Registration Dossier

Toxicological information

Carcinogenicity

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Administrative data

Description of key information

Based on the GLP compliant OECD TG 453 study, no excess tumours occurred at any dose level, therefore there was no indication of a carcinogenic potential.

All available data were assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not specified
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
1981
Qualifier:
according to
Guideline:
EPA OPP 83-5 (Combined Chronic Toxicity / Carcinogenicity)
Version / remarks:
1984
Qualifier:
according to
Guideline:
other: J-MAFF
Version / remarks:
1985
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Tif:RAIf (SPF), hybrids of RII/1 x RII/2 (Sprague-Dawley derived)
Details on species / strain selection:
The rat was selected as the test species as it is recognized by international guidelines as a preferred test species. The number of animals used was considered to be the minimum required to meet the scientific objectives of the study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 4-5 weeks
- Weight at study initiation: 134.7 to 224.7 g (males), 114.2 to 175.0 g (females)
- Housing: Carcinogenicity animals (50/sex/group) housed 5/cage in Macrolon type 4 cages. Remaining animals (30/sex/group) housed individually in Macrolon type 3 cages.
- Diet: Pelleted, certified standard diet (Nafag No. 8900 for GLP) ad libitum (except overnight prior to blood collection)
- Water: Drinking water ad libitum (except overnight during urine collection)
- Acclimation period: 11 days males; 10 days females

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55 ± 10%
- Air changes: 16-20 per hour
- Photoperiod: 12 hours light, 12 hours dark

IN-LIFE DATES: : 07 Aug 1995, End: 21 Aug 1997
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION AND ANALYSIS:
- The test substance was administered by mixture in Nafag No. 8900 FOR GLP diet.
- Fresh diets were prepared at four week intervals and stored in stainless steel containers at room temperature.
- The correct amount of the test substance was added to pulverised diet and homogeneously mixed together.
- Approximately 25% water was added before pelleting and the pellets subsequently air-dried.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Prepared pelleted samples of diet were analysed at the start of the study for analysis of stability and homogeneity. Samples were analysed periodically throughout the study for analysis of achieved concentration.
- Concentration analysis results: The mean test substance concentrations were 89.3-116.9% of nominal.
- Homogeneity results: Homogeneity varied between 93 and 101% of the nominal concentrations.
- Stability results: The test substance was stable in rodent diet at room temperature (22 ± 2°C). Concentrations were 96-103% of nominal after 5 weeks and 99-112% of nominal after 7 weeks.
Duration of treatment / exposure:
104 weeks
Frequency of treatment:
continuously
Dose / conc.:
10 ppm
Remarks:
Equivalent to 0.41 and 0.48 mg/kg bw/day for males and females, respectively
Dose / conc.:
30 ppm
Remarks:
Equivalent to 1.29 and 1.56 mg/kg bw/day for males and females, respectively
Dose / conc.:
500 ppm
Remarks:
Equivalent to 21.0 mg/kg bw/day for males.
Dose / conc.:
1 000 ppm
Remarks:
Equivalent to 50.3 mg/kg bw/day for females.
Dose / conc.:
1 500 ppm
Remarks:
Equivalent to 63.0 mg/kg bw/day for males.
Dose / conc.:
3 000 ppm
Remarks:
Equivalent to 155.0 mg/kg bw/day for females.
No. of animals per sex per dose:
80
Control animals:
yes, plain diet
Details on study design:
- Rationale for dose selection: Dose levels were based on the results of a 3 month dietary toxicity study in rats.
- Treatment: Groups of 80 male and 80 female Sprague-Dawley rats (strain Tif: RAIf, SPF) were administered the test substance (purity 98.6%) orally for 104 weeks. Twenty animals/sex/group (group II and III) were designated for clinical laboratory investigations and 50 animals/sex/group for the oncogenicity study (group I). Ten animals per sex and dose group were terminated after 12 months (Group IV). See Table 1 in "Any other information on methods and materials incl. tables" for more information.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
All animals were checked daily for mortality (am and pm weekdays, am only weekends and holidays).

DETAILED CLINICAL OBSERVATIONS
All animals were checked daily for signs of toxicity and findings were recorded at least once per week.

BODY WEIGHT
The body weight of each animal was recorded predose, weekly for 13 weeks and monthly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption for each cage was recorded weekly for 13 weeks and monthly thereafter. Food consumption ratios were calculated as g food/kg body weight/day. Test substance intake was calculated per group.

WATER CONSUMPTION
Water consumption for each cage was recorded monthly.

OPHTHALMOSCOPIC EXAMINATION
All animals of the carcinogenicity group were examined prior to dosing and at week 104. High dose and control carcinogenicity animals were examined after 6 and 12 months.

HAEMATOLOGY
- Haematology investigations were carried out on surviving animals of experimental groups II and III during weeks 13, 27, 53, 78 and 105. For terminal investigations at week 105 the animals were supplemented by animals of Group I to ensure 20 samples/sex/dose level for haematology and by animals of group III to ensure 10 samples/sex/group. The animals were fasted overnight prior to blood collection. Blood samples were collected under ether anaesthesia via the orbital sinus.
- The following parameters were measured: haemoglobin, mean cell haemoglobin concentration, haematocrit, haemoglobin concentration distribution width, red blood cell count, total white cell count, mean cell volume, differential white cell count, mean cell volume distribution width, thrombocyte count, mean cell haemoglobin, prothrombin time

CLINICAL CHEMISTRY
- Clinical chemistry investigations were carried out on surviving animals of experimental group II during weeks 13, 27, 53, 78 and 105. For terminal investigations at week 105 the animals were supplemented by animals of Group III to ensure 10 samples/sex/group. The animals were fasted overnight prior to blood collection. Blood samples were collected under ether anaesthesia via the orbital sinus.
- The following parameters were measured: urea, alkaline phosphatase activity, creatinine, aspartate aminotransferase activity, glucose, alanine aminotransferase activity, albumin, gamma-glutamyl transpeptidase activity, globulin, calcium, A/G ratio, chloride, total protein, sodium, cholesterol, potassium, total bilirubin, inorganic phosphorus, triglycerides

URINALYSIS
- Urinalysis carried out on surviving animals of experimental group II during weeks 13, 27, 53, 78 and 105. For terminal investigations at week 105 the animals were supplemented by animals of Group III to ensure 10 samples/sex/group. Urine was collected overnight and the individual animals were held in metabolism cages and food and water were withheld.
- The following parameters were measured: volume, glucose, colour, ketones, relative density, protein, pH, bilirubin, urobilinogen, blood
- In addition the spun deposit was stained and examined microscopically.
Sacrifice and pathology:
MACROSCOPIC EXAMINATION
At the end of the study, all control and treated animals were exsanguinated under ether anaesthesia and examined post mortem. This involved an external observation and an internal examination of all organs and structures. A complete necropsy with tissue preservation was performed on one 1250 ppm female which died on day 57.

ORGAN WEIGHTS
At necropsy, the following organs were removed, trimmed free of extraneous tissue and weighed: adrenal glands, ovaries, brain, spleen, heart, thyroid, kidneys, testes, liver, parathyroid

TISSUE SUBMISSION
The following tissues were removed and examined and fixed in an appropriate fixative: gross lesions including masses, ovary, adrenal gland, pancreas, aorta, peripheral nerve (sciatic), brain, pituitary gland, caecum, prostate gland, colon, rectum, duodenum, salivary gland (submandibular), epididymis, seminal vesicle, eyes (with optic nerve), skeletal muscle, extra-orbital lachrymal gland, spinal cord (cervical, thoracic, lumbar), femur (including joint), skin, heart, spleen, ileum, sternum with bone marrow, jejunum, stomach, kidney, testis, liver, thymus, lung, thyroid gland with parathyroid, lymph node - axillary, tongue, lymph node - mesenteric, trachea, mammary area, urinary bladder, muzzle, uterus, oesophagus, vagina, orbital gland, Zymbal gland

MICROSCOPIC EXAMINATION
All tissues listed above from all animals except the 20/sex/group designated for clinical pathology, were sectioned, stained and examined by light microscopy.
Statistics:
For body weight, food and water consumption, laboratory and organ weight data univariate statistical analyses were performed at each time point. Nonparametric methods were applied to allow for non normal as well as normal data distribution. Each treated group was compared to the control group by either Lepage’s or Wilcoxon’s two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere’s test for ordered alternatives. Survival analysis was performed by Cox’s regression model. Analysis of pathology data used SAS procedure MULTITEST. Neoplastic lesions were analysed by Peto’s mortality-prevalence test, non-neoplastic lesions by Cochran-Armitage’s linear trend test.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no treatment-related effects on mortality.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gains were significantly reduced in females at 3000 ppm. Weight gains of all male groups and female groups at up to 1000 ppm were unaffected by treatment. See Table 1 in "Any other information on results incl. tables" for more information.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- There were no effects on food consumption.
- Dose rates (based on analysed dietary levels of the test substance) were calculated in terms of mg test substance/kg body weight. Mean values calculated as a time-weighted average are shown below in Table 2 in "Any other information on results incl. tables".

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Mean water intake (weeks 1-102) was 13% higher than control in males in the 1500 ppm group.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related findings.

Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in haematology parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in blood chemistry parameters.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in urinalysis parameters.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects on organ weights at any dose level, at either 52 or 104 weeks. There were no significant differences from control values in absolute and relative organ weights among the males, with the exception of a minor negative trend in the relative thyroid weight of males at 1500 ppm. The trend was considered not to be of toxicological significance because the mean value was similar to historical control values and no related histological changes were evident. Mean absolute adrenal weights appeared elevated in males at 10 and 1500 ppm, but this difference was no longer apparent when animals with extremely high values were excluded. Female mean relative thyroid weights were higher than control values in all treated groups, but remained within the historical control range. The differences were judged to be without toxicological significance. The differences in adrenal weights between control and 10 and 1000 ppm females did not persist when animals with extreme values were excluded.
See Table 3 in "Any other information on results incl. tables" for more information.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 52 weeks: Treatment-related lesions at 52 weeks were increased incidences of renal tubular regenerative changes, chronic tubular lesion and tubular basophilic proliferation, in males at 500 and 1500 ppm. Minimally increased incidences of renal tubular and pelvic lymphocytic infiltration also occurred at 1500 ppm, but without tubular hyaline change. These alterations are considered to represent the sequelae of alpha-2µ-globulin mediated nephropathy. The kidneys of females at all dose levels were unaffected. There was a minimal increase in the severity of splenic haemosiderosis in females at 3000 ppm. In the liver, there were increased incidences of cholangiofibrosis in males at 10 ppm (5/10), 500 ppm (6/10) and 1500 ppm (4/10) compared to the controls (2/10), and increased incidence of inflammatory cell infiltration in males at 10 ppm (4/10), 30 ppm (3/10) and 500 ppm (5/10) relative to controls (1/10). These findings, which were not apparent after 104 weeks treatment, were considered incidental to treatment because of the lack of a dose relationship. Other non-neoplastic lesions occurred in animals that died during the first year or that were sacrificed at 52 weeks, but all commonly occur in this strain of rat, and the incidence, distribution, and/or morphology did not indicate an effect of treatment. See Table 4 in "Any other information on results incl. tables" for more information.
- 104 weeks: Treatment-related, non-neoplastic changes in the kidneys and liver occurred in animals sacrificed at 104 weeks or that died between weeks 53 and 104. In the kidneys, a slightly increased incidence of slight/moderate chronic nephropathy occurred in males at 1500 ppm accompanied by a minimal increase in incidence of lymphocytic infiltration of the renal cortex at 1500 ppm. Two animals also showed tubular hyaline change. In the liver, a treatment-related increase occurred in the incidence of slight/moderate focal cellular alteration, generally of the clear cell subtype, in females at 3000 ppm. See Table 5 in "Any other information on results incl. tables" for more information.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- 52 weeks: No treatment-related neoplastic lesions occurred in animals sacrificed at 52 weeks or that died during the first 52 weeks.
- 104 weeks: All neoplastic findings, both malignant and benign, occurring at 104 weeks were considered incidental to treatment with the test substance since the incidences in treated and control groups are similar, or the inter-group distribution shows no relationship to dose level, or the incidences are within historical control ranges and the lesions are known to occur spontaneously in aged rats.
Key result
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
>= 63 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: neoplastic
Dose descriptor:
NOAEL
Remarks:
carcinogenicity
Effect level:
>= 155 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: neoplastic
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
50.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
63 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOEL
Remarks:
systemic toxicity
Effect level:
1.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
155 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
155 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
21.3 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Table 1: Intergroup summary of cumulative body weight gain (g/animal) - selected time points

 

Week

Dietary concentration (ppm)

Males

Females

0

10

30

500

1500

0

10

30

1000

3000

12

263.1

261.4
(-0.6%)

266.2
(+1.2%)

266.7
(+1.4%)

264.2
(+0.4%)

131.0

133.0
(+1.5%)

129.9
(-0.8%)

130.9
(-0.1%)

121.5
(-7.3%)

54

430.3

438.5
(+1.9%)

443.2
(+3.0%)

458.1
(+6.5%)

466.8
(+8.5%)

226.3

234.5
(+3.6%)

223.7
(-1.1%)

232.4
(+2.7%)

197.5
(-12.7%)

103

479.8

484.3
(+0.9)

473.2
(-1.4%)

467.8
(-2.5%)

472.0
(-1.6%)

284.7

291.4
(+2.4%)

281.5
(-1.1%)

299.5
(+5.2)

248.8
(-12.6)

Differences from control are shown in parentheses

  

Table 2: Mean Dose Received (mg/kg/day)

Test substance (ppm)

10

30

500

1500

Males

0.41

1.29

21.0

63.0

Test substance (ppm)

10

30

1000

3000

Females

0.48

1.56

50.3

155

Dose rates (based on analysed dietary levels of the test substance) were calculated in terms of mg/kg body weight

  

Table 3: Organ weights

Organ

Males

Females

 

 

Week 53

Week 105

 

Week 53

 

Week 105

 

 

Dose [ppm]

Absolute weight [g]

Relative weight [%]

Absolute weight [g]

Relative weight [%]

Dose [ppm]

Absolute weight [g]

Relative weight [%]

Absolute weight [g]

Relative weight [%]

Liver (g)

0

21.23

35.89

21.98

35.02

0

11.69

33.78

15.55

38.75

 

10

18.16

31.25

22.27

35.53

10

10.54

33.25

15.74

38.46

 

30

18.28

33.08

22.04

36.06

30

11.67

33.09

15.58

39.31

 

500

20.62

33.10

22.74

37.61

1000

10.63

32.95

16.24

39.85

 

1500

19.72

35.63

22.80

37.88

3000

11.25

34.66

15.38

41.72+

Adrenals (mg)

0

70.17

0.12

112.7

0.18

0

69.65

0.20

87.3

0.22

 

10

65.19

0.12

322.7

0.56

10

73.84

0.23

107.0

0.27*+

 

30

66.53

0.12

105.1

0.18

30

73.99

0.21

95.8

0.25

 

500

70.27

0.11

169.1

0.32

1000

73.33

0.23

105.6*+

0.26*+

 

1500

73.71

0.14

251.8

0.48

3000

75.44

0.24

88.5

0.25*

Thyroids (mg)

0

30.20

0.05

111.3

0.17

0

37.30

0.11

45.59

0.11

 

10

32.34

0.06

73.8

0.12

10

33.99

0.11

49.50+

0.12

 

30

33.17

0.06

63.4

0.10

30

35.97

0.10

48.67

0.12+

 

500

30.67

0.05

69.4

0.12

1000

33.32

0.11

60.80*+

0.15*+

 

1500

34.22

0.06

61.7

0.10+

3000

36.32

0.11

49.82

0.14*+

* p≤ 0.01,Le Page test; +/- p ≤ 0.01 (Jonckheere)

  

Table 4: Incidence of non-neoplastic, treatment-related lesions at 52 weeks

 

 

Males

Females

Dose Level [ppm]

0

10

30

500

1500

0

10

30

1000

3000

Kidneys

No. exam.

10

10

10

10

10

10

10

10

10

10

chronic tubular lesion

 

2

2

2

4

6

1

1

1

2

0

tubular basophilic proliferation

 

0

1

0

2

1

0

0

2

0

0

total regenerative tubular lesions

 

 

2

 

3

 

2

 

6

 

7

 

1

 

1

 

3

 

2

 

0

lymphocytic infiltration

 

1

1

0

2

3

0

2

0

0

0

Renal Pelves

No. exam.

10

10

10

10

10

10

10

10

10

10

lymphocytic infiltration

 

1

0

0

1

3

0

1

0

0

0

Spleen

No. exam.

10

10

10

10

10

10

10

10

10

10

haemosiderosis

 

9

8

9

9

8

10

10

10

9

10

average grade

 

2.3

2.4

2.7

2.2

2.5

2.4

2.5

2.5

2.3

3.0

 

 

Table 5: Incidence of non-neoplastic, treatment-related lesions at 104 weeks

 

 

Males

Females

Dose Level [ppm]

0

10

30

500

1500

0

10

30

1000

3000

Kidneys

No. exam.

50

50

49

50

50

50

50

49

50

50

lymphocytic infiltration.

 

10

10

7

14

17

2

3

4

2

2

average grade

 

1.6

1.7

1.4

1.9

1.7

1.5

2.0

1.5

1.5

2.0

chronic nephropathy

 

30

35

32

37

42

12

10

8

6

10

average grade

 

2.4

2.6

2.4

2.5

2.7

2.8

2.9

2.0

2.2

2.5

tubular hyaline change

 

0

1

1

0

2

2

1

0

0

0

Liver

No. exam

50

50

49

50

50

50

49

49

50

50

focus of cellular alteration

20

21

15

21

20

10

21

12

15

26

average grade

 

2.2

2.3

1.6

2.0

2.0

1.9

1.6

1.5

1.4

2.1

amphophilic

 

3

3

1

5

1

3

2

1

1

3

basophilic

 

1

2

0

2

0

4

6

5

5

6

clear cell

 

13

10

11

11

14

3

5

2

5

15

eosinophilic

 

3

5

2

3

5

0

8

4

4

2

mixed

 

0

1

1

0

0

0

0

0

0

0

Conclusions:
Based on the GLP compliant OECD TG 453 study, no excess tumours occurred at any dose level, therefore there was no indication of a carcinogenic potential
Executive summary:

Groups of 70 male and 70 female Sprague-Dawley rats (strain Tif: RAIf, SPF) were administered with the test substance (purity 98.6%) orally for 104 weeks, by admixture in the diet, at concentrations of 0, 10, 30, 500 or 1500 ppm for males (0, 0.41, 1.29, 21.0 and 63.0 mg/kg bw/day) and 0, 10, 30, 1000 or 3000 ppm for females (0, 0.48, 1.56, 50.3 and 155 mg/kg bw/day). Twenty animals/sex/group were designated for clinical laboratory investigations and 50 animals/sex/group for the oncogenicity study. Additional groups of 10 animals/sex/group were similarly treated with the test substance for 52 weeks and sacrificed for interim evaluation. Clinical observations were made daily, body weights and food consumption recorded weekly for 13 weeks and monthly thereafter, and water consumption recorded monthly. The eyes of all animals in the carcinogenicity subgroup were examined pre-test and at 104 weeks. Control and high-dose animals were also examined at weeks 26, 52 and 77. Laboratory investigations were performed at weeks 13, 27, 53, 78 and prior to termination on 20 animals/sex/group for haematology and on 10 animals/sex/group for clinical chemistry and urinalysis. All animals were subjected to detailed necropsy and post mortem examination. Organ weights of all animals which survived until terminal sacrifice were recorded. Tissue/organ samples from all animals were preserved. Microscopic examination of tissues was performed on all animals, of all treatment and control groups, with the exception of the 20 animals/sex/group designated for laboratory investigations. Survival incidence was not affected by treatment, there were no clinical signs of an adverse effect of treatment and the incidences of single and multiple palpable masses were unaffected by treatment at all dose levels. Body weight development in all male groups was unaffected by treatment, but females at 3000 ppm had slightly depressed weight gain from week 3 until termination, at which time cumulative weight gain was depressed by 12.6%. Food consumption was unaffected by treatment in both sexes at all dose levels, but water consumption of males at 1500 ppm only was raised by 13%. Ophthalmological examinations revealed no evidence of ocular toxicity at any time point. There were no treatment-related changes in haematology, blood chemistry and urinalysis parameters. A myeloid leukaemia was identified in one male at 1500 ppm in week 53, but this occurs spontaneously at low incidence in rats of this source and strain, and was considered unrelated to treatment. There were no treatment-related changes in haematology, blood chemistry and urinalysis parameters. There were no treatment-related effects on organ weights. Post mortem examination of animals sacrificed after 52 and 104 weeks, and animals that died spontaneously between 52 and 104 weeks, revealed no treatment-related lesions. No treatment-related neoplastic lesions occurred in animals sacrificed at 52 or 104 weeks or that died during the study. Non-neoplastic, treatment-related lesions at 52 weeks were increased incidences of renal tubular regenerative changes, chronic tubular lesion and tubular basophilic proliferation, in males at 500 and 1500 ppm. Minimally increased incidences of renal tubular and pelvic lymphocytic infiltration also occurred at 1500 ppm, but without tubular hyaline change. These alterations were considered to represent the sequelae of alpha-2m-globulin mediated nephropathy. The kidneys of females at all dose levels were unaffected. There was a minimal increase in the severity of splenic haemosiderosis in females at 3000 ppm. In the liver, there were increased incidences of cholangiofibrosis in males at 10 ppm (5/10), 500 ppm (6/10) and 1500 ppm (4/10) compared to the controls (2/10), and increased incidence of inflammatory cell infiltration in males at 10 ppm (4/10), 30 ppm (3/10) and 500 ppm (5/10) relative to controls (1/10). These findings, which were not apparent after 104 weeks treatment, are considered incidental to treatment because of the lack of a dose relationship. Treatment-related, non-neoplastic changes in the kidneys and liver occurred in animals sacrificed at 104 weeks or that died between weeks 53 and 104. In the kidneys, a slightly increased incidence of slight/moderate chronic nephropathy occurred in males at 1500 ppm accompanied by a minimal increase in incidence of lymphocytic infiltration of the renal cortex at 1500 ppm. Two animals also showed tubular hyaline change. In the liver, a treatment-related increase occurred in the incidence of slight/moderate focal cellular alteration, generally of the clear cell subtype, in females at 3000 ppm. Based on no excess tumours occurring at any dose level, there was no indication of a carcinogenic potential. The no-observed-effect-levels (NOEL) for toxicity were 30 ppm (males) and 1000 ppm (females), equivalent to mean dose levels of 1.3 mg/kg bw/day (males) and 50.3 mg/kg bw/day (females), based on increased incidence of renal chronic tubular lesions and basophilic proliferation in males at ≥500 ppm, and foci of cellular alteration in the liver, and increased severity of splenic haemosiderosis, in females at 3000 ppm. Since the renal alterations observed in males at 500 and 1500 ppm are considered to be mediated by alpha-2µ-globulin, a mechanism specific to male rats, the observed renal toxicity has no relevance to human risk assessment. Therefore, it is concluded that a no-observed-adverse-effect-level (NOAEL) of >1500 ppm, equivalent to a dose level of >63.0 mg/kg bw/day, is applicable for males. Therefore, for both sexes, the overall NOAEL of relevance to human risk assessment is based on the female NOAEL of 50.3 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50.3 mg/kg bw/day
Study duration:
chronic
Species:
rat

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

24-Month carcinogenicity study

In a GLP compliant study (Bachmann, 1998), performed in accordance with OECD TG 453, groups of 70 male and 70 female Sprague-Dawley rats (strain Tif: RAIf, SPF) were administered with the test substance (purity 98.6%) orally for 104 weeks, by admixture in the diet, at concentrations of 0, 10, 30, 500 or 1500 ppm for males (0, 0.41, 1.29, 21.0 and 63.0 mg/kg bw/day) and 0, 10, 30, 1000 or 3000 ppm for females (0, 0.48, 1.56, 50.3 and 155 mg/kg bw/day). Twenty animals/sex/group were designated for clinical laboratory investigations and 50 animals/sex/group for the oncogenicity study. Additional groups of 10 animals/sex/group were similarly treated with the test substance for 52 weeks and sacrificed for interim evaluation. Clinical observations were made daily, body weights and food consumption recorded weekly for 13 weeks and monthly thereafter, and water consumption recorded monthly. The eyes of all animals in the carcinogenicity subgroup were examined pre-test and at 104 weeks. Control and high-dose animals were also examined at weeks 26, 52 and 77. Laboratory investigations were performed at weeks 13, 27, 53, 78 and prior to termination on 20 animals/sex/group for haematology and on 10 animals/sex/group for clinical chemistry and urinalysis. All animals were subjected to detailed necropsy and post mortem examination. Organ weights of all animals which survived until terminal sacrifice were recorded. Tissue/organ samples from all animals were preserved. Microscopic examination of tissues was performed on all animals, of all treatment and control groups, with the exception of the 20 animals/sex/group designated for laboratory investigations. Survival incidence was not affected by treatment, there were no clinical signs of an adverse effect of treatment and the incidences of single and multiple palpable masses were unaffected by treatment at all dose levels. Body weight development in all male groups was unaffected by treatment, but females at 3000 ppm had slightly depressed weight gain from week 3 until termination, at which time cumulative weight gain was depressed by 12.6%. Food consumption was unaffected by treatment in both sexes at all dose levels, but water consumption of males at 1500 ppm only was raised by 13%. Ophthalmological examinations revealed no evidence of ocular toxicity at any time point. There were no treatment-related changes in haematology, blood chemistry and urinalysis parameters. A myeloid leukaemia was identified in one male at 1500 ppm in week 53, but this occurs spontaneously at low incidence in rats of this source and strain, and was considered unrelated to treatment. There were no treatment-related changes in haematology, blood chemistry and urinalysis parameters. There were no treatment-related effects on organ weights. Post mortem examination of animals sacrificed after 52 and 104 weeks, and animals that died spontaneously between 52 and 104 weeks, revealed no treatment-related lesions. No treatment-related neoplastic lesions occurred in animals sacrificed at 52 or 104 weeks or that died during the study. Non-neoplastic, treatment-related lesions at 52 weeks were increased incidences of renal tubular regenerative changes, chronic tubular lesion and tubular basophilic proliferation, in males at 500 and 1500 ppm. Minimally increased incidences of renal tubular and pelvic lymphocytic infiltration also occurred at 1500 ppm, but without tubular hyaline change. These alterations were considered to represent the sequelae of alpha-2µ-globulin mediated nephropathy. The kidneys of females at all dose levels were unaffected. There was a minimal increase in the severity of splenic haemosiderosis in females at 3000 ppm. In the liver, there were increased incidences of cholangiofibrosis in males at 10 ppm (5/10), 500 ppm (6/10) and 1500 ppm (4/10) compared to the controls (2/10), and increased incidence of inflammatory cell infiltration in males at 10 ppm (4/10), 30 ppm (3/10) and 500 ppm (5/10) relative to controls (1/10). These findings, which were not apparent after 104 weeks treatment, are considered incidental to treatment because of the lack of a dose relationship. Treatment-related, non-neoplastic changes in the kidneys and liver occurred in animals sacrificed at 104 weeks or that died between weeks 53 and 104. In the kidneys, a slightly increased incidence of slight/moderate chronic nephropathy occurred in males at 1500 ppm accompanied by a minimal increase in incidence of lymphocytic infiltration of the renal cortex at 1500 ppm. Two animals also showed tubular hyaline change. In the liver, a treatment-related increase occurred in the incidence of slight/moderate focal cellular alteration, generally of the clear cell subtype, in females at 3000 ppm. Based on no excess tumours occurring at any dose level, there was no indication of a carcinogenic potential. The no-observed-effect-levels (NOEL) for toxicity were 30 ppm (males) and 1000 ppm (females), equivalent to mean dose levels of 1.3 mg/kg bw/day (males) and 50.3 mg/kg bw/day (females), based on increased incidence of renal chronic tubular lesions and basophilic proliferation in males at ≥500 ppm, and foci of cellular alteration in the liver, and increased severity of splenic haemosiderosis, in females at 3000 ppm. Since the renal alterations observed in males at 500 and 1500 ppm are considered to be mediated by alpha-2µ-globulin, a mechanism specific to male rats, the observed renal toxicity has no relevance to human risk assessment. Therefore, it is concluded that a no-observed-adverse-effect-level (NOAEL) of >1500 ppm, equivalent to a dose level of >63.0 mg/kg bw/day, is applicable for males. Therefore, for both sexes, the overall NOAEL of relevance to human risk assessment is based on the female NOAEL of 50.3 mg/kg bw/day.

Evaluation of the Human Health Relevance of Test substance-Related Mouse Liver Tumours

The test substance was shown to increase the incidence of mouse liver tumours in an 18-month study (Bachmann 1998a); however, the test substance was not hepatocarcinogenic in rats. The test substance is not genotoxic, and, given the late life generation of mouse liver tumours, suggests a time-related progression of key hepatic events that leads to the tumours. These key events were identified in a series of studies of up to 50 weeks that showed the time-dependent evolution of relatively mild liver dysfunction within 10 weeks of dosing, followed by frank signs of hepatotoxicity after 20 weeks, leading to cellular attrition and regenerative hyperplasia (see below). A metabolite, metabolite 3, was identified as generating the mild hepatic toxicity, and another metabolite, metabolite 2, exacerbated the initial toxicity by inhibiting inducible nitric oxide synthase. This combination of metabolite generated hepatotoxicity and increase in cell replication rates is postulated as the mode of action for the test substance-related mouse liver tumours. The relevance of these mouse-specific tumours to human health was assessed by using the framework and decision logic developed by ILSI-RSI. The postulated mode of action was tested against the Hill criteria and found to fulfil the comprehensive requirements of strength, consistency, specificity, temporality, dose-response, and the collective criteria of being a plausible mode of action that fits with known and similar modes of action. Whereas the postulated mode of action could theoretically operate in human liver, quantitation of the key metabolites in vivo and in vitro showed that mice, but not rats or humans, generate sufficient amounts of these metabolites to initiate the hepatic toxicity and consequent tumours. Indeed, rats fed 3000 ppm test substance for a lifetime did not develop hepatotoxicity or tumours. In conclusion, the coherence and extent of the database clearly demonstrates the mode of action for mouse liver tumorigenesis and also allows for the conclusion that the test substance does not pose a carcinogenic risk to humans.

In vitro and in vivo liver investigations in rats and mice

In the absence of cytotoxicity in rat and mouse primary hepatocytes in vitro (Bouis P, 1997), in in vivo oral toxicity studies with the test substance in the mouse, the liver was identified as the main target organ. In contrast, following oral administration to rats there was no indication of any effects on the liver (Noakes J, 2003b and Waechter F, 2003).

The test substance induces a number of changes in mouse liver which are believed to be key events in the development of the liver cancer seen in an 18 month feeding study. These include reductions in plasma cholesterol, single cell necrosis, increased apoptosis and increased cell replication rates (Persohn E, 1995; Weber E, 1999b; Weber E 2003; Soames T, 2003 and Green T, 2003a). When the 3 major metabolites were tested in feeding studies of up to 20 weeks duration, only 1 metabolite induced the same changes in mouse liver as the test substance. It is concluded that this metabolite has a key role in the development of the liver tumours seen in mice fed on diets containing the test substance since this is the only major metabolite identified which occurs in significantly greater quantities in mice than in rats and is unique to the test substance (Green T, 2003e and Noakes J, 2003a). The differences in production of this metabolite between rats and mice are consistent with the species differences in carcinogenicity (Green T, 2003c). The rates for each of the metabolic transformations of the test substance by its major pathways were significantly lower in human liver than in mouse liver and data suggest that metabolism of the test substance by these pathways in humans would be minimal at expected exposure levels (Green T, 2002).

Weaning mice were at least 2-fold less susceptible to the cholesterol lowering effects of the test substance than adult mice. Based on these animal studies there is no evidence to suggest that infants and children would be more susceptible than adults to the hepatotoxic effects of the test substance (Green T, 2003b).

Other non-key events consisted of induction of liver metabolising enzymes (Trendelenburg C, 1998), glycogenosis/fatty change and lipogenic pigmentation (Weber E, 1998). Transient cytoplasmic condensation of mostly periportal hepatocytes and suppression of the naturally high hepatocellular mitotic activity (Weber E, 1999a) and reduction in nitric oxide levels through inhibition of iNOS (Green T, 2003d).

Based on the differences in metabolism in human and mouse liver, the carcinogenicity observed in mice following oral treatment with the test substance is considered not relevant for the human situation and the risk assessment.

The oxadiazinane moiety of the test substance provides a slow release reservoir for the production of HCHO and potentially N-methylols as candidate hepatotoxicants and hepatocarcinogens with higher yields in mice than rats or humans. Final products of the test substance-oxadiazinane moiety metabolism are CLO and dm-test substance acting as nicotinic acetylcholine receptor agonists and insecticides (Nauen et al., 2003; Swenson T, 2013).

A systematic analysis of new in vitro human NR activity data on 309 environmental chemicals in relationship to their liver cancer-related chronic outcomes in rodents was conducted. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. The test substance was assigned to nuclear receptor group G. NRG G contains 96 chemicals with no activity on the nuclear receptors for RXR, SR, PPAR, PXR, CAR or AhR. The test substance was assigned to lesion progression group III (of VII) i.e. a group with intermediate promiscuity and potency (where group I has the highest) (Shah I, 2011).

Justification for classification or non-classification

Based on the available information, classification for carcinogenicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.