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: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 Oct 1987 to 27 Nov 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

1981

Deviations:

yes

Remarks:

1) Number of animals sacrificed after 16 days, 3 males and 3 females instead of 5 males and 5 females. 2) Number of animals for recovery 2 males and 2 females instead of 5 males and 5 females. Such deviations did not effect the integrity of the study.

GLP compliance:

yes (incl. QA statement)

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

Remarks:

SPF

Sex:

male/female

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

Mean airflow in the exposure chambers was 1.4 L/min/animal

Duration of treatment / exposure:

16 days

Frequency of treatment:

Exposed for 6 hours a day, 5 days a week for a total of 2 weeks (12 exposures). Recovery duration after the last exposure was 15 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.01 mg/L air (nominal)

Remarks:

Group 2, low

Doses / concentrations 2

Dose / conc.:

0.1 mg/L air (nominal)

Remarks:

Group 3, mid

Doses / concentrations 3

Dose / conc.:

1 mg/L air (nominal)

Remarks:

Group 4, high

No. of animals per sex per dose:

5

Control animals:

yes

Results and discussion

Effect levels

Dose descriptor:

NOAEL

Effect level:

0.01 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

food consumption and compound intake

haematology

histopathology: non-neoplastic

Remarks on result:

other: Equivalent to an actual dose of 0.017 mg/L, and equivalent to 4.9 mg/kg bw/day (6 hours exposure per day; 0.8 L air/kg/min)

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

0.1 mg/L air (nominal)

System:

other: Respiratory, hepatobiliary and immune system

Organ:

liver

lungs

spleen

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

ANALYTICAL VERIFICATION OF TEST SUBSTANCE 

Gravimetric determination of the aerosol concentrations revealed means of 0.017 mg/L (low dose), 0.099 mg/L (mid dose) and 1.026 mg/L (high dose).

Particle size determinations showed comparable distributions over time and dose. As a mean of all determinations 90 % of the particles were found to be smaller than 3.0 μm.

 

MORTALITY

Three females of the high dose group were found dead after the second exposure. Exposure of this group was stopped thereafter. The remaining animals had to be sacrificed on day 3 or day 6 for human reasons. No other deaths were observed.

 

CLINICAL SIGNS 

With all high dose animals’ slight hunched posture, laboured respiration, ruffled fur and weakness were observed. Ventral recumbence was observed in females only. In mid dose animals slight squealing, howling and crying was noted in one male and three females. The same symptoms were observed in 1 male and 2 females of the low dose group.

 

BODY WEIGHT AND FOOD CONDUMPTION

From day 1 to 16 there was a slight increase in food consumption in the males and a decrease in food consumption in the females. Over the same period there was a slight increase in body weight gain in males and a decrease in body weight gain in females. Mid-dose females showed a slightly reduced body weight gain throughout the study and reduced body weight at the end of the study (156.4 g versus 177.5 g in the control group).

 

HAEMATOLOGY

Analysis of WBCs revealed a dose dependent increase in both males and females after 3 weeks.

 

Table 1. 16-day inhalation rat – affected haematology parameters.

parameter	control	0.01 mg/L	0.1 mg/L	control	0.01 mg/L	0.1 mg/L
	males			females
WBC week 3	8.6	9.7	11.3	6.3	6.3	7.4
recovery			10.2			7.7

 

CLINICAL CHEMISTRY

Bilirubin levels were slightly reduced in male animals of the low and mid dose group. LDH and ALP activities were slightly lower in mid dose male (not statistically significant). In female animals, effects were only seen in the mid dose group, being related to liver toxicity, i.e. a slight to moderate increase of ASAT and ALAT levels and high LDH activity. None of these effects were statistically significant. After recovery they were all returned to normal levels.

 

Table 2. 16-day inhalation rat - affected parameters of clinical chemistry.

parameter	control	0.01 mg/L	0.1 mg/L	control	0.01 mg/L	0.1 mg/L
	males			females
bilirubin week 3	9.2	7.3*	7.6*	8.0	8.5	9.1
recovery			10.7			12.0
LDH week 3	204	181	157	158	190	373
recovery			160			75
ALP week 3	173	170	146	75.0	62.8	64.6
recovery			114			62.6
ASAT week 3	87.6	80.8	81.5	76.5	72.1	86.9
recovery			67.7			58.4
ALAT week 3	66.6	64.9	65.6	43.0	42.0	61.3
recovery			44.6			38.1

*) P<0.05

 

ORGAN WEIGHTS

In male animals there was a statistically significant increase in absolute liver and kidney weights in the low and the mid dose group and in relative liver and kidney weights in the mid dose group. In female animals there was only a slight increase of the relative liver weight in the mid dose group. Recovery data indicates reversibility of these effects.

 

Table 3. 16-day inhalation rat - affected organ weights.

parameter		control	0.01 mg/L	0.1 mg/L	control	0.01 mg/L	0.1 mg/L
		males			females
carcass	week 3	227	237	243	178	167	156*
	recovery			286			189
Liver	absolute week 3	6.27	6.90*	7.85**	5.53	5.08	5.73
	recovery			7.86			5.59
	relative to bw week 3	2.76	2.92	3.23**	3.11	3.05	3.66**
	recovery			2.75			2.95
kidney	absolute week 3	1.50	1.64*	1.75**	1.19	1.12	1.06
	recovery			1.78			1.23
	relative to bw week 3	0.66	0.69	0.72**	0.67	0.67	0.68
	recovery			0.62			0.65

*) P<0.05 **) P<0.01

 

MACROSCOPIC AND HISTOPATHOLOGY

No macroscopic findings were recorded.

For Histopathology, treatment-related findings were recorded in the respiratory tract, liver and spleen. In high dose animals (including intercurrent deaths), one male showed myonecrosis, slight squamous metaplasia of nasal epithelium was recorded in 2 females. The lungs were severely affected at the high dose with one male and 4 females showing signs of haemorrhage, 2 males and one female showing infiltration of macrophages, 3 males displaying perivascular cuffing and mineral deposition. Two females showed clear signs of thrombosis and emphysema.

Slight or moderate hepatocellular hypertrophy occurred in 4 males and 2 females at the high-dose, with minimal to marked fatty change of liver cells was recorded in 5 males and 4 females. Minimal or slight vacuolation of macrophages in the splenic pulp was recorded in 2 males and 2 females, and slight to moderate depletion of lymphocytes was evident in the spleen of 4 females treated at the high-dose.

Hepatocellular hypertrophy was also recorded in 2 males and 1 female of the middose group and one male of the low-dose group. At the end of the recovery period, hepatocellular hypertrophy was not recorded in the two males and two females of the 0.1 mg/L recovery group. Haemosiderosis was recorded in the spleen of 3 females.

 

Table 4. 16-day inhalation rat - histopathological findings of intercurrently died animals.

parameter	control	1 mg/L	control	1 mg/L
	male		female
Nasal epithelium
Squamous metaplasia				2/5
Liver
Hepatocellular hypertrophy		4/5
Fatty change	1/5	5/5	1/5	4/5
Spleen
Vacuolation of macrophages		2/5		2/5
Depletion of lymphocytes				4/5

 

Table 5. 16-day inhalation rat - histopathological findings of survivors.

parameter	control	0.01 mg/L	0.1 mg/L	control	0.01 mg/L	0.1 mg/L
	male			female
Liver
Hepatocellular hypertrophy week 3		1/3	2/3			1/3
Spleen
Haemosiderosis		0/3	0/3		0/3	3/3
Recovery						1/3

Applicant's summary and conclusion

Conclusions:

The aerosol concentration of 1 mg/L caused substantial mortality in male and female rats. The liver, the respiratory tract and the spleen were the target organs at this excessive level. At lower concentrations the liver is indicated as target organ, since changes in liver weights were accompanied by slight changes in liver enzymes and hepatocellular hypertrophy. There were also changes in the weight of the kidney but these were not associated with any changes in clinical chemistry and only slight mineralisation observed histopathologically. Histopathological examination revealed effects at the highest dose and haemosiderosis in the females at the mid-dose. Since the weak effects observed at the lowest concentration represent the kind of effects from which the animals recovered completely at the higher concentration of 0.1 mg/L, this nominal concentration of 0.01 mg/L corresponding to an achieved concentration of 0.017 mg/L is considered to represent the NOAEL of the study, equivalent to 4.9 mg/kg bw/day (6 hours exposure per day; 0.8 L air/kg/min).

Executive summary:

In this 16-day inhalation toxicity study conducted according to OECD TG 412 following GLP, the test substance was administered via inhalation on five days per week to SPF-bred Wister rats in a nose-only exposure system. The study was comprised of four groups, each containing 5 male and female rats who recieved a nominal dose level of 0, 0.01, 0.1 or 1.0 mg/L air. Mean airflow in the exposure chambers was 1.4 L/min/animal. Recovery duration after the last exposure was 15 days. Aerosol concentration was determined gravimetrically using glass fibre filters once pre-test and daily thereafter during exposure periods. Particle size distribution was determined at least once daily in the high dose and in one of the lower doses using a stage cascade impactor. The animals were observed twice daily for mortality and once for clinical signs of toxicity. Food consumption was recorded weekly and individual body weights were recorded twice during the acclimatisation period and weekly thereafter. Blood samples for haematology and clinical chemistry were collected from all animals of the control and low dose groups and from 3 males and 3 females of the mid dose group on day 17 and from 2 males and 2 females of the mid dose group following the 15 day recovery period. At treatment termination or after recovery period, animals were sacrificed and subjected to a detailed necropsy.

Three females of the high dose group were found dead after the second exposure. Exposure of this group was stopped thereafter. The remaining animals had to be sacrificed on day 3 or day 6 for human reasons. No other deaths were observed. With all high dose animals’ slight hunched posture, laboured respiration, ruffled fur and weakness were observed. Ventral recumbence was observed in females only. In mid dose animals slight squealing, howling and crying was noted in one male and three females. The same symptoms were observed in 1 male and 2 females of the low dose group. From day 1 to 16 there was a slight increase in food consumption in the males and a decrease in food consumption in the females. Over the same period there was a slight increase in body weight gain in males and a decrease in body weight gain in females. Mid-dose females showed a slightly reduced body weight gain throughout the study and reduced body weight at the end of the study (156.4 g versus 177.5 g in the control group). Analysis of WBCs revealed a dose dependent increase in both males and females after 3 weeks. Bilirubin levels were slightly reduced in male animals of the low and mid dose group. LDH and ALP activities were slightly lower in mid dose male (not statistically significant). In female animals, effects were only seen in the mid dose group, being related to liver toxicity, i.e. a slight to moderate increase of ASAT and ALAT levels and high LDH activity. None of these effects were statistically significant. After recovery they were all returned to normal levels. In male animals there was a statistically significant increase in absolute liver and kidney weights in the low and the mid dose group and in relative liver and kidney weights in the mid dose group. In female animals there was only a slight increase of the relative liver weight in the mid dose group. Recovery data indicates reversibility of these effects. No macroscopic findings were recorded. For Histopathology, treatment-related findings were recorded in the respiratory tract, liver and spleen. In high dose animals (including intercurrent deaths), one male showed myonecrosis, slight squamous metaplasia of nasal epithelium was recorded in 2 females. The lungs were severely affected at the high dose with one male and 4 females showing signs of haemorrhage, 2 males and one female showing infiltration of macrophages, 3 males displaying perivascular cuffing and mineral deposition. Two females showed clear signs of thrombosis and emphysema. Slight or moderate hepatocellular hypertrophy occurred in 4 males and 2 females at the high-dose, with minimal to marked fatty change of liver cells was recorded in 5 males and 4 females. Minimal or slight vacuolation of macrophages in the splenic pulp was recorded in 2 males and 2 females, and slight to moderate depletion of lymphocytes was evident in the spleen of 4 females treated at the high-dose. Hepatocellular hypertrophy was also recorded in 2 males and 1 female of the middose group and one male of the low-dose group. At the end of the recovery period, hepatocellular hypertrophy was not recorded in the two males and two females of the 0.1 mg/L recovery group. Haemosiderosis was recorded in the spleen of 3 females.

Since the weak effects observed at the lowest concentration represent the kind of effects from which the animals recovered completely at the higher concentration of 0.1 mg/L, this nominal concentration of 0.01 mg/L corresponding to an achieved concentration of 0.017 mg/L is considered to represent the NOAEL of the study, equivalent to 4.9 mg/kg bw/day (6 hours exposure per day; 0.8 L air/kg/min).