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Toxicological information

Carcinogenicity

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Description of key information

The test material was determined to have no carcinogenic effects according to rat and hamster studies performed in line with EPA Guideline 83-2. The test material was determined to show evidence of tumourigenic potential that was considered to be based on toxicologically significant non-neoplastic hepatic changes rather than any effect of the test material in a further study performed in line with EPA Guideline 83-2.

In addition, two in vitro cell transformation screens performed using BALB/c-3T3 cells (performed to a method similar to the cell transformation assay validated by ECVAM) demonstrated that the test material was inactive in the transformation assay.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 April 1980 to 30 November 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
EPA OPP 83-2 (Carcinogenicity)
Deviations:
not specified
GLP compliance:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: five weeks old
- Weight at study initiation: 82-102 g (male); 74-92 g (female)
- Housing: individual feeding cages
- Diet: ad libitum
- Water: city water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 1 °C
- Humidity: 55 ± 5 %
- Air changes: 20 times per hour
- Photoperiod: 12 hours light; 12 hours dark
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
110 weeks (10 animals of each sex sacrificed from each group at weeks 52 and 78).
Frequency of treatment:
Daily
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
400 ppm (nominal)
Dose / conc.:
3 200 ppm (nominal)
No. of animals per sex per dose:
Seventy
Control animals:
yes, plain diet
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (at scheduled sacrifice and sacrifice of moribund animals).
- Organ weight was measured at weeks 52, 78 and 110 for brain, heart, lungs, liver, kidneys, adrenals, spleen, testes, ovaries, thyroid, thymus and pituitary. Ratio of organ weight to body weight was calculated.

HISTOPATHOLOGY: Yes
- for organs detailed under Gross Pathology and spinal cord, eyes, salivary gland, lungs, trachea, oesophagus, stomach, large and small intestine, pancreas, prostate, uterus and cervical canal, lymph nodes, femur, femoralis muscle, skin, sciatic nerve and mammary gland.
Statistics:
Incidence of tumours was analysed by Peto's analysis method.
Relevance of carcinogenic effects / potential:
The test material has no obvious carcinogenic effect.
Key result
Dose descriptor:
other: NOEL
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Toxicity. 50 ppm is equal to 3.18 mg/kg bw/day for males and 3.16 mg/kg bw/day for females

At the 3200 ppm dose level organic disturbances attributable to pathological changes due to the test material were observed as chronic renal disturbances in many males and in less than half of the females. Furthermore swelling of parathyroid, renal osteodystrophy and calcification in bloods vessels and gastric mucosa were observed in both sexes. The changes were more appreciable in the males and from around week 78 up to week 102 all males died at this dose level. Diffuse development in the increase in amount of lipids in liver tissues was noted. Some animals, both males and females, demonstrated aggregated acidophile "oxyphilic cells" near Langerhans islets in the pancreas. At the 400 ppm dose level some animals showed the same chronic renal disturbances and aggregated "oxyphilic cells". At the 50 ppm dose level no changes attributable to the test material were noted.

Selected information on tumours can be found in Tables 1 to 3.

Information on historical control data versus results from the study can be found in Table 4. The incidence rate of hepatic adenoma in females is slightly higher than lab and breeder historical controls but within historical controls for NTP (US National Toxicology Program). This supports the claim that the adenomas seen are not treatment related but are on the high side of spontaneous range for hepatic adenomas. The incidence rate for carcinomas is well within the historical control range. When adenomas and carcinomas are considered concurrently, the incidence rate remains the same (4.0 %).

Table 1: Selected histopathological findings at week 52 (number of findings per group of 10 animals)

 Dose level (ppm) 0  50  400  3200
 Sex  M/F  M/F  M/F  M/F
 Organ  Finding        
 Liver b.t.  + Adenoma  1 ' / 0  0 / 0  0 / 0  0 / 0
 Testis  + Interstitial cell tumour  0 / 0  0 / 0  0 / 0  1 ' / 0

Grade of findings: 0 = normal; ' = slight

+ = non-neoplastic tumour

Table 2: Selected histopathological findings at week 78 (number of findings per group of 10 animals)

 Dose level (ppm)  0  50  400  3200
 Sex  M/F  M/F  M/F  M/F
 Organ  Finding        
 Lung  + Adenoma  0 / 0  0 / 0  1 ' / 0  0 / 0
 Kidney  * Adenocarcinoma  0 / 0  0 / 0  0 / 0  1 ' / 0
 Adrenal  + Pheochromo-cytoma  1 ' / 0  0 / 0  0 / 0  0 / 0
 Liver b.t.  * Hepatocellular carcinoma  0 / 0  0 / 0  0 / 0  0 / 1 '
 Spleen  * Malignant lymphoma  0 / 0  0 / 0  1 ' / 0  0 / 0
 Thyroid  + Adenoma  1 ' / 0  0 / 0  1 ' / 0  0 / 0
 Testis  + Interstitial cell tumour  4 / 0  5 ' / 0  6 ' / 0  6 ' / 0
 Pituitary  + Adenoma  1 ' / 0  0 / 1 '  1 ' / 0  1 ' / 1 '
 Bone  + Osteoma  0 / 0  0 / 0  0 / 0  1 ' / 0
 Uterus  + Leiomyoma  0 / 1 '  0 / 0  0 / 0  0 / 1 1'
   + Fibrosarcoma  0 / 1 '  0 / 0  0 / 0  0 / 0

Grade of findings: 0 = normal; ' = slight

+ = non-neoplastic tumour; * neoplastic tumour

Table 3: Selected histopathological findings at week 110

 Dose level (ppm)  0  50  400  3200
 Sex  M/F  M/F  M/F  M/F
 Organ  Finding        
 Number of animals examined    47/47  47/47  47/47  50/47
 Lung  + Adenoma  0 / 0  0 / 0  0 / 1 '  0 / 0
 Number of animals examined    45/47  46/45  44/47  50/47
 Thymus  * Malignant lymphoma  0 / 0  0 / 0  0 / 0  0 / 1 '
 Number of animals examined    47/47  47/47  47/47  50/47
 Kidney  * Adenocarcinoma  0 / 0  0 / 0  0 / 0  2 ' / 1 '
   + Hamartoma  0 / 0  0 / 0  0 / 0  1 ' / 0
 Number of animals examined    47/47  47/46  47/47  50/47
 Adrenal  + Pheochromocytoma  6 ' / 1 '  6 ' / 1 '  6 ' / 0  0 / 0
 Number of animals examined    47/47  47/47  47/47  50/47
 Liver / b.t.  + Adenoma  0 / 0  0 / 0  0 / 2 '  4 ' / 6 '
   + Leiomyoma  2 ' / 0  0 / 0  0 / 0  0 / 0
   * Hepatocellular carcinoma  0 / 0  0 / 0  0 / 0  1 ' / 1 '
 Number of animals examined    46/47  47/47  46/47  50/47
 Spleen  * Malignant lymphoma  1 ' / 2 '  0 / 0  1 ' / 3 '  0 / 1 '
   * Angiosarcoma  0 / 0  1 ' / 0  0 / 0  0 / 0
 Number of animals examined    47/47  47/47  47/47  50/47
 Islets  + Islet cell adenoma  1 ' / 1 '  2 ' / 1 '  0 / 1 '  0 / 0
   * Squamous cell carcinoma  0 / 0  0 / 0  0 / 0  0 / 1 '
 CNS  * Astrocytoma  0 / 0  0 / 0  1 ' / 0  0 / 0
 Number of animals examined    47/47  47/47  46/47  50/47
 Stomach  + Papilloma 0 / 0 1 ' / 0   0 / 0  2 ' / 0
 Number of animals examined    47/47  47/47  47/47  50/47
 Small intestine  * Undifferentiated sarcoma  0 / 0  1 ' / 0  0 / 0  0 / 0
   * Malignant lymphoma  0 / 0  0 / 0  0 / 0  1 ' / 0
 Number of animals examined    45/47  47/46  47/47  47/47
 Lymph  * Malignant lymphoma  2 ' / 3 '  1 ' / 1 '  2 ' / 3 '  0 / 6 '
 Number of animals examined    44/47  45/47  46/47  48/46
 Thyroid  + C-cell adenoma  1 ' / 5 '  5 ' / 2 '  3 ' / 1 '  0 / 2 '
   + Adenoma  4 ' / 0  1 ' / 0  0 / 1 '  2 ' / 1 '
   * Adenocarcinoma  0 / 0  0 / 0  0 / 0  0 / 1 '
 Number of animals examined    47/47  47/46  47/46  48/46
 Skin / sc  + Keratocanthoma  5 ' / 3 '  1 ' / 0  2 ' / 0  2 ' / 1 '
   + Fibroma  2 ' / 1 '  1 ' / 1 '  1 ' / 1 '  1 ' / 0
   * Malignant fibrous histiocytoma  0 / 0  0 / 2 '  1 ' / 1 '  0 / 0
   + Papilloma  0 / 1 '  0 / 0  0 / 0  1 ' / 1 '
   * Undifferentiated sarcoma  0 / 0  1 ' / 0  1 ' / 0  0 / 1 '
   * Basal cell carcinoma  0 / 1 '  0 / 2 '  0 / 0  0 / 0
   + Leiomyoma  1 ' / 1 '  0 / 0  0 / 0  0 / 0
   + Lipoma  0 / 0  0 / 0  1 ' / 0  0 / 0
 Number of animals examined    46/47  47/47  47/45  50/46
 Marrow  * Malignant lymphoma  0 / 0  0 / 2 '  0 / 3 '  0 / 0
 Number of animals examined    47/0  47/0  47/0  50/0
 Testis  + Interstitial cell tumour  38 ' / 0  33 ' / 0  43 ' / 0  35 ' / 0
 Number of animals examined    2/0  1/0  2/0  1/0
 Epididymis  * Mesothelioma  1 ' / 0  1 ' / 0  2 ' / 0  1 ' / 0
 Number of animals examined    24/44  23/44  30/46  33/47
 Mammary  + Fibroadenoma  0 / 3 '  0 / 5 '  0 / 7 '  0 / 3 '
   * Adenocarcinoma  0 / 2 '  0 / 1 '  1 ' / 0  0 / 0
   * Adenocanthoma  0 / 0  1 ' / 0  0 / 1 '  0 / 0
   + Fibroma  1 ' / 0  0 / 0  0 / 0  0 / 0
   * Carcinosarcoma  0 / 1 '  0 / 0  0 / 0  0 / 0
 Number of animals examined    47/47  46/46  47/46  49/46
 Pituitary  + Adenoma  11 ' / 14 '  4 ' / 11 ' 7 ' / 10 '  0 / 7 '
   * Adenocarcinoma  0 / 5 '  0 / 1 '  0 / 1 '  0 / 0
 Number of animals examined    47/0  47/0  47/0  50/0
 Prostate  * Squamous cell carcinoma  1 ' / 0  0 / 0  0 / 0  0 / 0
 Number of animals examined    45/47  47/47  47/47  50/47
 Bladder  * Transitional cell carcinoma  0 / 0  0 / 0  0 / 1 '  0 / 0
 Number of animals examined    46/47  47/47  47/46  50/46
 Bone  * Osteogenic sarcoma  0 / 0  1 ' / 0  0 / 0  0 / 0
 Number of animals examined    0/1  3/1  2/1  2/5
 Blood  * Leukemia  0 / 1 '  3 ' / 1 '  2 ' / 1 '  2 ' / 5 '
 Number of animals examined    0/47  0/47  0/46  0/47
 Uterus  + Polyp  0 / 2 '  0 / 3 '  0 / 0  0 / 0
   * Fibrosarcoma  0 / 1 '  0 / 0  0 / 1 '  0 / 2 '
   * Fibromyxoma  0 / 1 '  0 / 1 '  0 / 1 '  0 / 0
   + Leimyoma  0 / 0  0 / 1 '  0 / 0  0 / 0
   * Adenocarcinoma  0 / 0  0 / 0  0 / 1 '  0 / 0
 Number of animals examined    0/0  0/0  0/0  0/1
 Vagina  * Undifferentiated sarcoma  0 / 0  0 / 0  0 / 0  0 / 1 '

Grade of findings: 0 = normal; ' = slight

+ = non-neoplastic tumour; * neoplastic tumour

Table 4: Overview of historical control data versus test material

 Laboratory  AN-PYO  Charles River  NTP  ARegDoc 12564
  No. animals/total no. examined  % No. animals/total no. examined  % No. animals/total no. examined  % No. animals/total no. examined %
 Adenoma                        
 Male  5/569  0.9  8/956  0.8  23/1002  2.3  0/50

 0.0

 Female

 2/573

 0.3

 8/954

 0.8

 4/1000

 0.4

 2/50

 4.0

 Carcinoma                        

 Male

 3/569

 0.5

 6/956

 0.6

 7/1002

 0.7

 0/50

 0.0

 Female

 1/573

 0.2

 2/954

 0.2

 1/1000

 0.1

 0/50

 0.0

 Combined                        

 Male

 8/569

 1.4

 14/956

 1.5

 30/1002

 3.0

 0/50

 0.0

 Female

 3/573

 0.5

 10/954

 1.0

 5/1000

 0.5

 2/50

 4.0

 Overall range

 0 -7.7%

 0 -4.3%

 0 -10%

 4%

 

Conclusions:
Under the conditions of the test, there was no effect upon the total tumours observed, a negative trend being apparent in 21/40 tumour types tested. The test material has no obvious carcinogenic effect. A slight excess of tumours occurred only at the notably toxic 3200 ppm dose level.
Executive summary:

In a GLP compliant study conducted in line with standardised guideline EPA OPP 83-2 (as part of a combined chronic toxicity/carcinogenicity study), the effects of the carcinogenicity of the test material over 52, 78 and 110 weeks was determined in the rat.

Rats were administered the test material by oral (diet) at 0, 50, 400 and 3200 ppm. There was no effect upon the total tumours observed, a negative trend being apparent in 21/40 tumour types tested. The test material has no obvious carcinogenic effect. A slight excess of tumours occurred only at the notably toxic 3200 ppm dose level.

The NOEL for chronic toxicity was determined to be 50 ppm (3.18 mg/kg bw/day for males; 3.16 mg/kg bw/day for females).

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 December 1987 to 27 October 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 83-2 (Carcinogenicity)
Deviations:
yes
Remarks:
(at necropsy stage, two group 3 males were found to be transposed. It was not certain at which time point transposition occurred. As both animals are from the same group, all group mean and incidence data remain unaltered)
Qualifier:
according to
Guideline:
other: OECD 'Short Term and Long Term Toxicology Group Guidelines' published in the Chemical Regulations Reporter, 14th August 1981
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Requirements for safety evaluation of agricultural chemicals published in 59 Nohsan No. 4200, Ministry of Agriculture, Forestry and Fisheries dated 28th January 1985
Deviations:
not specified
GLP compliance:
yes
Species:
hamster, Syrian
Strain:
other: Bio F1D Alexander
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
88 weeks (males); 80 weeks (females)
Frequency of treatment:
Daily
Dose / conc.:
5 ppm
Dose / conc.:
26 ppm
Dose / conc.:
132 ppm
Dose / conc.:
675 ppm
No. of animals per sex per dose:
100 controls (male and female) and 132 and 675 ppm for males; 50 for all other test groups
Control animals:
yes, plain diet
Description (incidence and severity):
No apparent clinical signs of a reaction to treatment were noted. There were no apparent treatment-related ante-mortem clinical signs.
Description (incidence):
In both sexes, the administration of test material was not considered to have affected the distribution or rate of mortalities.
Description (incidence and severity):
During the period of maximal growth (weeks 0 to 6) males receiving 675 mg/kg diet showed slightly lower than control body weight gain. Subsequently, during the body weight decline/plateau phase after week 8, animals receiving 675 mg/kg diet showed a higher rate of mean body weight loss, when compared with the controls; females receiving 132 mg/kg diet were similarly affected, but the difference did not achieve statistical significance. Additionally, the overall body weight change (weeks 0 to 88) of males receiving 675 mg/kg diet was lower than control (see Table 2).
Description (incidence and severity):
During the first 6 weeks of treatment lower food consumption values were obtained for males receiving 675 mg/kg diet. Subsequently, the values obtained for this group were comparable with the controls. The food consumption values of the remaining treated groups were considered to be unaffected by treatment (see Table 3). The overall (weeks 1 to 8) food utilisation efficiency was considered to be unaffected by treatment. The achieved intakes followed the expected pattern for a fixed dietary level study.
Description (incidence and severity):
Examination of the blood smears for differential white cell counts did not reveal any treatment-related effects.
Description (incidence and severity):
Analysis of organ weights obtained from females killed after 80 weeks revealed slightly higher than control liver weights in the high dose females. In males killed after completion of 88 weeks of treatment, there were no statistical differences from the control (see Table 4).
Description (incidence and severity):
Macroscopic pathology of animals killed or dying during the treatment period, or killed at termination, revealed pitting of the liver of terminal females receiving 675 mg/kg diet. A reduction of adipose tissue was also noted in the decedent males. In addition, the following findings were also noted in the high dose group, but were unassociated with notable microscopic pathological change: uniform cortical scarring in the kidneys of the terminal females and pallor of the kidneys and small testes in the terminal males.
Description (incidence and severity):
Examination of animals killed or dying during the treatment period, or killed at termination, revealed notable non-neoplastic findings in females only (see Table 6):
• Liver: the number of animals involved is small and the toxicological importance of this finding is unclear.
• Adrenals: the toxicological importance of this finding is unclear.
Description (incidence and severity):
Microscopic pathology revealed that there was no evidence of tumourigenic potential in this study (see Table 5).
Relevance of carcinogenic effects / potential:
There was no evidence of tumorigenic potential at the dose levels used in this study.
Key result
Dose descriptor:
NOAEL
Effect level:
132 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
gross pathology
other: Lower bodyweight and feed consumption in high dose males, with a reduction in adipose tissue of decedent males. Pitted liver and centrilobular hepatocyte enlargement and cortical hyperplasia in the adrenals of high dose females.

Table 1: Mortality

     Dose group (ppm)
 Sex  Week  0  5  26  132  675
 Male  4  0/100  0/50  0/50  0/100  0/100
   41  2/100  1/50  1/50  0/100  0/100
   60  2/98  0/49  0/49  1/100  4/100
   70  8/96  3/49  6/49  4/99  5/96
   80  14/88  6/46  6/43  5/95  4/91
   88  24/74  10/40  13/37  8/90  8/87
 Total dead / % mortality    50/50  20/40  26/52  18/36  21/42
 Female  4  0/100  1/50  0/50  0/50  0/50
   40  3/100  0/49  0/50  0/50  1/50
   60  3/97  2/49  3/50  3/50  2/49
   70  22/94  5/47  14/47  9/47  7/47
   75  10/72  9/42  6/33  10/38  7/40
   80  20/62  11/33  6/27  13/28  5/33
 Total dead / % mortality    58/58  28/56  29/58  35/70  22/44

Table 2: Statistical analysis of body weight gain (g)

     Dose group (ppm)
 Sex  Week range  0  5  26  132  675
 Male  0-6  31.7  35.5  31.3  30.8  26.6**
   0-8  31.5  32.7  33.3  32.3  31.2
   8-80  -11.1  -11.2  -12.3  -12.8  -27.8**
   0-80  20.1  20.9  20.3  19.5  3.2**
   8-88  -14.1  -9.8  -13.8  -14.6  -30.1**
   0-88  16.6  21.9  19.5  17.7  0.7**
 Female  0-6  43.3  41.8  43.7  44.9  42.9
   0-8  40.8  42.2  41.7  42.1  45.7**
   8-80  -9.8  -12.1  -11.6  -18.5  -20.1**
   0-80  30.0  29.7  26.8  19.7  24.4

**P<0.01 in comparison to control

Table 3: Statistical analysis on food consumption (g/hamster/period)

     Dose group (ppm)
 Sex  Week range  0  5  26  132  675
 Male  1-6  473  460*  448**  461**  435**
   7-80  4545  4514  4539  4727  4464
   1-80  5018  4978  4984  5189  4899
   7-88  5006  4955  5065  5224  4871
   1-88  5482  5414  5507  5689  5307*
 Female  1-6  479  471  466  470  486
   7-80  4739  4690  4581  4727  4940**
   1-80  5213  5159  5032  5198  5422**

*P<0.05 in comparison to control; P<0.01 in comparison to control

Table 4: Mean group organ weights, absolute (g)

     Dose group (ppm)
 Sex  Organ  0  5  26  132  675
 Male  Brain  1.055  1.067  1.052  1.063  1.038
   Heart  0.894  0.929  0.902  0.861  0.850
   Liver  6.87  6.62  6.74  6.66  6.64
   Kidneys  1.721  1.786  1.867  1.667  1.551
   Adrenals  0.0285  0.0240  0.0286  0.0243  0.0243
   Testes  3.411  3.713  3.280  3.525  2.482
 Female  Brain  1.043  1.070  1.060  1.036  1.040
   Heart  0.977  0.971  1.019  0.944  0.886
   Liver  8.67  9.19  7.89  8.41  9.77**
   Kidneys  1.995  1.952  1.976  1.933  1.886
   Adrenals  0.0214  0.0207  0.0178  0.0233  0.0219

** P<0.01 in comparison to control

Table 5: Microscopic pathology - inter-group comparison of tumour incidence

     Dose group (ppm)
 Sex  Type of tumour  0  5  26  132  675
 Animals completed (decedent / terminal)  51 / 49  20 / 30  26 / 24  20 / 30  22 / 28
 Number of tumour bearing animals      21 / 21  10 / 8  11 / 12  4 / 6  7 /13
 Males  Malignant  10 / 6  7 / 3  6 / 5  3 / 4  4 / 4
   Benign  15 / 16  5 / 6  6 / 9  2 / 2  4 / 10
   Multiple  4 / 8  4 / 2  2 / 3  2 / 0  1 / 2
   Single  17 / 13  6 / 6  9 / 9  2 / 6  6 / 11
   Multiple malignant  0 / 0  1 / 0  0 / 0  1 / 0  0 / 1
   Multiple benign  1 / 7  1 / 1  1 / 1  0 / 0  0 / 0
   Metastatic  2 / 0  1 / 0  0 / 1  0 / 0  1 / 1
 Animals completed (decedent / terminal)     58 / 42  28 / 22  32 / 18  36 / 14  24 / 26
 Number of tumour bearing animals     17 / 14  7 / 6  12 / 2  17 / 4  10 / 13
 Female  Malignant  10 / 6  3 / 2  6 / 0  7 / 3  3 / 5
   Benign  8 / 10  5 / 4  9 / 2  12 / 1  7 / 9
   Multiple  1 / 4  4 / 0  4 / 1  4 / 0  1 / 4
   Single  16 / 10  3 / 6  8 / 1  13 / 4  9 / 9
   Multiple malignant  0 / 0  1 / 0  0 / 0  1 / 0  0 / 0
   Multiple benign  0 / 3  2 / 0  1 / 1  1 / 0  1 / 3
   Metastatic  0 / 0  1 / 0  1 / 0  1 / 2  1 / 1

Table 6: Incidence of centrilobular hepatocyte enlargement with or without rarefraction; incidence of adrenal cortical hyperplasia and adrenal cortical tumours (females)

     Dose group (ppm)
 Findings  0  5  26  132  675
 Number of animals examined  100  50  50  50  50
 Liver  Centrolibular hepatocyte enlargement (A)  2  2  0  0  4
   Centrolibular hepatocyte enlargement rarefraction (B)  1  1  1  1  6
   (A) and (B)  2 (3)  3 (6)  1 (2)  1 (2)  10 (20)
 Number of animals examined  99  50  49  49  49
 Adrenals  Cortical hyperplasia  10 (10)  7 (17)  3 (6)  6 (12)  8 (16)
   Cortical tumours  48 (48)  25 (52)  23 (58)  29 (58)  36 (72)

Numbers in brackets are the findings expressed as a percentage of the total number of animals examined.

Conclusions:
Under the conditions of the study, there was no evidence of tumourigenic potential of the test material. Treatment related changes were primarily confined to the high dose level. In the high dose males these changes included initial impairment of body weight gain together with associated lower food consumption, and lower overall body weight change together with the macroscopic pathological finding of a reduction in the adipose tissue in the male decedents. Although there were no pathological findings of clear toxicological importance, the changes in the liver (slightly higher weights, pitted liver, and centrilobular hepatocyte enlargement) and adrenals (cortical hyperplasia) of the high dose females are of note. The NO(A)EL for toxicity has been determined to be 132 mg/kg diet.
Executive summary:

In a GLP compliant study conducted in line with standardised guideline EPA OPP 83-2, the carcinogenicity of the test material over 80 weeks (females) and 88 weeks (males) was determined in the hamster.

Hamsters were administered the test material orally (via diet) at 0, 5, 26, 132 and 675 ppm. There was no evidence of tumourigenic potential of the test material. Treatment related changes were primarily confined to the high dose level; in males these changes included initial impairment of body weight gain together with associated lower food consumption, and lower overall body weight change together with the macroscopic pathological finding of a reduction in the adipose tissue in the male decedents. Although there were no pathological findings of clear toxicological importance, the changes in the liver (slightly higher weights, pitted liver, and centrilobular hepatocyte enlargement) and adrenals (cortical hyperplasia) of the high dose females are of note.

The NO(A)EL (for toxicity) has been determined to be 132 mg/kg diet.

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 February 1989 to 10 February 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPP 83-2 (Carcinogenicity)
Deviations:
yes
Remarks:
(during week 23, a female in group 2 was found to be pregnant and in the cage of a male from group 3. The male was re-housed and food and body weight data for weeks 18-22 excluded. The female was sacrificed and all data from week 18 onwards excluded).
Qualifier:
according to
Guideline:
other: OECD 'Short Term and Long Term Toxicology Group Guidelines' published in the Chemical Regulations Reporter, 14th August 1981
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Requirements for safety evaluation of agricultural chemicals published in 59 Nohsan No. 4200, Ministry of Agriculture, Forestry and Fisheries dated 28th January 1985
Deviations:
not specified
GLP compliance:
yes
Species:
hamster, Syrian
Strain:
other: Bio F1D Alexander
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
91 weeks (males); 78 weeks (females)

Termination was initiated when mortality in the respective control group attained 50 %
Frequency of treatment:
Daily
Dose / conc.:
675 mg/kg diet
Dose / conc.:
1 500 mg/kg diet
Dose / conc.:
3 375 mg/kg diet
No. of animals per sex per dose:
100 (controls); 50 (all treatment groups)
Control animals:
yes, plain diet
Description (incidence and severity):
There were no clinical signs of a reaction considered attributable to treatment throughout the treatment period. There were no apparent treatment-related ante-mortem clinical signs.
Description (incidence):
The administration of the test material was not considered to have affected the distribution or incidence of mortalities in the males. The females of the highest dose group showed a lower mortality than the other groups, possibly as a consequence of their reduced food intake (see Table 1).
Description (incidence and severity):
During the first two weeks of treatment, group mean body weight loss occurred amongst animals receiving 3375 mg/kg diet and males receiving 1500 mg/kg diet, whilst lower weight gain was recorded for all other treatment animals when compared to controls. Lower weight gain persisted up to week 18 for females receiving 3375 mg/kg diet. Subsequent to these initial effects group mean body weight changes were essentially comparable with controls although parity with controls was not regained at any treatment level (see Table 2).
Description (incidence and severity):
Cumulative group mean food intake was slightly but consistently lower than controls for all treated males, although not dose-related in degree, and for females receiving 3375 mg/kg diet throughout the treatment period. Food consumption was also reduced during the first two weeks of treatment for females receiving 1500 mg/kg diet; thereafter being comparable with controls. Food intake of females receiving 675 mg/kg diet was considered to be unaffected by treatment (see Table 3). The efficiency for food utilisation (weeks 1-18) was innpaired for all treated groups during the first two weeks of treatment. During weeks 3 to 18 food utilisation was marginally improved for males receiving 3375 mg/kg diet, but remained impaired for females receiving 3375 mg/kg diet, whilst values for animals receiving 675 or 1500 mg/kg diet were comparable with concurrent controls. The mean achieved intakes over the treatment period were 51, 117 and 277 mg/kg bw/day for males and 55, 121 and 277 mg/kg bw/day for females with both sexes receiving 675, 1500 and 3375 mg/kg diet.
Description (incidence and severity):
Examination of the blood smears for differential white cell counts did not reveal any treatment-related effects.
Description (incidence and severity):
There was a dose-related increase in liver weight for all treated groups; attaining statistical significance (when adjusted for body weight) in all treated female groups and for males at 1500 and 3375 mg/kg diet, when compared with controls. Slightly lower than control testes plus epididymides weights were noted for all treated male groups; attaining statistical significance (when adjusted for body weight) in males at 1500 and 3375 mg/kg diet. A dose-related decrease in heart weights was also noted in all treated groups. However, in the absence of corroborative macropathological significance or micropathological changes these differences were considered attributable to the inter-group differences in body weight. (see Table 4).
Description (incidence and severity):
Macroscopic pathology revealed enlargement of the liver in both sexes receiving 3375 mg/kg diet, together with a slightly increased incidence of liver masses in males receiving the same dose. A slight increase in the incidence of pale areas on the liver together with a reduction in adipose tissue was also noted in all treated male groups. In addition, increased incidences of the following findings were noted in the high dose male group when compared with controls, but were unassociated with any notable microscopic pathological change: decreased size and pallor of the spleen, urinary bladder distension and protrusion of the penis.
Description (incidence and severity):
Non-neoplastic findings considered to be of toxicological relevance were recorded for all treatment levels, and all confinded to the liver (see Table 7).
Description (incidence and severity):
Neoplastic findings during microscopic pathology revealed that treatment resulted in an increased incidence of benign liver cell tumours in males receiving 1500 or 3375 mg/kg diet (see Tables 5 and 6).
No liver cell tumours were reported in females.
Description (incidence and severity):
Several other incidental and spontaneous pathological findings were reported in control and treated animals of both sexes, however were considered to be of no toxicological significance.

No treatment related findings were seen among the factors contributory of death reported in decedent males and females.
Relevance of carcinogenic effects / potential:
Dietary administration of the test material at 675, 1500 and 3375 mg/kg diet for 78 weeks in females and 91 weeks in males resulted in evidence of tumourigenicity amongst males receiving 3375 mg/kg diet manifest as increased incidence of benign liver tumours. A single malignant tumour was also recorded for males at this treatment level, whilst a single benign liver tumour was also recorded amongst males receiving 1500 mg/kg diet. It was considered that the occurrence of tumours amongst males receiving 1500 or 3375 mg/kg diet resulted from the spectrum of toxicologically significant non-neoplastic hepatic changes rather than any direct effect of the test material.
Key result
Dose descriptor:
NOEL
Effect level:
675 mg/kg diet
Based on:
test mat.
Sex:
male
Basis for effect level:
other: equivalent to 51 mg/kg bw/d
Key result
Dose descriptor:
NOEL
Effect level:
3 375 mg/kg diet
Based on:
test mat.
Sex:
female
Basis for effect level:
other: equivalent to 266 mg/kg bw/d

Table 1: Mortality

      Dose group (mg/kg diet)
 Sex  Week  0  675  1500  3375
 Male  5  0/100  0/50  0/50  1/50
   39  2/100  2/50  0/50  0/49
   60  0/98  0/48  0/50  1/49
   70  1/98  1/48  1/50  3/48
   78  5/97  9/47  12/49  10/45
   91  42/92  14/38  14/37  9/35
 Total dead / % mortality    50/50  26/52  27/54  24/48
 Female  5  1/100  0/50  0/50  0/50
   39  2/99  2/50  0/50  1/50
   60  8/97  4/48  4/50  1/49
   70  12/89  6/44  7/46  2/48
   74  11/77  8/38  4/39  2/46
   78  16/66  3/30  6/35  5/44
 Total dead / % mortality    50/50  23/47  21/42  11/22

Table 2: Statistical analysis of body weight gain (g)

       Dose group (mg/kg diet)
 Sex  Week range  0  675  1500  3375
 Male  0-2  5.2  2.1**  -1.3**  -16.7
   2-18  14.8  14.9  13.3  16.5
   18-78  -14.4  -19.5  -16.3  -6.7*
   0-78  5.5  -2.0**  -5.4**  -5.6**
   18-91  -7.7  -14.2  -15.3  -9.7
   0-91  11.8  3.8*  -4.3**  -7.8**
 Female  0-2  5.7  4.5  2.5**  -2.8**
   2-18  17.3  17.2  16.9  14.0*
   18-78  2.0  -6.5  -4.6  5.5
   0-78  25.0  16.5**  15.8**  17.4**

*P<0.05 in comparison to control; **P<0.01 in comparison to control; all week 0-2 value have Kruskal-Wallis analysis of mean ranks applied

Table 3: Statistical analysis on food consumption (g/hamster/period)

     Dose group (mg/kg diet)
 Sex  Week range  0  675  1500  3375
 Male  1-2  134  122**  120**  129**
   3-18  1055  1007**  1014**  941**
   19-78  3970  3709**  3811**  3570**
   1-78  5159  4842**  4947**  4639**
   19-91  4805  4524**  4536**  4377**
   1-91  5990  2654**  5661**  5456**
 Females  1-2  142  138  131**  129**
   3-18  1085  1089  1075  1042**
   19-78  4316  4201  4231  4097**
   1-78  5536  5417  5438  5269**

*P<0.05 in comparison to control; P<0.01 in comparison to control (Williams test); males in weeks 1-2 and 3-18 had Kruskal-Wallis analysis of mean ranks applied; analysis on females weeks 3-18 was performed in log-transformed data

Table 4: Mean group organ weights, absolute (g) during 78/91 week feeding study

     Dose group (mg/kg diet)
 Sex  Organ  0  675  1500  3375
 Male  Brain  1.059/1.049  1.056/1.056  1.032/1.040  1.021/1.032
   Heart  0.940  0.885*  0.855**  0.841**
   Liver  6.33/5.95  6.22/6.17  6.52/6.75  8.54/9.08**
   Kidneys  1.640/1.551  1.581/1.580  1.473/1.548  1.475/1.576
   Adrenals  0.0205  0.0206  0.0216  0.0205
   Testes and epididymides  1.199/1.160  1.126/1.125  0.944/1.026*  0.961/1.004**
 Female  Brain  1.055/1.046  1.048/1.054  1.036/1.042  1.054/1.058
   Heart  0.964  0.916  0.899  0.864**
   Liver  7.70/7.34  7.73/7.98*  8.80/9.02*  11.57/11.70**
   Kidneys  2.041  1.900  1.854  2.095
   Adrenals  0.0184  0.0181  0.0183  0.0181

*P<0.05 in comparison to control; ** P<0.01 in comparison to control (Williams test)

Table 5: Microscopic pathology - inter-group comparison of tumour incidence

     Dose group (mg/kg diet)
 Sex  Type of tumour  0  675  1500  3375
 Animals completed (decedent / terminal)    55 / 45  27 / 23  28 /22  26 /240
 Number of tumor bearing animals    14 / 20  3 / 6  8 /5  9 / 7
 Male  Malignant  5 / 5  1 / 2  1 / 0  3 / 2
   Benign 10 / 15   2 / 4  7 / 5  6 / 6
   Multiple  1 / 2  0 / 0  0 / 0  2 / 3
   Single  13 / 18  3 / 6  8 / 5  7 / 4
   Multiple malignant  0 / 0  0 / 0  0 / 0  1 / 2
   Multiple benign  0 / 2  0 / 0  0 / 0  1 / 2
   Metastatic  0 / 0  0 / 1  1 / 0  0 / 0
 Animals completed (decedent / terminal)    50 / 50  23 / 26  21 / 29  11 / 39
 Number of tumour bearing animals    12 / 18  10 / 7  3 / 6  3 / 9
 Female  Malignant  7 / 7  4 / 3  3 / 1  1 /2
   Benign  5 / 11  6 / 4  0 / 5  3 / 7
   Multiple  1 / 0  1 / 0  0 / 0  1 / 1
   Single  11 / 18  9 / 7  3 / 6  2 / 8
   Multiple malignant  1 / 0  0 / 0  0 / 0  0 / 0
   Multiple benign  0 / 0  1 / 0  0 / 0  0 / 1
   Metastatic  1 / 0  1 / 0  0 / 0  0 / 0

Table 6: Incidence of liver cell tumours in males

   Dose group (mg/kg diet)
   0  675  1500  3375
 Benign liver cell tumour  0  0  1  6
 Malignant liver cell tumour  0  0  0  1
 Total examined  100  50  50  50

Table 7: Microscopic pathology - incidence of liver findings

     Dose group (mg/kg diet)
 Sex  Finding  0  675  1500  3375
 Male  Eosiniophilic hepatocytes  0  0  1  5
   Minimal centrolibular hepatocyte enlargement  7  15  10  14
   Hepatitis  1  1  2  5
   Pigmented giant cells  10  4  5  20
   Pigmented sinusoidal cells  5  1  3  9
   Brown pigment in hepatocytes  9  7  16  19
   Total examined  100  50  50  50
 Female  Minimal centrolibular hepatocyte enlargement  7  5  5  11
   Hepatitis  0  0  3  4
   Pigmented sinusoidal cells  7  4  2  9
   Brown pigment in hepatocytes  1  1  3  10
   Total examined  100  50  50  50
Conclusions:
Under the conditions of the test, dietary administration of the test material at 675, 1500 or 3375 mg/kg diet for 78 weeks in females and 91 weeks in males resulted in evidence of tumourigenicity amongst males receiving 3375 mg/kg diet, manifest as an increased incidence of benign liver tumours. A single malignant tumour was also recorded for males at this treatment level, whilst a single benign liver tumour was also recorded amongst males receiving 1500 mg/kg diet. There was no evidence of tumourigenicity amongst males receiving 675 mg/kg diet or any treated females. Non-neoplastic changes were evident in the liver. It was considered that the occurrence of tumours amongst males receiving 1500 or 3375 mg/kg diet resulted from the spectrum of toxicologically significant non-neoplastic hepatic changes rather than any direct effect of the test material. In addition to hepatotoxicity, evident at all treatment levels, toxicity was also manifest as impaired body weight gain and food intake amongst all treated males and for females receiving 3375 mg/kg diet. Initial impaired body weight gain was also evident for females receiving 1500 mg/kg diet. Thus, 675 mg/kg diet for males (equalling 51 mg/kg bw/day) and 3375 mg/kg diet for females (equalling 277 mg/kg bw/day) was considered to be the no observable effect level in respect of tumourigenicity, however, none of the treatment levels investigated was considered to be a no observable effect level.

Additional data on the incidence of spontaneously occurring tumours in hamsters of the Bio FiD Alexander Syrian strain, supplied by Bio Breeders Inc (USA) were obtained in a separate study. This information was for the provision of background data in support of two previous studies in this strain of test animal, performed at Huntingdon Research Centre Ltd., to assess the potential tumourigenic effects of the test material. Thus, the conditions and procedures used in this study were designed to duplicate those of the aforementioned investigations.
Executive summary:

In a GLP compliant study conducted in line with standardised guideline EPA OPP 83-2, the carcinogenicity of the test material over 78 weeks (females) and 91 weeks (males) was determined in the hamster.

Hamsters were administered the test material by oral (diet) at 0, 675, 1500 and 3375 ppm. Evidence of tumourigenicity amongst males receiving 3375 mg/kg diet, manifest as an increased incidence of benign liver tumours was observed. A single malignant tumour was also recorded for males at this treatment level, whilst a single benign liver tumour was also recorded amongst males receiving 1500 mg/kg diet. There was no evidence of tumourigenicity amongst males receiving 675 mg/kg diet or any treated females. Non-neoplastic changes were evident in the liver. It was considered that the occurrence of tumours amongst males receiving 1500 or 3375 mg/kg diet resulted from the spectrum of toxicologically significant non-neoplastic hepatic changes rather than any direct effect of the test material. In addition to hepatotoxicity, evident at all treatment levels, toxicity was also manifest as impaired body weight gain and food intake amongst all treated males and for females receiving 3375 mg/kg diet. Initial impaired body weight gain was also evident for females receiving 1500 mg/kg diet. Thus, 675 mg/kg diet for males (equalling 51 mg/kg bw/day) and 3375 mg/kg diet for females (equalling 277 mg/kg bw/day) was considered to be the no observable effect level in respect of tumourigenicity, however, none of the treatment levels investigated was considered to be a no observable effect level.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a 110 week feeding study in rats, dose levels of 50, 400 and 3200 mg/kg food did not have an effect upon total tumours observed, a negative trend being apparent in 21/40 tumour types tested. The test material was considered to have no carcinogenic effect with a slight excess of tumours observed only at 3200 mg/kg food which is a notably toxic dose level.

In a 88-week hamster study no evidence of tumourigenic potential was found at any dose levels. Treatment-related changes were primarily confined to the high dose level, i.e. 675 mg/kg food. In the males these changes included initial impairment of body weight gain together with associated lower food consumption, and lower overall body weight change together with the macroscopic pathological finding of a reduction in the adipose tissue in the male decedents. Although there were no pathological findings of clear toxicological importance, the changes in the liver (slightly higher weights, pitted liver and centrilobular hepatocyte enlargement) and adrenals (cortical hyperplasia) of the high dose females are of note. The dose level of 132 mg/kg diet, equal to 9.39 mg/kg bw for males and 9.20 mg/kg bw for females, should be regarded as the NO(A)EL for toxicity.

In a second study with hamsters at dose levels of 675, 1500 or 3,75 mg/kg diet for 78 weeks in females and 91 weeks in males it was confirmed that 675 mg/kg diet is a Maximum Tolerated Dose for the test material in this species. This was evidenced by hepatotoxicity at all treatment levels and was also manifest as impaired body weight gain and food intake amongst all treated males and for females receiving 3375 mg/kg diet. Initial impaired body weight gain was also evident for females receiving 1500 mg/kg diet. There was no evidence of tumourigenicity amongst males receiving 675 mg/kg diet or any treated females. The occurrence of tumours amongst males receiving 1500 or 3375 mg/kg diet resulted from the spectrum of toxicologically significant non-neoplastic hepatic changes rather than any direct effect of the test material. Thus, 675 mg/kg diet for males (equalling 51 mg/kg bw/day) and 3375 mg/kg diet for females (equalling 277 mg/kg bw/day) was considered to be the no observable effect level in respect of tumourigenicity. However, none of the treatment levels investigated in this study was considered to be a no observable effect level.

Two additional in vitro screening studies performed with BALB/c-3T3 demonstrated that the test material did not transform BALB/c-3T3 cells both in the presence and absence of metabolic activation. The methods performed in both screens were almost identical, except that one was performed with metabolic activation (with a shorter incubation time) and the other performed in the absence of metabolic activation (with a longer incubation time). In both studies, After an initial preliminary toxicity screening test, tissue cultures were treated with doses of 667.0, 2667, 4000.0, 6000.0 and 7,500.0 µg/mL in the presence of metabolic activation and 240, 480, 720, 900 and 1125 µg/mL in the absence of metabolic activation administered in media. After sufficient incubation, the cell monolayers were assessed for foci and compared with negative and positive control cultures. The test material, did not induce a significant number of transformed foci in either of the two screens. The results of both screening studies are in accordance with the in vivo carcinogenicity data.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substancec is considered to be unclassified for carcinogenicity.