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

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Remarks:

Liver cell proliferation study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

This 4–week dietary study was used to determine time-dependent effects of the test substance on liver cell proliferation during continuous application of the test substance through dietary admixture to rats and mice. On days 1, 2, 3, 4, 7, 14, 21 and 28 days, groups of 5 animals at each dosage level, were administered a timed intraperitoneal (i.p.) injection of 100 mg/kg bromodeoxyuridine (BrdU) which is taken up by cells actively synthesising new DNA (S-phase label), and sacrificed 2 hours later. Five control animals were sacrificed on day 0 (day of start of treatment).

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

carcinogenicity

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-chloro-phenyl)-3-cyclopropyl-1-[1,2,4]triazol-1-yl-butan-2-ol

Cas Number:

94361-06-5

Molecular formula:

C15H18ClN3O

IUPAC Name:

2-(4-chloro-phenyl)-3-cyclopropyl-1-[1,2,4]triazol-1-yl-butan-2-ol

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

1, 2, 3, 4, 7, 14, 21 and 28 days

Frequency of treatment:

Continuous

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 ppm

Remarks:

Dietary equivalent to 2.2 mg/kg bw/day (mean)

Doses / concentrations 2

Dose / conc.:

350 ppm

Remarks:

Dietary equivalent to 13.8 mg/kg bw/day (mean)

Doses / concentrations 3

Dose / conc.:

1 400 ppm

Remarks:

Dietary equivalent to 28.2 mg/kg bw/day (mean)

Results and discussion

Details on results:

There was no mortality during the study; all animals survived through to their scheduled sacrifice time. Dietary administration of the test substance to rats at levels of up to 1400 ppm (or about 80 mg/kg/day intake) caused moderate body weight and hepatotoxicity with slight increases in plasma enzymes. Marked increases in liver weight were accompanied histopathologically by centrilobular hepatocyte enlargement and vacuolation, but with no increase in hepatocyte proliferation even though the rats were subjected to hepatotoxic doses of the test substance. There was no consistent evidence for an increase in cell proliferation during the exposure period as individual animal data was highly variable.
Treatment-related findings at necropsy were confined to the liver. Accentuated lobular pattern of the liver occurred in rats as a function of administered dose and exposure time. These findings were only present in the high dose group and first occurred after 3 days of treatment and thereafter at each sacrifice date. After 28 days of treatment all 5 animals of the treatment group showed accentuated lobular pattern of the liver.
The effect of the test substance on hepatocyte proliferation indices showed a later significant decrease in the high dose only. There was no evidence of reactive or inflammatory hepatic pathology, which might have interfered with hepatocyte labelling index determination. The mean labelling index for the high dose group showed a distinct peak on day 7. However, this did not attain statistical significance, and this value was largely due to a high index in a single rat (out of a group of 5). A smaller peak was also observed on day 2 (again due to one animal being higher than the rest). The index showed a rapid decrease by day 14, giving statistically significant decreases in the mean index on days 14, 21 and 28. No significant differences were detected at the 350 ppm or 20 ppm dosages.
Histopathology showed time- and dose-related centrilobular hepatocyte enlargement and hepatocyte vacuolation. In the rat study at 1400 ppm, the enlargement was first detected on day 2 and progressed to moderate on day 7, remaining at this degree until day 28. At 350 ppm, minimal enlargement was first detected on day 7 and remained at this degree until day 28. At 20 ppm, moderate enlargement was found in a single rat on day 14. However, as this change was not detected in any subsequent kills, it seems unlikely to be of toxicological importance and 20 ppm was considered the no effect level.

Any other information on results incl. tables

Dietary Consumption:

Analysis of the diets confirmed adequate homogeneity with respect to formulation and test substance content. Food consumption by rats receiving 1400 ppm was lower than that of the controls throughout the study, the effect being most marked during the first week of treatment. Rats receiving 350 ppm showed a slightly reduced food consumption during week 1 which returned to levels similar to controls thereafter. There was no effect on food consumption at the 20 ppm dose level.

As a measure of food utilisation, food conversion ratios (FCR, food consumed divided by body weight gain) were calculated for rats. The efficiency of food utilisation by animals receiving 1400 ppm was impaired during weeks 1 (net body weight loss) and 2; from week 3 onwards there was no further impairment. Rats on the 350 ppm dose showed impaired food utilisation during week 1 only. It should be noted that the intake of rats receiving 1400 ppm were much higher in week 2 than during the rest of the study.

 

Changes in Body Weight:

Interpretation of the changes in body weights is compounded by the fact that at different time points there are differing numbers of animals. There are only 5 animals for the 1, 4 and 28 day groups at each dosage, whereas there are much more available for the other time points in the study (days 7; n = 20, 14; n = 15, and 21; n = 10). Rats receiving the highest dose of 1400 ppm lost weight, and rats on the 350 ppm dose showed a retarded weight gain during the first week of the study. During the second week of treatment rats of the high dose level gained weight at a reduced rate in comparison with controls, while weight gain by rats receiving 350 ppm had returned to control levels. For the remainder of the study, weight gain of all groups of treated rats was similar to that of the controls. There was no effect at 20 ppm.

 

Blood Chemistry:

On day 2 substantial haemolysis was seen in rat blood samples. This was considered attributable to sampling technique. Due to this haemolysis, there was some elimination of animal enzyme data due to the highly variable and unreliable results obtained. Samples collected at all time were heparinised (plasma). Haemolysis appeared to have a marked influence on many of the parameters determined (bilirubin content and enzyme activity). Even though levels varied up and down throughout the study, there were no dramatic changes in enzyme activity values sufficient in magnitude suggestive of marked, pathological hepatotoxicity and cell damage. Rats at the high dose level did not show elevations of SDH, LDH, AL-P, ALT and AST values from day 7 onwards, rather they peaked at day 7 and declined or showed variable results thereafter. In addition, some of these values are similar to or slightly less than those for the 0 ppm controls. The significance of these high values is questionable, as cell damage would predict much higher values relative to normal controls, typically 10-fold or more. There was little change in the total bilirubin content save for small increases due to haemolysis of some samples. GGT activities were very low and quite variable – the highest activities recorded for the mid dose of 350 ppm at days 4 and 7.

 

Necropsy:

Treatment-related findings at necropsy were confined to the liver. Accentuated lobular pattern of the liver occurred in both rats and mice as a function of administered dose and exposure time. In rats these findings were only present in the high dose group and first occurred after 3 days of treatment and thereafter at each sacrifice date. After 28 days of treatment all 5 animals of the treatment group showed accentuated lobular pattern of the liver.

 

Liver Weights:

From day 4, a dosage-related increase in liver weight is apparent affecting the mid- and high dose animals. Data from the high dose group shows a continuous high liver weight from day 7 onwards until study termination. At the mid dose, the effect was less with a plateau effect from days 7 to 21 before rising again until the end of the study.

 

Histopathology:

The effect of the test substance on hepatocyte proliferation indices showed a later significant decrease in the high dose only. There was no evidence of reactive or inflammatory hepatic pathology, which might have interfered with hepatocyte labelling index determination. The mean labelling index for the high dose group showed a distinct peak on day 7. However, this did not attain statistical significance, and this value was largely due to a high index in a single rat (out of a group of 5). A smaller peak was also observed on day 2 (again due to one animal being higher than the rest). The index showed a rapid decrease by day 14, giving statistically significant decreases in the mean index on days 14, 21 and 28. No significant differences were detected at the 350 ppm or 20 ppm dosages.

Histopathology showed time- and dose-related centrilobular hepatocyte enlargement and hepatocyte vacuolation. In the rat study at 1400 ppm, the enlargement was first detected on day 2 and progressed to moderate on day 7, remaining at this degree until day 28. At 350 ppm, minimal enlargement was first detected on day 7 and remained at this degree until day 28. At 20 ppm, moderate enlargement was found in a single rat on day 14. However, as this change was not detected in any subsequent kills, it seems unlikely to be of toxicological importance and 20 ppm was considered the no effect level.

 

 

Table 1. Average feed consumption over the course of the study.

g/animal/day	control	low dose	mid dose	high dose
	0 ppm	20 ppm	350 ppm	1400 ppm
week 1	23.0	23.1	20.4	14.2
2	23.8	23.6	23.7	22.0
3	24.0	23.2	23.6	18.7
4	23.2	22.8	22.1	19.6

 

Table 2. Summary of active substance intake over the course of the study

mg/kg bw/day	control	low dose	mid dose	high dose
	0 ppm	20 ppm	350 ppm	1400 ppm
week 1	0.0	1.6	24.6	68.0
2	0.0	1.5	28.1	116.4
3	0.0	1.4	26.1	93.6
4	0.0	1.3	22.7	93.9
week 1–4 mean	0.0	1.5	24.9	78.7

 

Table 3. Summary of body weight changes over the course of the study.

mg/kg bw/day	control	low dose	mid dose	high dose
	0 ppm	20 ppm	350 ppm	1400 ppm
week 1	23.5	24.5	11.1**	-17.8**
week 1-3 mean	59.7	69.5	54.3	11.1
week 1–4 mean	68.5	85.5	71.4	22.6

 

 Table 4. Rat high dose (1400 ppm) plasma enzyme activities (means).

Day	SDH (IU/L)	LDH (IU/L)	AL-P (IU/L)	ALT (IU/L)	AST (IU/L)
1	22.1	581.6	103.0	35.90	64.84
2*	23.2	999.0	83.0	27.70	60.30
3	18.2	793.4	75.8	78.26	83.14
4	27.1	477.4	95.4	39.94	64.82
7	110.6	1179.5	124.8	103.33	121.05
14	47.6	555.4	115.8	55.78	79.26
21	43.0	559.0	106.2	51.26	76.08
28	31.5	595.8	86.2	46.32	80.76

* Unreliable data, too few samples or interference from haemolysis

 

Table 5. Main necropsy results for rats and mice over the 28 day period

Day*	Dose	Liver ALP**	Liver discoloration
3 4 7 14 21 21 28	1400 1400 1400 1400 1400 350 1400	2/5 1/5 4/5 3/5 2/5 1/5 5/5	-- -- 3/5 1/5 1/5 -- 4/5

 * earliest day of appearance.
** Accentuated Lobular Pattern of the liver (no. of animals affected / group total)

 

Table 6.Major histopathological findings in the liver of rat.

Rat: days post dosage:	day 1	day 2	day 3	day 4	day 7	day 14	day 21	day 28
Animals examined:	5	5	5	5	5	5	5	5
Centrilobular hepatocyte
enlargement
control
20 ppm						0/1
350 ppm					5/0	5/0	5/0	5/0
1400 ppm		1/0	2/0	5/0	4/1	0/5	0/5	0/4
Centrilobular hepatocyte vacuolation control 20 ppm 350 ppm 1400 ppm	2/0 1/0 4/0	2/0 1/0 1/1 1/0	2/0 1/0 1/0 3/0	2/0 3/0 2/0 2/0	2/0 3/0 4/0	1/0   3/0	1/0 2/0	1/0 3/0 3/0 1/0
Midzonal hepatocyte vacuolation
control
20 ppm
350 ppm					2/0		2/0
1400 ppm				3/0			2/0	1/1
Midzonal/periportal hepatocyte vacuolation					1/4		1/0 0/3	1/1
control 20 ppm 350 ppm 1400 ppm						1/0   2/1

Figures indicate number of animals (out of 5) affected at a specific degree of effect, the position of that number indicates severity of that effect; the first position designates trace or minimal severity; second position designates a moderate severity

Applicant's summary and conclusion

Conclusions:

In relation to hepatocyte proliferation a later decrease below control levels was observed - there was however no clear evidence of increased cell proliferation. The low dose group of 20 ppm (1.5 mg/kg/day) was found to be the no-effect level, even though at this dose moderate enlargement was found in a single rat on day 14. As this change was not detected in any subsequent kills this finding was unlikely to be of any toxicological importance and 20 ppm be set as the no-effect level. Some minimal centrilobular hepatocyte vacuolation was observed in rats from the low dose group (as was also observed in control animals) but no specific pattern is evident.

Executive summary:

Dietary administration of the test substance to male Wistar rats (291 ± 15 g) took place over a 4-week period. Rats were exposed to dietary levels of 0 (controls), 20, 350 and 1400 ppm. On days 1, 2, 3, 4, 7, 14, 21 and 28 days, groups of 5 animals at each dosage level, were administered a timed intraperitoneal (i.p.) injection of 100 mg/kg bromodeoxyuridine (BrdU) which is taken up by cells actively synthesising new DNA (S-phase label), and sacrificed 2 hours later by CO2 asphyxiation. Five control animals were sacrificed on day 0 (day of start of treatment). The acclimatisation period was for 12 days. Animals were housed individually and were approximately 12 weeks old at the start of the treatments. All animal weights were within the range ± 20% of the mean body weight for each species. Food and water were available ad libitum. Animals were housed in air-conditioned rooms at 23 ± 2 °C; relative humidity, 50 – 80 %; with 12 – hour light / dark cycles. 10 rats were designated for a batch health check during the acclimatisation period, sacrificed and subjected to a full necropsy. Mortality and gross ill-health were checked twice daily (only once per day at weekends and holidays). Animals were subjected to a detailed symptom check including palpation once per week. Body weights were recorded from week-1 prior to commencement of the treatment, at the start of the study, weekly thereafter and on the day of sacrifice. Mean food consumption was determined weekly and on the day of sacrifice.

There was no mortality during the study; all animals survived through to their scheduled sacrifice time. Treatment-related findings at necropsy were confined to the liver. No other organ systems appeared to be adversely affected even with high doses of the test substance. High levels of dietary test substance cause moderate body weight alterations and some hepatotoxicity. The marked increases in liver weights appear to be a result of hepatocyte hypertrophy rather than hyperplasia. These changes were accompanied histopathologically by centrilobular hepatocyte enlargement and vacuolation in both species. Blood chemistry measurements were inconclusive but did indicate an adaptive response to a chemical insult at high dose levels. In addition, any references to statistical significance must be treated with caution, as it does not necessarily indicate biological significance; in some cases inappropriate statistical tests were employed or there were insufficient sample numbers to arrive at a conclusion. In relation to hepatocyte proliferation a later decrease below control levels was observed - there was however no clear evidence of increased cell proliferation.

The low dose group of 20 ppm (1.5 mg/kg/day) was found to be the no-effect level, even though at this dose moderate enlargement was found in a single rat on day 14. As this change was not detected in any subsequent kills this finding was unlikely to be of any toxicological importance and 20 ppm be set as the no-effect level. Some minimal centrilobular hepatocyte vacuolation was observed in rats from the low dose group (as was also observed in control animals) but no specific pattern is evident.