cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Carcinogenicity

Description of key information

- The NOAEL for carcinogenicity and toxicity was established at 15 ppm (1.84 mg/kg bw/day and 2.56 mg/kg bw/day in male and female mice, respectively), based on incidence of hepatologic neoplasia (combined carcinomas and adenomas); OECD 451, Warren 1989

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:

29 Oct 1986 to 22 Jul 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

1981

Deviations:

yes

Qualifier:

according to guideline

Guideline:

EPA OPP 83-2 (Carcinogenicity)

Version / remarks:

1982

Qualifier:

according to guideline

Guideline:

EU Method B.32 (Carcinogenicity Test)

Version / remarks:

1988

GLP compliance:

yes

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: average weight of males ranged from 24 – 34 g, the average weight of females ranged from 18 – 28 g
- Housing: Individually, in size 1 Makrolon cages with autoclaved sifted sawdust provided as bedding.
- Diet: standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 Oct 1986 To: 22 Jul 1988

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
One hundred and 200 ppm diets were prepared from a 1% premix, which was prepared freshly on exhaustion of the previous batch by direct dilution of powdered and sieved test material with diet. This 1% premix was also used to prepare a premix II of 1000 ppm, which was then used to prepare diets of 5 or 15 ppm by direct dilution.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of diets mixed were analysed for homogeneity and stability prior to study commencement. During the study, samples of each premix were analysed for accuracy of mixing at monthly intervals, and samples of each final diet analysed at least every two months.

Duration of treatment / exposure:

Males: 81 weeks; females: 88 weeks

Frequency of treatment:

Continuously

Dose / conc.:

5 ppm

Remarks:

Group A. Mean 0.69 and 1.03 mg/kg bw/day for males and females, respectively.

Dose / conc.:

15 ppm

Remarks:

Group B. Mean 1.84 and 2.56 mg/kg bw/day for males and females, respectively.

Dose / conc.:

100 ppm

Remarks:

Group C. Mean 13.17 and 17.65 mg/kg bw/day for males and females, respectively.

Dose / conc.:

200 ppm

Remarks:

Group D. Mean 27.85 and 36.30 mg/kg bw/day for males and females, respectively.

Dose / conc.:

200 ppm

Remarks:

Group D1. Mean 33.06 and 43.30 mg/kg bw/day for males and females, respectively.

No. of animals per sex per dose:

Main: 50. Satellite: 10 (control and high dose)

Control animals:

yes, plain diet

Details on study design:

- Satellite groups: To one of the control groups and to the 200 ppm group, 10 males and 10 females were added as a satellite group for interim sacrifice after 13 weeks, although one female animal of this satellite study was used to replace a mis-sexed main group animal at the start of the study leaving only 9 in the high-dose female group.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: twice daily
- Cage side observations checked: mortality, gross signs of ill health or reactions to the treatment

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: weekly up to the week 65, from week 66 to 78 every two weeks; from week 78 up weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption determined at the same intervals by calculating the difference in weight of food containers at the start and end of each week.
- Group mean and individual calculated achieved intakes of test material (mg/kg/day) were derived by computer using the weekly body weight and food consumption data, and nominal dietary concentrations.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 52 and 78 weeks, and (for females) prior to termination, blood smears were prepared from all surviving animals following superficial venesection of the tail.
- How many animals: All surviving animals
- Parameters checked: Differential white blood cell counts (banded and segmented neutrophils, lymphocytes, monocytes, eosinophils and basophils) were only performed on the smears of all animals from the control and the high dose groups

Sacrifice and pathology:

GROSS PATHOLOGY:
After 13 weeks of treatment all animals of the satellite groups were sacrificed (by CO2 asphyxiation) to assess liver effects. The remaining, surviving, animals were sacrificed when survival in the group with the highest mortality approached 30%. Accordingly, males were terminated after 81 weeks when survival in the second control group was 32% and females were terminated after 88 weeks, when survival in first control group was 34%. All sacrificed animals were subjected to a detailed gross pathological examination and examined externally both visually and by palpation. A macroscopic examination was performed after opening the cranial, thoracic and abdominal cavities. Organs and tissues were examined carefully in situ and after removal; at terminal sacrifice, lungs were inflated with formalin and examined. Any abnormalities were carefully recorded, with precise details of the location, colour, shape and size.

Following organs were weighted: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, exsanguinated body

HISTOPATHOLOGY: Yes, following tissues were examined: adrenals, aourta, brain, caecum, colon, duodenum, epididymes, oesophagus, eyes with optic nerve, femur with joint, gall bladder, sternum with bone marrow, heart, ileum, jejunum, kidneys, liver, lung, mammary area, lymph nodes, skeletal muscle, peripheral nerve (scatic n.), ovaries, pancreas, pituitary, prostate, rectum, salivary glands, seminal vesicle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid and parathyroid, tongue, trachea, urinary bladder, uterus and cervix, gross lesions/masses

Statistics:

A computer programme automatically subjects all measured values to a parametric or non-parametric statistical analysis. The particular analysis chosen depends on the distribution of values under test.
a. Parametric analysis: The programme tests the homogeneity of the variances then proceeds to the non-parametric analysis if the variances are not homogenous. If the variances are homogeneous, the programme performs a one-way analysis of variance, followed by a multiple comparison to controls using the Dunnett test. This test is only performed if the analysis of variance shows a significant difference between the groups. If, however, there is only one treated group, the t-test is performed.
b. Non-parametric analysis: The Kruskal-Wallis test is used when more them one treated group is present. If a significant difference exists between the groups, the programme performs a multiple comparison to the control group using the Dunn-Bonferroni test. When only one treated group exists the Mann-Whitney U test is performed for samples of four or more animals per group; with three or fewer animals per group the t-test is used.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related symptoms were observed. The majority of symptoms recorded were signs of ill-health preceding death.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality among mice receiving 100 or 200 ppm appeared less than that among the controls or among mice receiving 5 or 15 ppm. Otherwise, no treatment-related effect on mortality was detected. There were no decedents among the 39 mice allocated in the satellite groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight development of males receiving 100 or 200 ppm was notably retarded after only 13 weeks of treatment. After one year of treatment mean body weight was about 8% below control level at these two dose groups. Mean body weight gain at week 26 was reduced by 33% at both 100 and 200 ppm, relative to controls. By the end of the study (week 81), mean body weight gain was reduced to only 63% of control level, at both higher dose levels. The male satellite group (200 ppm) sacrificed at week 13 of treatment also showed a significantly reduced body weight gain (-34%).
Body weight development in females was also somewhat retarded at these two dose levels, though not to the same extent as in males, and the effects were only really evident after 26 – 52 weeks on test (at week 52 body weight was reduced by about 7%, relative to controls at the
two higher dose levels). Body weight gain in females at 100 and 200 ppm was reduced by 17% and 15% at week 26 and by 21% and 19% at week 52, respectively. By the end of the study, body weight gain in females at the two lowest dose levels also appeared to be reduced (relative to control group K) but a similar reduction was observed among females of the second control group (group P).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant greater food consumption among male mice receiving 5 ppm was not thought to be of toxicological significance.

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):

Evaluation of blood smears prepared from control and high dose animals at different time points revealed no treatment-related differences.

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serology samples drawn during week 46, as a response to an unexpected incidence of sudden mortality in an adjacent room, showed the presence of antibodies to Mouse Hepatitis Virus (MHV) in a proportion of the 6 mice tested from each room. However, there were no symptoms of ill health observed in the study and the putative infection could only be shown to be present as a result of the seroconversion. Samples of blood were drawn from mice killed in extremis during the weeks 71 to 74, and compared with samples from male mice killed at termination in week 82, to see if virus antibody titres increased in mice dying spontaneously which might have indicated an adverse effect on survival due to the infection. These samples, from a total of 29 mice, again revealed seroconversion to MHV, in 19/29 mice tested. Seroconversion of 16/29 mice to Mycoplasma pulmonis, and of 1/29 mice to Sendai virus, was also noted. There was however no pattern of incidence of seroconversion between mice killed in extremis or killed at termination, nor between mice in the two different rooms in which the study was housed. It was concluded that the presence of seroconversion to the agents detected did not correlate with an adverse effect on the study due to these pathogens.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative liver weights of the satellite groups (200 ppm) terminated after 13 weeks of treatment were significantly increased in males (41% and 53%) and in females (45% and 46%).
At terminal sacrifice relative liver weights of males and females which had received 100 or 200 ppm were again significantly increased; in males at 100 ppm by 22% and at 200 ppm by 42%, and in females by 29% and 59%, respectively. Absolute liver weights were increased by 26% in high dose males and by 24% and 49% in females at 100 and 200 ppm, respectively. Since the presence of liver nodules might be considered to have an effect on the liver weights, an additional analysis was performed excluding the weights of all organs with a nodule greater than 10 mm diameter. This analysis confirmed increased relative liver weights at 200 ppm (males 37%, females 56%) and at 100 ppm (males 18%, females 27%), which therefore indicates an increase in liver weight that is not a consequence of the nodules.
The apparent significant decrease in absolute kidney weights in both sexes at 100 and 200 ppm, relative to controls were not considered to be treatment related, as effects were no longer evident after correction for body weight (relative kidney weight).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the main study there was a significant increase in the incidence of hepatic accentuated lobular pattern and of hepatic masses in the 15, 100 and 200 ppm male and 100 and 200 ppm female groups when compared to each of the two control groups. The hepatic masses corresponded to the treatment-related increases in hepatic adenomas and carcinomas in both sexes. There was also an increased incidence in ‘areas of hepatic change and enlargement’ in high dose females, and a slight increase in the incidence of granular liver in the high dose male group. In the satellite group, sacrificed at 13 weeks, one high dose male and one high dose female also had accentuated lobular livers, and two high dose females had pale livers, but none of these findings were statistically significant.
In the gastrointestinal tract of the 100 and 200 ppm male groups there were reduced incidences of those with abnormal contents in all regions examined apart from the stomach, which was unaffected. This treatment-related effect was not seen in females.
Renal findings, which may relate to treatment, were restricted to a reduction in the incidence of granular kidneys in the 200 ppm female group.
There were increased incidences of skin ulceration in all treated groups of both sexes, but only statistically significant in high dose females. Reductions were seen in the incidences of oedema of the subcutis in male mice of the 100 and 200 ppm dose groups.
There were increased incidences of flaccid testes in the 15 and 200 ppm male groups, though a dose-response relationship was not apparent. These increased incidences corresponded to an increased testicular germinal epithelial deficit in male mice of the 200 ppm dose group only.
Other statistically significant effects included a reduced incidence of excess thoracic fluid in the 100 and 200 ppm male groups and a reduction in the incidence of apparent gall bladder enlargement in the 200 ppm female group.

Neuropathological findings:

not examined

Description (incidence and severity):

Non-neoplastic findings – satellite group: Examination of the satellite group revealed that treatment-related effects following 13 weeks of the test substance administration were confined to the liver. The incidence of periacinar hepatocytic hypertrophy was significantly increased in both sexes at 200 ppm, relative to controls. In addition there was a statistically significant increase in periacinar hepatocytic vacuolation in males and in non-zonal hepatocytic fat vacuolation in females at the high dose level.

Non-neoplastic findings – main study: Consistent with the findings in the satellite group, the major non-neoplastic microscopic changes following long-term administration of the test substance, occurred in the liver. There were a group of toxic changes (focal inflammation, single cell necrosis, hypertrophy, vacuolation) at the two greater dosage levels (100 and 200 ppm) in both sexes, although the male mice were more severely affected, in terms of the numbers affected. A statistical evaluation of histopathological hepatotoxic effects, utilising non-pooled control group incidences, gave essentially very similar results as the pooled results. When using control group 1 alone, there was a loss of significance of focal inflammation in male mice at 100 ppm, of single cell necrosis in the 100 ppm female group, of focal hepatocytic hyperplasia at 200 ppm in females and of centriacinar vacuolation in both sexes. Using control group 2 as the sole reference there were losses of significance as with group 1 as control, plus the loss of significance of periacinar hepatocytic vacuolation at 200 ppm in females.
The following findings also achieved statistical significance at the highest or the two highest dose levels in males: epididymal aspermia (100 and 200 ppm), testicular germinal epithelial deficit (100 and 200 ppm), and testicular amyloid (200 ppm). Other treatment-related, nonneoplastic changes in male mice were an increased incidence (relative to controls) of skin ulceration (200 ppm) and cellulitis (100 and 200 ppm), optic nerve gliosis (200 ppm), and amyloidosis of the ileum (200 ppm) and salivary glands (200 ppm). Long-term test substance administration in males also caused a statistically significant reduction in the incidence of some effects (relative to controls), including pancreatic oedema (200 ppm), interstitial degeneration of the salivary gland (100 and 200 ppm), subcutaneous oedema (200 ppm) and amyloidosis of the spleen (100 and 200 ppm).
Additional treatment-related, non-neoplastic changes in female mice were an increased incidence (relative to controls) of aortic arteritis (200 ppm) and lymphoid hyperplasia in the mesenteric lymph modes (200 ppm). Findings for which the treatment appeared to diminish the incidence of in females were: caecal submucosal oedema (100 and 200 ppm), subcutaneous oedema (100 and 200 ppm), spinal cord compression (200 ppm) and amyloidosis of the kidney interstitium (100 and 200 ppm), the liver (100 and 200 ppm), the spleen (100 and 200 ppm) and the heart (15, 100 and 200 ppm).

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No treatment related neoplasms were found in any of the animals at interim sacrifice at 13 weeks. In the main study, treatment-related neoplastic effects were confined mainly to the liver and comprised a significant increase in the incidence of hepatocytic adenoma (in males at 100 and 200 ppm; in females at 200 ppm) and hepatocytic carcinoma (in males at 15 and 100 ppm; in females at 200 ppm). Thirteen percent of females that died prior to study termination had hepatic adenomas and 22% had hepatic carcinomas (compared to 0% in controls). In males, the hepatic neoplasms only became evident in animals by the end of the study. A statistical evaluation of hepatocytic neoplasia, utilising non-pooled control group incidences, gave essentially very similar results though the significance of the association with treatment of hepatocytic carcinoma in the 15 and 100 ppm male groups was lost.
There were a number of other neoplasms present in animals of the 200 ppm groups that had a low incidence (1/50 or 1/49), were not statistically significant and thus considered not to be treatment-related. They included, in male mice, an oligodendroglioma of the CNS, a cutaneous haemangiosarcoma, a urinary bladder papilloma; in females there was an adrenal medulla adenoma, ileal lymphoma, a cutaneous basal cell tumour and histiocytic sarcoma.

Relevance of carcinogenic effects / potential:

Since the test substance was devoid of mutagenic activity, the induction of liver tumours in mice is not likely to be caused by a genotoxic mechanism. Investigative studies suggest a cytotoxic mode of action in mice by which continuous treatment with the test substance leads to a well-defined sequence of events, starting with a perturbation of hepatic homeostasis (strongly increased liver weight, hepatocyte enlargement, hepatocyte proliferation and liver enzyme induction). In addition, hepatocellular necrosis is observed with the likely consequence of slight regenerative cell proliferation. Upon sustained and strong perturbation of hepatic homeostasis, preneoplastic lesions (foci of cellular alteration) and finally hepatocellular tumours are formed.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

Dietary equivalent to 13.2 and 17.7 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

Carcinogenicity

Effect level:

15 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other: Non-genotoxic mechanism of oncogenesis; tumorigenesis as a secondary effect of cytotoxicity and chronic inflammation

Remarks:

Dietary equivalent to 1.8 and 2.6 mg/kg bw/day in males and females, respectively

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Table 2. Mean and maximum achieved daily dietary intake of test substance in mice administered for 81 weeks (males) or 88 weeks (females).

Mean and highest intake of test substance (mg/kg bw/day)
Dose (ppm)	Group	Males		Females
		Mean	Highest	Mean	Highest
5	A	0.69	0.90	1.03	1.15
15	B	1.84	2.68	2.56	3.42
100	C	13.17	17.60	17.65	23.17
200	D	27.85	35.24	36.30	46.64
200	D1	33.06	35.88	43.30	48.22

 

Table 3. Mortality data (number of decedents/number of animals at start of period) for mice administered the test substance in their diet for 81 weeks (males) or 88 weeks (females).

Dose (ppm)	Group No.	Male Decedents	Female Decedents
0	K	31/50	33/50
0	P	34/50	27/50
5	A	30/50	26/50
15	B	25/50	30/50
100	C	21/50	14/50
200	D	14/50	23/50

 

Table 4. Mean body weights (g) of male and female mice administered the test substance in their diets for the periods indicated.

Dose (ppm)	Group	Body weight (g)
		Male					Female
		Week 0	Week 13	Week 26	Week 52	Week 81	Week 0		Week 13	Week 26	Week 52	Week 88
0	K	30	38	42	44	46	22	27		29	32	35
0	P	30	38	42	44	44	22	27		29	32	33
0	K1	29	38	-	-	-	21	27		-	-	-
5	A	30	37	40	42	44	22	27		29	31	31
15	B	29	37	40	43	44	22	27		30	32	33
100	C	30	35	38	40	40	22	27		28	30	31
200	D	30	36	38	41	40	22	26		28	30	31
200	D1	29	35	-	-	-	21	26		-	-	-

Table 5. Cumulative body weight gain (in grams and as a % of control group 1 (groupK)) at various time points in mice administered the test substance in their diet for 18 months.

			Bodyweight gain (g) and % of controls
			Male				Female
Dose (ppm)	Group		Week 0-13	Week 0-26	Week 0-52	Week 0-81	Week 0-13	Week 0-26	Week 0-52	Week 0-88
0	K		8	12	14	16	5	7	10	13
0	P		8 (100%)	12 (100%)	14 (100%)	14 (88%)	5 (100%)	7 (100%)	10 (100%)	11 (85%)
0	K1		9 (113%)	-	-	-	6 (120%)
5	A		7 (88%)	10 (83%)	12 (86%)	14 (88%)	5 (100%)	7 (100%)	9 (90%)	9 (70%)
15	B		8 (100%)	11 (92%)	14 (100%)	15 (94%)	5 (100%)	8 (114%)	10 (100%)	11 (85%)
100	C		5 (63%)	8 (67%)	10 (71%)	10 (63%)	5 (100%)	6 (86%)	8 (80%)	9 (70%)
200	D		6 (75%)	8 (67%)	11 (79%)	10 (63%)	4 (80%)	6 (86%)	8 (80%)	9 (70%)
200	D1		6 (75%)	-	-	-	5 (100%)

Table 6. Notable changes in terminal body weight, absolute organ weights and relative organ weights (% of bw) of mice administered the test substance in their diet for the periods indicated.

Organ	Sacrifice time	Dose (ppm)
		0(K)	0(P)	5	15	100	200
Males
Terminal Bodyweight (g)	Week 13 Week 81	33.8 46.8	- 44.6	- 45.2	- 46.0	- 41.3**	31.3 41.1**
Absolute Liver Weight (g)	Week 13 Week 81	1.355 2.554	- 2.252	- 2.193	- 2.426	- 2.770	1.913** 3.228**
Absolute Liver Weight (excluding animals with nodules) (g)	Week 81	2.213	2.127	2.164	2.322	2.335	2.696
Relative Liver Weight (%)	Week 13 Week 81	3.988 5.488	- 5.108	- 4.885	- 5.332	- 6.682**	6.097** 7.789**
Relative Liver Weight (excluding animals with nodules) (g)	Week 81	4.859	4.822	4.824	5.207	5.747*	6.674**
Absolute Kidney Weight (g)	Week 13 Week 81	0.679 0.967	- 0.946	- 0.880	- 0.895	- 0.832**	0.642 0.830**
Relative Kidney Weight (%)	Week 13 Week 81	2.042 2.117	- 2.170	- 1.958	- 1.975	- 2.028	2.056 2.027
Females
Terminal Bodyweight (g)	Week 13 Week 81	23.88 34.9	- 33.8	- 31.5	- 35.2	- 33.5	23.19 32.4
Absolute Liver Weight (g)	Week 13 Week 81	1.045 1.73	- 1.648	- 1.536	- 1.828	- 2.145**	1.511** 2.570**
Absolute Liver Weight (excluding animals with nodules) (g)	Week 81	1.73	1.654	1.536	1.685	2.121**	2.493**
Relative Liver Weight (%)	Week 13 Week 81	4.410 4.963	- 4.895	- 4.878	- 5.257	- 6.407**	6.425** 7.897**
Relative Liver Weight (excluding animals with nodules) (g)	Week 81	4.963	4.932	4.878	4.765	6.322**	7.747**
Absolute Kidney Weight (g)	Week 13 Week 81	0.396 0.585	- 0.571	- 0.533	- 0.542	- 0.508**	0.378 0.498**
Relative Kidney Weight (%)	Week 13 Week 81	1.673 1.681	- 1.699	- 1.700	- 1.556	- 1.530	1.634 1.541

*P<0.05, **P<0.01

Table 7. Notable macroscopic changes in mice administered the test substance in their diet for 81 weeks (males) or 88 weeks (females) (data is for all animals, combining those that died or were sacrificed in extremis during the study, and those that were terminated at the end of the study; n=50)

Finding	Dose (ppm)
	0 (K)	0 (P)	5	15	100	200
No of tissues examined	50	50	50	50	50	50
Male
Liver            - accentuated lobular pattern	3	3	5	10*	13***	23***
- masses	5	5	5	8	17***	20***
- areas of hepatic change	2	3	4	3	3	7
- large appearance	1	3	0	1	2	5
- granular	0	1	0	0	1	4*
Gastrointestinal tract - caecum, abnormal content - colon, abnormal content	12 12	10 10	9 10	5 5	3* 3*	16 1**
- rectum, abnormal content	11	11	10	5	3*	1**
- duodenum, abnormal content - ileum, abnormal content - jejunum, abnormal content - stomach, abnormal content	12 12 12 8	11 11 11 11	10 10 10 12	7 8 8 6	3* 3* 3* 7	4* 4* 4* 5
Skin                             -ulceration - oedema of subcutis	6 8	3 12	7 12	7 11	7 3*	10 2*
Testes                - flaccid appearance	1	3	4	9**	6	9**
Thorax                        - excess fluid	10	12	10	8	4*	3*

Female
Liver	- accentuated lobular pattern	3	4	4	3	12**	21***
	- masses	1	2	1	3	5	18***
	- areas of hepatic change	3	3	3	0	1	9*
	- large appearance	2	2	1	1	6	10**
	- granular	1	1	0	1	0	3
Gastrointestinal tract - caecum, abnormal content - colon, abnormal content		8 9	7 6	7 5	6 6	2 2	5 6
- rectum, abnormal content		7	6	4	4	2	5
- duodenum, abnormal content - ileum, abnormal content - jejunum, abnormal content - stomach, abnormal content		7 7 8 9	6 6 5 7	7 6 7 7	5 5 6 8	3 2 2 5	5 5 5 6
Skin	- ulceration - oedema of subcutis	2 6	4 4	6 3	4 6	6 0*	9* 5
Kidney	- granular appearance	21	18	21	17	17	10*
Gall bladder	- enlarged appearance	7	6	2	3	10	0*

Table 8. Notable microscopic changes in mice administered the test substance in their diet for 13 weeks (satellite group; n = 10).

Finding		Dose (ppm)
		0	200	0	200
		Male		Female
No. of tissues examined		10	10	10	9
Liver	- periacinar hepatocytic hypertrophy - periacinar hepatocytic vacuolation - non-zonal hepatocytic fat vacuolation (large vacuole) - focalinflammation	4	10*	2	8**
		0	5*	2	1
		0	4	0	8***
		0	3	2	0

*P<0.05,**P<0.01,***P<0.001 (compared to control K1)

Table 9. Notable microscopic changes in mice administered the test substance in their diet for 81 weeks (males) or 88 weeks (females) (data is for all animals, combining those that died or were sacrificed in extremis during the study, and those that were terminated at the end of the study;

Finding			Dose (ppm)
			0 (K)	0 (P)	5	15	100	200
No of tissues examined			50	50	50	50	50	50
Male
Liver		- focal inflammation	1	1	1	4	5*	8**
		- single cell necrosis	0	2	2	3	14***	25***
		- diffuse hepatocytic hypertrophy	4	10	4	6	26***	36***
		- centriacinar hepatocytic vacuolation	0	0	0	0	1	3*
		- periacinar hepatocytic hypertrophy	1	0	2	5*	4*	2
		- periacinar hepatocytic vacuolation	0	1	4*	3	1	1
		- amyloid	10	13	4*	12	4*	8
Skin		- ulceration	4	2	5	4	6	9*
		- cellulitis	4	1	5	6	8*	8
		- subcutaneous oedema	7	11	5	12	3	2*
Optic Nerve		- gliosis	0	0	2	2	2	3*
Salivary gland		- amyloid	2	8	2	13*	11	16**
		- interstitial degeneration	8	2	6	1	0*	0*
Ileum		- amyloid	20	22	24	32*	28	34**
Gall bladder		- amyloid	0	0	1	0	0	5**
Pancreas		- oedema	3	9	12	8	1	0*
Spleen		- amyloid	13	13	7	10	3**	1***
Epididymides		- aspermia	10	15	20	15	26**	21*
Testes		- germinal epithelium deficit	22	23	31	29	34**	33*
		- amyloid	18	17	20	22	25	27*
Female
Liver	- focal inflammation		1	6	5	9	5	4
	- single cell necrosis		0	0	3*	2	4*	9***
	- diffuse hepatocytic hypertrophy		5	8	6	6	7	20***
	- centriacinar hepatocytic vacuolation		0	0	0	0	4*	3*
	- periacinar hepatocytic vacuolation		0	1	0	1	17***	6**
	- amyloid		13	19	19	16	6**	3***
	- periacinar hepatocytic hypertrophy		0	0	1	0	0	3*
Skin	- ulceration		1	3	4	3	5	4
	- cellulitis		1	3	4	3	5	6
	- subcutaneous oedema		6	11	4	6	1*	1*
Heart	- amyloid		12	15	8	5*	2***	4**
	- aortic arteritis		1	0	0	0	2	4*
Kidneys	- interstitial amyloid		18	18	20	20	5***	1***
Caecum	- submucosal oedema		7	12	7	8	3*	0**
Mesenteric Lymph node - lymphoid hyperplasia			3	7	2	1	2	13*
Spleen	- amyloid		11	18	13	14	1***	1***
Spinal chord	- compression		5	6	1	5	2	0*

*P<0.05,**P<0.01,***P<0.001              (compared to pooled control K &P)

 

Table 10. Notable neoplastic changes in mice administered the test substance in their diet for 18 months.

Finding	Time of death/sacrifice	Dose (ppm)
		0(K)	0(P)	5	15	100	200
Males
Hepatocytic adenoma	During study Study termination All animals	1/31 2/19 3/50	0/34 3/16 3/50	2/30 2/20 4/50	1/25 4/25 5/50	0/21 12/29* 12/50**	2/14 10/36 12/50**
Hepatic carcinoma	During study Study termination All animals	0/31 0/19 0/50	0/34 0/16 0/50	0/30 0/20 0/50	2/25 1/25 3/50*	2/21 1/29* 3/50*	1/14 0/36 1/50

Females
Hepatocytic adenoma	During study Study termination All animals	0/33 0/17 0/50	0/27 0/23 0/50	0/26 0/24 0/50	0/30 0/20 0/50	0/14 2/36 2/50	3/23* 3/27 6/50**
Hepatic carcinoma	During study Study termination All animals	0/33 0/17 0/50	0/27 0/23 0/50	0/26 0/24 0/50	0/30 0/20 0/50	0/14 0/36 0/50	5/23** 2/27 7/50***

Table 11. Statistical analysis of combined liver adenoma and carcinoma in all mice (excluding satellite group) administered the test substance in their diet for 18 months.

Parameter	Dose levels (ppm)
	0	5	15	100	200	0	5	15	100	200
	Males					Females
No. of animals	100	50	50	50	50	100	50	50	50	50
No. of affected animals	6	4	8	15	13	0	0	0	2	13
Statistical significance: (not adjusted for age)	###	n/s	*	***	***	###	n/s	n/s	n/s	***
Statistical significance: (age-adjusted analysis)	##	n/s	n/s	**	n/s	###	n/s	n/s	n/s	***

## significant trend over affected groups, P < 0.01;

### significant trend over affected groups, P < 0.001

*significant pairwise comparison, p < 0.05;

** significant pairwise comparison, p < 0.05;

*** significant pairwise comparison, p <0.001. n/s not significant pairwise comparison, p > 0.05

Conclusions:

The principal treatment-related effects were seen in the liver, and included toxic and neoplastic changes. Increased incidences of combined adenomas and carcinomas were found in males and females at 200 ppm, and in males down to 15 ppm. Appropriate statistical analysis revealed that the apparent increase in hepatocytic neoplasia seen at 15 ppm was due to increased survival among these mice. The NOAEL for carcinogenicity and toxiciy was established at 15 ppm (1.84 and 2.56 mg/kg bw/day in male and female mice, respectively).

Executive summary:

The purpose of this study was to characterise the carcinogenic potential the test substance following long-term exposure to mice. The study was designed to comply with OECD testing guideline 451(Carcinogenicity Studies) and with EPA pesticide assessment guideline 83-2 (Oncogenicity Study). Groups each of 50 male and 50 female CD-1 mice were offered diets containing 5, 15, 100, or 200 ppm of the test substance (dietary equivalent to 0, 0.69, 1.84, 13.17 27.85 mg/kg bw/day and 0, 1.03, 2.56, 17.65, 36.30 mg/kg bw/day for males and females, respectively). Two groups each of another 50 males and 50 females received untreated diets, as duplicate control groups. An additional 10 males and 10 females were added to one of the control groups, and to the 200 ppm group (dietary equivalent of 33.06 and 43.30 mg/kg bw/day for males and females, respectively), for interim sacrifice after 13 weeks to demonstrate an effect on the liver. A mortality check was performed twice daily, and body weight and food consumption measured weekly. Symptomology was performed once each week. Animals found dead or moribund were subjected to a full necropsy. The study was continued until mortality in any one group, treating each sex separately, was approaching 70 % (actually after completion of 81 weeks of treatment in males, and 88 weeks in females). Blood smears were evaluated at weeks 53, 79, and at (in females only) termination. At terminal sacrifice a full necropsy was performed, and organ weights evaluated. Histopathology was performed on a full spectrum of tissues of all animals.

At 200 ppm reduced body weight gain (-29 % in males, -18 % in females after 26 weeks) occurred and survival was better than that in the controls. At necropsy, increased liver weights were found after 13 weeks and at termination, and an increase in macroscopic liver nodules was found in the later decedents and at termination. Histopathologically, an increased incidence of liver tumours (combined adenomas and carcinomas) was found. Treatment-related pathology also included focal hepatocytic inflammation, single cell necrosis and diffuse hepatocytic hypertrophy. At 100 ppm, reduced body weight gain occurred and survival was better than that of the controls in both sexes. At necropsy, an increased liver weight and an increased incidence of macroscopic liver nodules was found. Histopathologically, an increased incidence of liver tumours (adenomas and carcinomas) was found in male mice only. Focal hepatocytic inflammation, single cell necrosis and diffuse hepatocytic hypertrophy was found in both sexes; an increased incidence of periacinar hepatocytic vacuolation was seen in females.

At 15 ppm, a slightly increased incidence of liver tumours (adenomas and carcinomas) was found in males only in comparison with controls. Statistical analysis according to the method of Peto showed that this increase was a consequence of increased survival, therefore is considered not and effect level. At 5 ppm, no reaction to treatment was found.

The test substance caused an increased liver tumour incidence (combined benign and malignant) at levels of 100 ppm or over. Males were more affected than females. The dose level of 15 ppm (1.84 and 2.56 mg/kg/day for males and females, respectively) was established as the no adverse effect level (NOAEL). Doses of 100 or 200 ppm clearly met MTD (Maximum Tolerated Dose) body weight gain requirements.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1.84 mg/kg bw/day

Study duration:

chronic

Species:

mouse

Quality of whole database:

OECD 451 study performed in compliance with GLP.

System:

hepatobiliary

Organ:

liver

Carcinogenicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

As reported in the repeated dose oral toxicity studies by Warren, (1995) and Trendelenberg, (1995), the test substance induced significantly higher liver cell proliferation in mice at all dose levels compared to controls. In rats no significant hepatocyte proliferation was found, and the no-adverse effect level (NOAEL) was set at 20 ppm. In a re-evaluation of histopathological effects (Weber, 1999) found in previous studies the reported histological changes included centrilobular cytoplasmic vacuolation and dose-dependent liver weight increase in exposed mice. The author concluded that the test substance induces changes similar to ones observed in animals treated with phenobarbital. There were no significant qualitative differences between these substances and quantitative differences generally reflected the different degrees of induced hepatomegaly.

The study by Dorobek, (1995), found that the test substance induces enzymes of phase I and II metabolism by factors of approximately 1.5 times that of controls in rats. It also proves to be a particularly strong inducer of the CYP2B family of cytochrome P450 isozymes in rats (determined as PROD activity). The test substance induces enzymes of phase I and II metabolism by factor of approximately 4.0 times that of controls in mice. CYP1A mediated activity remains greater than CYP2B activity (by about 10-fold) even though there is a higher level of induction of CYP2B with the test substance treatment. There are differences in other enzyme systems too, but the ratio of the CYP2B to CYP1A activities would probably have the greatest influence on the pattern of the resulting metabolites of the test substance. This finding is further corroborated by the results of study by Trendelenberg, (2001), where the test substance was found to be a dose-dependent, strong phenobarbital-type inducer of xenobiotic-metabolising enzymes in rat and mouse liver. In the mouse this was comprised of an induction of cytochrome P450 isoenzymes of subfamily CYP2A, CYP2B and CYP3A and the phase II enzymes microsomal epoxide hydrolase, microsomal UDP-glucuronosyltransferase and cytosolic glutathione S-transferase. Likewise, a mode of action of the test substance as a polycyclic aromatic hydrocarbon- or peroxisome-type inducer can be excluded as cytochrome P450 isoenzymes of subfamily CYP1A and CYP4A were only slightly induced if at all. The phase II enzyme induction was lower in rats than in mice according to the study by Dorobek, (1995).

 

There appears to be a clear species difference in the oncogenic potential of the test substance. Supplementary investigative studies suggest a cytotoxic mode of action in mice by which continuous treatment with the test substance leads to a well-defined sequence of events, starting with a perturbation of hepatic homeostasis and resulting in degenerative lesions with subsequent liver cell proliferation leading to preneoplastic lesions and finally hepatocellular tumours. Although several of these events were also observed in rats treated with the test substance, the incidence of liver tumours did not increase. The mechanistic evidence provided suggests that the mechanism of tumour induction (through activation of CAR and downstream tumourigenesis similar to phenobarbital), as observed in the mouse, was not relevant to man. In a study by Elcombe, 2011a, cultured human hepatocytes have been exposed to the test substance for 96 h and examined for cytochrome P450 2B and 3A expression induction as well as the induction of cell proliferation. Similarly to the control substance, phenobarbital, the test substance induced CYP2B/3A without affecting cell proliferation. In a parallel study with cultured mice hepatocytes the effect on cell proliferation has been reported (Elcombe, 2011b) for both, the test substance and phenobarbital. These contrasting findings suggest that the effects of exposure to the test substance seen in the mouse model are not relevant to humans.

The involvement of CAR activation and subsequent liver tumorigenesis was examined in two studies by Milburn, (2011a,b), where wild-type and CAR null strains of mice were exposed either to the test article or phenobarbital in order to assess the hepatotoxicological effects of these substances. Findings in both studies were similar: administration of either substance to the wild-type mice resulted in a strong pleiotropic effect on the liver, including liver size changes, stimulation of hepatocyte cell cycle and low level liver damage at high doses. Contrarily to the wild-type animals, the CAR null mice were barely affected by the exposure to either phenobarbital or test article. The CAR activation plays a critical role in liver carcinogenesisin mice, however in humans CAR activation does not induce liver cancer.

Justification for classification or non-classification

The mechanistic evidence provided suggests that the mechanism of tumour induction (through activation of CAR and downstream tumourigenesis similar to phenobarbital), as observed in the mouse, was not relevant to man. Based on the available data classification for carcinogenicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

Additional information

Carcinogenicity in mice

The purpose of this key study (Warren 1989) was to characterise the carcinogenic potential the test substance following long-term exposure to mice. The study was designed to comply with OECD testing guideline 451 (Carcinogenicity Studies) and with EPA pesticide assessment guideline 83-2 (Oncogenicity Study). Groups each of 50 male and 50 female CD-1 mice were offered diets containing 5, 15, 100, or 200 ppm of the test substance (dietary equivalent to 0, 0.69, 1.84, 13.17 27.85 mg/kg bw/day and 0, 1.03, 2.56, 17.65, 36.30 mg/kg bw/day for males and females, respectively). Two groups each of another 50 males and 50 females received untreated diets, as duplicate control groups. An additional 10 males and 10 females were added to one of the control groups, and to the 200 ppm group (dietary equivalent of 33.06 and 43.30 mg/kg bw/day for males and females, respectively), for interim sacrifice after 13 weeks to demonstrate an effect on the liver. A mortality check was performed twice daily, and body weight and food consumption measured weekly. Symptomology was performed once each week. Animals found dead or moribund were subjected to a full necropsy. The study was continued until mortality in any one group, treating each sex separately, was approaching 70 % (actually after completion of 81 weeks of treatment in males, and 88 weeks in females). Blood smears were evaluated at weeks 53, 79, and at (in females only) termination. At terminal sacrifice a full necropsy was performed, and organ weights evaluated. Histopathology was performed on a full spectrum of tissues of all animals.

At 200 ppm reduced body weight gain (-29 % in males, -18 % in females after 26 weeks) occurred and survival was better than that in the controls. At necropsy, increased liver weights were found after 13 weeks and at termination, and an increase in macroscopic liver nodules was found in the later decedents and at termination. Histopathologically, an increased incidence of liver tumours (combined adenomas and carcinomas) was found. Treatment-related pathology also included focal hepatocytic inflammation, single cell necrosis and diffuse hepatocytic hypertrophy. At 100 ppm, reduced body weight gain occurred and survival was better than that of the controls in both sexes. At necropsy, an increased liver weight and an increased incidence of macroscopic liver nodules was found. Histopathologically, an increased incidence of liver tumours (adenomas and carcinomas) was found in male mice only. Focal hepatocytic inflammation, single cell necrosis and diffuse hepatocytic hypertrophy was found in both sexes; an increased incidence of periacinar hepatocytic vacuolation was seen in females.

At 15 ppm, a slightly increased incidence of liver tumours (adenomas and carcinomas) was found in males only in comparison with controls. Statistical analysis according to the method of Peto showed that this increase was a consequence of increased survival, therefore is considered not and effect level. At 5 ppm, no reaction to treatment was found.

The test substance caused an increased liver tumour incidence (combined benign and malignant) at levels of 100 ppm or over. Males were more affected than females. The dose level of 15 ppm (1.84 and 2.56 mg/kg/day for males and females, respectively) was established as the no adverse effect level (NOAEL). Doses of 100 or 200 ppm clearly met MTD (Maximum Tolerated Dose) body weight gain requirements.

 

Carcinogenicity in rats 

In a supporting study (Warren 1988), the test substance was administered as a dietary admixture to KFM Wistar (HAN Wistar origin) rats at target doses of 0, 20, 50 or 350 ppm (dietary equivalent to 0, 1.0, 2.2, 15.6 mg/kg bw/day and 0, 1.2, 2.7, 21.8 mg/kg bw/day for males and females, respectively) over a period of 118-119 weeks (males) or 121-122 weeks (females). Seventy animals per sex were used for each dose level (50 animals in the main study, 20 in the satellite groups). The treatment diets were prepared weekly by appropriate dilution of the premix with untreated powdered fodder. Animals had free access to the dietary admixture and tap water during the course of the study. Control animals received untreated diet. Achieved concentrations of test substance in the dietary admixtures were determined prior to treatment commencement and at monthly intervals during the study. Animals were observed twice daily for clinical signs, morbidity and mortality throughout the study. More detailed examinations of skin, fur, eyes, mucous membranes, respiratory and circulatory activity, and palpation of masses (if indicated) were conducted bi-weekly. Body weight and food consumption were determined weekly up to 13 weeks of treatment, then for weeks 15, 17, 19 and 21, then reverted to weekly measurement. An ophthalmoscopic examination was performed on all surviving rats of the control group and the high dose group during weeks 98 and 99. Blood samples were obtained from all animals in week 13 for measurement of haematology and clinical chemistry parameters. Haematology and clinical chemistry determinations were performed at 6 treatment intervals (week 14, 26, 52, 78, 105 and at treatment termination), using 10 animals per sex per group. Plasma gamma-GT and creatinine phosphokinase levels were measured at treatment intervals 14, 26, 52 and 78 only and corticosterone levels were measured at week 52 only. Urinalyses were also performed on all animals during week 14, 26, 52, 78, 105 and at treatment termination. Animals dying before scheduled investigations were replaced with another animal of the same group to ensure that the appropriate number of samples per group was examined on each occasion. In addition, samples of liver from rats killed at the 52-week interim sacrifice, were frozen and analysed for: glycogen content, glucose-6-phosphatase activity and fructose-1,6- diphosphatase activity. Following 52 and 78 weeks of treatment, 10 males and 10 females from each group were sacrificed (by CO2 asphyxiation) for interim chronic toxicity investigations. The remaining, surviving, animals were sacrificed at treatment termination. At each scheduled sacrifice, all animals were subjected to a detailed gross pathological examination and the kidneys, liver gonads, heart, spleen and adrenals were weighed. The pituitaries were additionally weighed at terminal sacrifice. All major organs and tissues of each test animal were prepared for standard histological examination. Additional liver slides were stained with Sudan III or were prepared for electron microscopy, in order to confirm a diagnosis of lipid vacuolation. Gross lesions were examined microscopically for neoplastic changes. Parametric or non-parametric statistical analyses were performed where appropriate to check for significant differences between treated animals and controls.

Administration of test substance at target dietary doses of 0, 20, 50 or 350 ppm to KFM Wistar rats for a period of 2 years did not result in any treatment-related mortalities or clinical signs of toxicity. Body weight gain in high dose animals however, was clearly impaired by treatment from week 2 onwards, the effect being more pronounced in females (final body weights of males and females from the high dose group were 5.1% and 13.3% lower than controls, respectively). A reduction in food consumption was not observed in any test group, in fact consumption actually increased in high dose females during the first 13 weeks by 7% compared to the control group (though this apparent increase may partially be due to food scattering). The liver appears to be a target organ for test substance in rats, with hepatic effects occurring in both sexes at the high dose level (350 ppm). The principle effects occurring in males were an increase in the incidence and severity of fatty change, which occurred in all three sacrifice groups (weeks 52, 78 and 118). These fatty changes were not associated with degenerative hepatic lesions. Associated clinical chemistry alterations in high dose males included decreased bilirubin levels, increased gamma-GT activity and increased ALAT and ASAT. In addition, relative liver weights of high dose males were slightly increased (up to 10% increase, relative to controls at week 78) but not statistically significant. In females, hepatic effects included an increased incidence of hepatocellular hypertrophy which was found in 44% (4/9 animals) of high dose females of the interim sacrifice (week 78) and a statistically significant increase in relative liver weights (all sacrifice groups). The absolute liver weights of high dose females were also increased relative to controls at week 78 and at terminal sacrifice but the increase was not statistically significant. These hepatic changes in high dose females were accompanied by decreased bilirubin levels, increased gamma-GT activity and increased cholesterol levels. Another possible target organ in females appears to be the heart - an increase in the incidence (and a slight increase in severity) of necrosis/fibrosis of the heart was noted in females from all treatment groups at week 78 and at study termination. At study termination, the pattern of increase appeared to be dose related. There was also an increase in relative heart weight in high dose females at both of these sacrifice time-points, though absolute heart weights did not appear to be affected. In addition, a statistical increase in total proteins and total globulins was observed in high dose females at sacrifice week 78 and at study termination and total proteins also were significantly increased at 50 ppm in females at study termination. These increases in plasma proteins and globulins may represent an increase in acute phase proteins following cardiotoxic changes (Woodman, 1996). The toxicological significance of these findings is uncertain however. CPK levels (usually a marker for cardiotoxicity) were actually reduced in high dose females at week 78, though not to a statistically significant extent (though standard deviations in CPK levels in general were very large). LDH levels were not measured. Myocardial necrosis/fibrosis is a common finding among aged rats and even the highest incidence in females (74%) were still comparable to the incidence of this finding in males, where the mean incidence ranged from 70% - 78% across all dose groups, including controls (though historical control data provided by the company indicates that the incidence is normally between 1.2 and 4.8 times higher in males than in females anyway). In this instance, given the weight of evidence available, it is assumed that only the cardiac effects at the highest dose level (myocardial necrosis/fibrosis and an increase in relative heart weight) may be treatment related - the apparent increased incidence at 15 and 100 ppm are not considered to be of significant toxicological relevance. There was no evidence of an effect of test substance on the endocrine system. Corticosterone levels measured at week 52 were unaffected and there was no disturbance of the tumour profile in endocrine tissues that might have resulted from a disturbed endocrine status.

There was also no evidence of treatment-related tumourigenesis. It is, therefore, concluded that, under the conditions employed, test substance is not carcinogenic in Han Wistar rats. Based on reduced body weight gain, liver changes and cardiac effects at 350 ppm, the NOAEL in this study is considered to be 50 ppm corresponding to 2.22 and 2.73 mg/kg bw/day in males and females, respectively.