses.
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption: Yes - weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of the test and again prior to termination

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of the study
- Anaesthetic used for blood collection: Yes, carbon dioxide
- Animals fasted: Yes
- How many animals: all
- Parameters checked: haemoglobin, erythrocyte count, absolute and percent reticulocytes, platelets, prothrombin time, activated partial thromboplastin time, total and differential leucocyte counts, hematocrit, MCV, MCH and MCHC.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the study
- Animals fasted: Yes
- How many animals: all
- Parameters checked: sodium, potassium, chloride, calcium, phosphorus, glucose, urea nitrogen, cholesterol, total bilirubin, total protein, albumin, globulin, albumin/globulin ratio, creatinine, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase and sorbitol dehydrogenase.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to termination of the study
- Dose groups that were examined: all
- Battery of functions tested: motor activity / functional observational battery
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- The following organs were weighed: adrenals, brain, kidneys, liver, heart, lungs, pituitary, ovaries, prostate, spleen, testes, thymus, thyroids and uterus.

HISTOPATHOLOGY: Yes
- The following organs/tissues were sampled: adrenals, aorta, sternum with bone marrow, femur with bone marrow, bone marrow smears, brain, caecum, colon, duodenum, epididymis, eyes, harderian gland, heart, ileum, jejunum, kidneys, lacrimal gland, larynx, liver, lungs, mandibular and mesenteric lymph nodes, mammary gland (females only), nasal tissues, oesophagus, optic nerves, ovaries, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerves, seminal vesicle, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus, vagina and all gross lesions.
Statistics:
Levene's test was used to assess homogeneity of group variances. If Levene's test was not significant (p≥0.01)< Dunnett's test was used to compare each treatment group with the control group. If Levene's test was significant (p<0.01), comparisons with the control group were made using Welch's t-test with Bonferroni correction.

Chi-square test for homogeneity was used to analyse Functional Observational Battery results except for continuous data.

Log transformation was performed on leucocyte counts prior to analysis.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test material related clinical observations seen in the study.
Mortality:
no mortality observed
Description (incidence):
No unscheduled deaths occurred during the course of the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no clear test material effect on the body weight. The mean weight for the females in the highest dose group was statistically lower than the control mean weights. However, this decrease appeared to be due to one animal, therefore this is not clearly test material related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no test material related effect on food consumption. The statistically significant differences that occurred in some weeks were sporadic and showed no relationship to dose.
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 test material related Ophthalmoscopic effects were seen.
Haematological findings:
no effects observed
Description (incidence and severity):
When compared to controls, no toxic effects on haematology were observed after 28 days for male or female rats exposed up to the highest dose level.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related toxic effects on clinical chemistry parameters were observed for males or females exposed up to the highest dose level. Any observed alterations were not dose dependent and were considered related to inter-day collection variety.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Motor activity evaluations revealed no test material related effects. There was no test material related effect seen on any of the functional observational categories of activity/arousal, neuromuscular, sensorimotor, autonomic, or physiological measurements. The only statistically significant differences seen in the highest dose group was in hind limb grip strength and body temperature in the males. No difference was seen in fore limb strength and neither of these differences was seen in the females. These differences were therefore considered to be spurious and not related to the test material.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test material related organ weight changes occurred in either sex.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test material related macroscopic observations were noted in either sex.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test material related microscopic effects noted in either sex.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
12 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test material related effects were seen.
Critical effects observed:
not specified

Table 1: Mean body weights (g)

     Dose group (mg/mm3)
 Time (weeks)  Sex  0.0  2.3  5.1  12.0
 -1  M  183.0  192.2*  194.3*  180.1
 1  M  233.8  237.1  240.0  229.7
 2  M  282.9  278.8  280.5  277.6
 3  M  318.9  309.6  309.3  312.6
 4  M  337.2  324.5  326.0  333.0
 -1  F  169.4  173.7  172.3  169.3
 1  F  192.4  188.3  192.8  185.3
 2  F  209.5  201.9  207.0  198.5
 3  F  226.1  214.2  222.1  209.9*
 4  F  232.1  222.7  228.3  216.8

*significantly different from control (P<0.05)

Table 2: Selected haemotology values

     Dose group (mg/m3)
 Parameter  Sex  0.0  2.3  5.1  12.0
 Platelets (K/mm3)  M  1205.0  1013.2  921.1**  1045.7
   F  1033.3  1089.5  879.9  1139.1
 APTT (sec)  M  30.18  19.38**  19.58**  31.00
   F  23.44  16.93**  17.47**  21.50
 Prothrombin time (sec)  M  17.67  14.80**  14.65**  17.27
   F  13.98  14.64  14.14  13.66

**significantly different from control (P<0.01)

Table 3: Selected clinical chemistry values

     Dose group (mg/m3)
 Parameter  Sex  0.0  2.3  5.1  12.0
 Sodium (mEq/L)  M  148.2  151.2**  150.8**  149.0
   F  146.3  148.6  149.2*  147.5
 Potassium (mEq/L)  M  7.18  6.11  5.99*  6.98
   F  7.44  6.52  5.97**  7.50
 Sorbital dehydrogenase (U/L)  M  16.11  23.58**  23.99**  15.77
   F  16.51  23.25*  21.87  16.88
 Cholestoerol (mg/dL)  M  47.1  57.9  59.2*  48.3
   F  53.8  66.2  7.1**  60.6
 Glucose (mg/dL)  M  10.64  90.8*  100.3  110.6

*significantly different from control (P<0.05)

**significantly different from control (P<0.01)

Table 4: Selected organ weight change values

     Dose group (mg/m3)
 Parameter  Sex  0.0  2.3  5.1  12.0
 Thymus (g)  M  0.51  0.41*  0.52*  0.48
 Thymus/body weight %x10  M  1.70  1.39*  1.75  1.63
 Thymus/brain weight %x10  M  2.87  2.22*  2.83  2.67
 Ovary/bodyweight %x10  F  7.18  6.28  5.96*  6.80
Conclusions:
Under the conditions of the test, the test material had no toxic effect at 2.3, 5.1 and 12 mg/m³. The NOEL was determined to be 12 mg/m³.
Executive summary:

In a GLP compliant repeat dose (inhalation) study conducted in line with standardised guideline EU Method B.8, the effects of the repeat dose of the test material in rats was determined.

Under the conditions of the test, no toxic effect was seen at 2.3, 5.1 and 12 mg/m3 and the NOEL was determined to be 12 mg/m3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
12 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 March 1995 to 13 December 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Froxfield Farms (UK) Limited,
- Age at study initiation: 13 to 15 weeks
- Housing: individually in metal cages
- Diet: SDS Rabbit Diet SQC ad libitum
- Water: ad libitum
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature: 16.5-22 °C
- Humidity: 40-68 %
- Air changes: approximately 19 per hours
- Photoperiod: 12 hours light/12 hours dark

IN-LIFE DATES: From: 15th March 1995 To: 20th June 1995
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: 12 x 8 cm
- % coverage: approximately 10%
- Type of wrap if used: impervious bandage consisting of gauze covered with 'Elastoplast' elastic adhesive dressing backed with impervious 'Sleek' plaster.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 6 hours on each day
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
Daily for 21 consecutive days
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Five
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: three times daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing on day 1 and then weekly intervals

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: packed cell volume, haemoglobin, red blood cell count, mean corpuscular haemoglobin concentration, mean corpuscular volume, platelet counts, total white blood cell count, thrombotest, differential white blood cell count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to termination
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked: glucose, total protein, albumin, globulin, albumin/globulin ratio, urea nitrogen, creatinine, alkaline phosphatase, glutamic-pyruvic transaminase, gutamic-oxalaxetic transaminase, total bilirubin, sodium, potassium calcium, chloride, inorganic phosphorus, cholesterol

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- The following organs were weighed: adrenals, brain, kidneys, liver, ovaries, spleen, testes.

HISTOPATHOLOGY: Yes
- The appearance of the following tissues was recorded: adrenals, aorta, brain, caecum, colon, duodenum, eyes, gall bladder, heart, ileum, jejunum, kidneys, larynx, liver, lungs, cervical and mesenteric lymph nodes, mammary glands, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, salivary gland, sciatic nerve, skeletal muscle, skin, spleen, sternum, stomach, testes and epididymides, thymus, thyroid/parathyroid, tongue, trachea, urinary bladder, uterus, vagina and any macroscopically abnormal tissue.
Statistics:
Where appropriate, the following sequence of statistical tests was used:
- if the data consisted predominantly of one particular value, the proportion of values different from the mode was analysed by Fisher's exact test followed by Mantel's test for a trend in proportions. Otherwise:
- Bartlett's test was applied to test for heterogeneity of variance between treatments.
- If no significant heterogeneity was detected, one way ANOVA was performed followed by Williams' test
- If significant heterogeneity was detected and could not be removed by log transformation, Kruskal-Wallis analysis of ranks was used followed by non-parametric equivalent of Williams' test (Shirley's test).

Covariate of analysis of organ weight data was performed using adjusted organ weights where a correlation between organ weight and body weight was established at the 10% level of significance.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment related clinical signs.
Dermal irritation:
no effects observed
Description (incidence and severity):
There were no dermal reactions that were considered to be treatment related. In two animals from the control group, spots were observed during the last few days of the study.
Mortality:
no mortality observed
Description (incidence):
There were no treatment related mortalities.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences from the control group noted for body weight gains in any of the treatment groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The food consumption for low and high dose group females was slightly higher than control; these differences were however not considered to be treatment related.
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):
There were no differences from the controls in the haematological parameters measured that were related to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean values of significantly different biochemistry values were recorded. Cholesterol levels were higher than control for females of the high dose group. However, individual values were within the expected background range and in the absence of any related biochemical or pathological changes in the liver, this finding was not considered to be of toxicological importance.
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):
There were no differences from controls in organ weights that were considered to be related to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed at termination revealed no changes attributable to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
All of the lesions observed by microscopic pathology were minor in nature and not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
100 other: mg/kg/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Elevated cholesterol levels in high dose females (considered to be equivocal)
Key result
Dose descriptor:
NOAEL
Effect level:
300 other: mg/kg/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Elevated cholesterol levels in high dose females (considered to be equivocal)
Critical effects observed:
not specified

Table 1: Group mean values body weights (g)

     Dose group (mg/kg/day)
 Time (weeks)  Sex  Control  100  300  1000
 0  M  2823  2929  2954  3029
   F  3144  3206  3083  3189
 1  M  2911  3067  3102  3146
   F  3212  3319  3186  3370
 2  M  3060  3230  3261  3326
   F  3363  3474  3352  3564
 3  M  3200  3341  3337  3489
   F  3475  3631  3485  3631
 Weight gain  M  377  412  383  460
   F  331  425  402  442

Table 2: Group mean values of food consumption (g/animal/week)

     Dose group (mg/kg/day)
 Time (weeks)  Sex  Control  100  300  1000
 1  M  1289  1339  1257  1237
   F  1240  1358  1208  1409
 2  M  1277  1364  1357  1355
   F  1228  1416  1376  1397
 3  M  1210  1289  1217  1262
   F  1258  1357  1321  1313
 Cumulative value  M  3776  3992  3931  3855
   F  3725  4131  3905  4119

Table 3: Significantly different biochemistry values

     Dose group (mg/kg/day)
 Parameter  Sex  Control  100  300  1000
 Glucose (mg/dL)  M  128  139  140  147*
 Protein (g/dL), total  F  5.7  5.9  5.8  6.2*
 Glob  F  2.2  2.4  2.3  2.5*
 Creatinine (mg/dL)  F  1.3  1.2  1.3  1.0*
 GPT (mU/mL)  M  42  34  31*  31*
 GOT (mU/mL)  F  10  16***  16***  9
 Cholesterol (mg/dL)  F  43  50  47  64**

*significantly different from control (P<0.05), Williams' test

**significantly different from control (P<0.01), Williams' test

***significantly different from control (P<0.05), Student's t test

Table 4: Significantly different organ weights

     Dose group (mg/kg/day)
 Organ  Sex  Control  100  300  1000
 Adrenals  M  252  195***  237  232
 Brain  F  9.42  8.85***  8.77***  9.18
 Ovaries  F  0.223  0.350  0.267  0.365*

*significantly different from control (P<0.05), Williams' test

***significantly different from control (P<0.05), Student's t test

Conclusions:
Under the conditions of the test, the test material had no toxic effect at any of the doses tested. The NOAEL was determined to be 1000 mg/kg/day and the NOEL was determined to be 300 mg/kg/day.
Executive summary:

In a GLP compliant repeat dose (dermal) study conducted in line with standardised guidelines OECD 401 and EPA OPP 82-2, the effects of the repeat dose of the test material in rats was determined.

Under the conditions of the test, no toxic effect was seen at any of the doses tested. The NOAEL was determined to be 1000 mg/kg/day and the NOEL was determined to be 300 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rabbit

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Short-term toxicity

Two 90-day studies have been performed in rats resulting in a lowest NOEL of 2.5 mg/kg bw/day (50 ppm in food). Liver and kidney appear to be primary target organs following exposure at higher dose levels.

Newer GLP-compliant 90-day studies in the hamster and mouse resulted in NOAEL of 41 and 25 mg/kg food respectively. In the hamster, liver and prostate weights, as well as certain blood parameters, were impaired whereas effects on liver and testis weight were decisive in the mouse.

90-day and one year studies in dogs determined NOAELs of 50 mg/kg food based on increased liver weight in the 90-day study. The same NAOEL was determined in the one year study based on slight deleterious effects consisting of increased enzyme activity of serum alkaline phosphatase, serum glutamic-pyruvic transaminase, glucose-6-phosphatase and glucose-6-phosphate dehydrogenase, indications of an increased occurrence of red blood cells in the urine of females, slightly increased weight of the liver and thyroid and slight hepatic changes. In a more recent one year dog study, the NOAEL was found to be 1 mg/kg bw.

In a 28-day inhalation toxicity study in rats, 6 hour exposures to the test material via nose-only inhalation for five days per week for four weeks at levels of 2.3, 5.1 and 12 mg/m3 had no toxic effect. The highest dose was considered to be the NOEL.

The dose rate of 1000 mg/kg bw/day in the 21 -day dermal toxicity study in rabbits was considered to represent the NOAEL as the only possible treatment-related effect seen at the highest dose level was not of toxicological importance. The intermediate dose of 300 mg/kg/day was considered to represent the NOEL.

Long-term toxicity

In a 112 week chronic toxicity study in rats, no effects were seen up to the highest dose of 100 mg/kg food.

In a second 110 week feeding study in rats at dose levels up to 3200 mg/kg food, a NOEL of 50 mg/kg food was found (equal to 3.18 mg/kg bw/day for males and 3.16 mg/kg bw/day for females based on the measured concentration of the test material in food and measured food intake). The 400 mg/kg food level was the minimum effective dose (equal to 29 mg/kg bw/day for males and 26.3 mg/kg bw/day for females). The high dose level of 3200 mg/kg food (equal to 241 mg/kg bw/day for males and 248 mg/kg bw/day for females) was a notably toxic dose level with almost all effects being corollaries of a primary adverse effects on liver and kidney.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

No study selected as four key studies identified.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to repeated dose toxicity.