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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 Oct 1984 to 18 Mar 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Deviations:

yes

Remarks:

There was no measure of blood clotting time or potential.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1988

GLP compliance:

yes

Limit test:

no

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/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 179 g, females: 149 g
- Housing: Individually in cages (size 3) with wood chips
- Diet: ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 7 days

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

IN-LIFE DATES: 17 Oct 1984 to 21 Jan 1985

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: Weekly
- Mixing appropriate amounts with: 50 g of the test substance was mixed with 4950 g rat fodder to produce 5 kg of 1% premix, monthly

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance contents of premix (made monthly) and final diets (made weekly) were analysed prior to study initiation and at monthly intervals during the study. Diet and premix are mixed in a Turbula radial mixer for approximately 1 hour to ensure homogeneity.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

20 ppm

Remarks:

Low dose. Equivalent to mean intake of 1.5 and 1.9 mg/kg bw/day for males and females, respectively

Doses / concentrations 2

Dose / conc.:

80 ppm

Remarks:

Mid dose. Equivalent to mean intake of 6.4 and 7.0 mg/kg bw/day for males and females, respectively

Doses / concentrations 3

Dose / conc.:

320 ppm

Remarks:

High dose. Equivalent to mean intake of 23.8 and 31.1 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

15 animals

Control animals:

yes, plain diet

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Daily
- Examinations: Inspection of skin, fur, faeces, eyes, mucous membranes, respiration, circulatory and neurological activity

BODY WEIGHT:
- Time schedule for examinations: Daily

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY:
- Time schedule animals 13 weeks of treatment: Week 4, 8 and 13
- Time schedule animals 13 weeks for treatment and 4 weeks of recovery : Week 14 and 18
- Animals fasted: Yes, 16 - 18 hours prior to collection of blood
- How many animals: 10 animals per sex per dose
- Parameters: Haematocrit, haemoglobin, erythrocytes, mean cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, leukocytes (total and differential), platelets and reticulocytes

CLINICAL CHEMISTRY:
- Time schedule animals 13 weeks of treatment: Week 4, 8 and 13
- Time schedule animals 13 weeks for treatment and 4 weeks of recovery : Week 14 and 18
- Animals fasted: Yes, 16 - 18 hours prior to collection of blood
- How many animals: 10 animals per sex per dose
- Parameters: Hemolytic score, glucose, urea, bilirubin, albumin, total protein, sodium, potassium, calcium, chloride, total cholesterol, glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, alkaline phosphatase and creatinine

URINALYSIS:
- Time schedule for collection of urine: Week 4, 8 and 13
- Time schedule animals 13 weeks for treatment and 4 weeks of recovery: Week 14 and 18
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters: Volume, specific gravity, pH, protein, glucose, ketones, occult blood, urobilinogen and bilirubin and urine sediments.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all survivors were sacrificed through CO2 asphyxiation and the organ/body weight ratios were calculated for the following organs: kidneys, livers, testes/ovaries, heart, brain, spleen and adrenals

HISTOPATHOLOGY: Yes, all major organs and tissues of each test animal were preserved in 4.0% buffered formaldehyde (10% formalin) dehydrated and embedded in paraffin wax. Sections were cut at approximately 5 microns, stained with haematoxylin and eosin and studied microscopically.

OTHER:
Post mortem investigations:
- Sacirifce method: CO2 asphyxiation
- How many animals: All survivors

Statistics:

Body weight, body weight change, food consumption, substance intake, haematology, clinical chemistry, organ weights and organ weight/body weight ratios were analysed statistically for differences between treated and control group mean values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 320 ppm, an increased incidence of piloerection was seen among male rats only, with 14/30 rats affected.
At 80 ppm, an increased incidence of piloerection was also noted among male rats only, with 8/15 rats being affected.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- At 320 ppm, there appeared to be a marginal impairment of body weight gain among male rats from week 6 of treatment. Females of this level did not appear to show any clear sign of a treatment effect on body weight gain. Although mean body weights of main group females were noted to be significantly lower (P< 0.05) than controls at week 13 there was no corresponding change among females of the withdrawal group.
- At 80 ppm, males also showed marginally impaired body weight gain during the latter part of the study. Females at this treatment level, and males and females treated at 20 ppm, showed no sign of a treatment-related disturbance of body weight gain.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 320 ppm, a lower food consumption during week 1 was seen among withdrawal group male rats. Main group male rats did not appear similarly affected; there was no evidence of impaired food consumption among females at this dosage level. It was not considered that food consumption by males or females receiving 80 or 20 ppm was disturbed by treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematocrit values among male rats receiving 320 or 80 ppm were noted to be slightly lower than those of the controls at weeks 4 and 8. There was no corresponding change at week 13 among these rats, nor was there any sign that females might be affected. Values for RBC and HG remained undisturbed, and thus an increase in MCHC was seen among these rats. Depression of haematocrit among rats of one sex only with no other change in red cell parameters or morphology is not considered to represent a finding of toxicological significance. There was no other haematological disturbance indicative of a reaction to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Creatinine levels among female rats receiving 320 or 20 ppm were higher than those of the corresponding controls at weeks 4, 8 and 13. Values among males receiving 320 or 20 ppm were also higher than those of the corresponding controls at weeks 8 and 13. Possibly associated with this observation was an apparent depression of calcium levels among females receiving 320 or 20 ppm at week 4, among males receiving these levels at week 13, and among females receiving 320 or 80 ppm at week 13. It was also noted that sodium levels were significantly higher than those of the controls at week 13 among males and females receiving 320 ppm and among males receiving 80 or 20 ppm; however, at week 4 investigations, sodium levels among females receiving 320 or 20 ppm appeared lower than those of the controls.
It is noted that the results are inconsistent and show no dosage-related trend.
Elevated GOT values among females receiving 320 or 80 ppm were noted at week 13. The magnitude of change was not one of toxicological significance, and values among male rats appeared undisturbed by treatment.

Among withdrawal group rats at week 14 (end of treatment period) elevated blood urea values were noted among treated males and females. Creatinine levels were elevated among males, and sodium values among males and females. Calcium values were similar to those of the controls. Following four weeks of withdrawal, creatinine and sodium values appeared to have recovered. Values for urea among rats previously treated with the test substance remained higher than those of the control rats; however, in the absence of an effect on urea seen among main group rats during the study the significance of a constant difference in urea levels among withdrawal group rats during withdrawal is obscure.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

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):

Increased absolute liver weights were seen among females treated at 320 ppm. Liver weights of males receiving 320 or 80 ppm were also slightly heavier than those of the controls, but these differences did not achieve a level of statistical significance. Calculation of organ weights relative to body weight demonstrated a highly significant increase (P < 0.01) in liver weight, among both males and females treated at 320 ppm. It was not considered that relative liver weights of males or females treated at 80 or 20 ppm were disturbed by treatment. No other differences in organ weights were noted which were considered attributable to treatment with the test substance. Liver weights of rats previously treated with the test substance had returned to control values at necropsy after four weeks of withdrawal.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: Vacuolated hepatocytes with single large or several small vacuoles predominantly centrilobular are present in 6 of 15 males treated at 320 ppm. This represents reversible lipid storage and is treatment­ dependent. Five males and four females treated at 320 ppm show a distinct lobular pattern probably due to enlarged, activated cytoplasm. This is secondary to increased functional demands, and is reversible. Other hepatic changes including vacuoles in hepatocytes, predominantly peripherolobar, and mononuclear focal mild hepatitis are comparable in frequency and degree in control and all treated groups or in single animals only. There is no treatment-related change in male and female recovery rats.

Kidneys: Nutritional tubular calcification at the corticomedullary junction in females is observed with comparable frequency and degree in the control and all treated groups. Other renal lesions occurred sporadically without relation to treatment. There was no treatment-related renal change.

Other organs and tissues: Sporadic changes unrelated to group or treatment are present in some or single organs or tissues of rats of all groups. Increased functional demands in some C dose livers (5 of 15 males, 4 of 15 females) is manifested by a distinct lobular pattern probably due to mild cytoplasmic enlargement. Enlarged vacuolated hepatocytes (lipid storage) are a treatment related change in some C males (6 of 15). These changes are of a mild degree and reversible. They are not present in recovery rats. There is no other treatment related lesion in the liver or any other organ

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

80 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

Dietary equivalent to 6.4 and 7.0 mg/kg bw/day in males and females, respectively

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

320 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Table 1. Summary of body weight gain in rats over 13 week feeding study

		Body weight gain [g] (% difference to control)
Sex	Treatment	Week 0-4	Week 4-8	Week 8-13	Week 0-13	Week 14-17
Males	Control 320 ppm	129   127.5 (-1.2%)	68   61 (-4.7%)	51   41 (-18.1%)	248   232 (-6.2%)	22   20 (-9.1%)
Females	Control   320 ppm	51   48.5 (-4.9%)	24.5   24.5 (-0%)	21.5   15.9 (-24.6%)	99.8   87.4 (-12.5%)	10   10 (-0%)

Table 2. The group mean achieved intakes of the test substance

Dose (ppm)	Males (mg/kg)	Females (mg/kg)
20	1.5	1.9
80	6.4	7.0
320	23.8	31.1

Table 3. Affected haematology parameters

Parameter		Control	20 ppm	80 ppm	320 ppm
Males
Hct	Week 4 Week 8 Week 13 Week 14 (recov. grp.) Week 18 (recov. grp.)	48.5 50.1 48.9 47.6 49.0	48.7 51.5 46.8*	46.5* 45.7** 48.0	45.8** 46.1** 48.37 47.7 48.6
MCHC	Week 4 Week8 Week 13 Week 14 (recov. grp.) Week 18 (recov. grp.)	35.6 35.3 36.0 36.4 35.6	35.6 34.6 36.1	36.5** 37.7** 36.2	36.7** 37.5** 36.4 36.1 36.1**
MCV	Week 4 Week 8 Week 13 Week 14 (recov. grp.) Week 18 (recov. grp.)	54.6 52.9 50.2 50.1 50.8	55.3 53.8 50.7	54.1 50.2** 50.0	53.6 49.7** 49.6 50.0 51.7

*) P<0.05 **) P<0.01

Table 4. Affected absolute and relative organ weights

Organ		Control	20 ppm	80 ppm	320 ppm
Males
Liver	Absolute (g): Week 13 Week 18 Rel. to bw: Week 13 Week 18	11.6 11.1 2.86 2.71	11.6   2.82	12.2   2.94	12.4 11.1 3.18** 2.65
Females
Liver	Absolute (g): Week 13 Absolute (g): Week 18 Rel. to bw week 13 Rel. to bw week 18	7.2 7.0 3.05 2.98	7.3   3.12	7.2   3.17	8.0* 6.8 3.60** 2.90

*) P<0.05 **) P<0.01

Applicant's summary and conclusion

Conclusions:

The NOAEL is 80 ppm (equivalent to a mean daily intake if 6.4 and 7.0 mg/kg bw in males and females, respectively)

Executive summary:

The test substance was orally administered to groups of 15 male and 15 female Han Wistar rats by ad libitum dietary admixture for 13 weeks, at the following dietary levels: 0, 20, 80 and 320 ppm (dietary equivalent to 0, 1.5, 6.4, 23.8 mg/kg bw/day and 0, 1.9, 7.0, 31.1 mg/kg bw/day for males and females, respectively). This study was performed according to OECD TG 408 and GLP principles. Two 4-week recovery groups of 15 male and 15 female rats, one control and one high dose treatment, were included in the study.The animals were daily assessed for clinical signs. Body weight and food consumption were recorded weekly. Laboratory examinations were performed at week 4, 8 and 13 for the main groups and at week 14 and 18 for the recovery groups. After the treatment, or recovery period, animals were sacrificed and subjected to complete gross port-mortem examinations and histopathological examinations.

Results showed at 320 ppm, An increase incidence of piloerection in male rats only. Body weight gain among male rats showed marginal impairment, from week 6. Impairment of food consumption was noted, on the first week of treatment only, among some male rats. A slight depression of haematocrit was noted among male rats at weeks 4 and 8 but not at week 13. Elevated creatinine levels were noted among females at week 4 and among males and females at weeks 8 and 13. Calcium levels were lowered among females at week 4, and among males and females at week 13. Sodium levels among males and females were elevated at week 13. Elevated blood urea levels were noted among rats allocated to withdrawal, but not among main group rats, at week 14; this finding persisted until the end of withdrawal. Lower calcium levels and elevated sodium levels did not persist until the end of withdrawal. At necropsy, liver weights of males and females were heavier than those of the controls after 13 weeks of treatment; this finding regressed by the end of the withdrawal period. Histopathologically, after 13 weeks of treatment, predominantly centrilobular vacuolation of hepatocytes was seen in 6/15 males. 5/15 males and 4/15 females showed a distinct lobular pattern. These changes were reversible and no treatment-related abnormalities were found at the end of the withdrawal period. At 80 ppm, an increased incidence of piloerection was noted among male rats only. Body weight gain of male rats was marginally impaired. A slight depression of haematocrit was noted among male rats at weeks 4 and 8. Calcium levels among females were lower than those of the controls at week 13. Higher sodium values were noted among male rats at week 13. At 20 ppm, elevated creatinine levels were seen among female rats at week 4, and among male and female rats at weeks 8 and 13. Lower calcium levels were seen among females at week 4 and among males at week 13. Sodium levels among males at week 13 were higher than those of the controls. These changes were not dose related or consistent through time and are biologically irrelevant.

Based on these results, at a dietary level of 320 ppm produced a number of toxic changes which appeared readily reversible after a withdrawal period of 4 weeks. A dietary level of 20 ppm was considered to represent a no-effect level. The NOAEL is 80 ppm (equivalent to a mean daily intake if 6.4 and 7.0 mg/kg bw in males and females, respectively)