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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 Jul 1985 to 14 Aug 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 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

1982

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: "Teratogenicity study", Guidelines for Registering Pesticides in the U.S.A., U.S. Environmental Protection Agency, Hazard Evaluation: Human and Domestic Animals. U.S. Federal Reg­ ister, Vol. 43, paragraph 163.83-3, adopted November

Version / remarks:

1982

Deviations:

no

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:

other: Wistar/HAN rat (Kfm: WIST, outbred, SPF quality)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 11 weeks, minimum
- Weight at study initiation: 180 -236 g
- Housing: The animals were housed individually in Makrolon cages type-3 with wire mesh tops and standardized granulated softwood bedding.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days under test conditions, after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2
- Humidity (%): 55 ±10
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 8 Jul 1985 to 14 Aug 1985

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed into a glass beaker on a tared precision balance and the vehicle added (w/v). The mixtures were prepared using a homogenizer. During the daily administration period, homogeneity was maintained using a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance/vehicle mixture were prepared daily prior to administration.

VEHICLE
- Concentration in vehicle: 4% CMC
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of concentration, homogeneity and stability of the test article in the vehicle/test article mixtures were performed once during the treatment period. Samples were taken immediately after mixture preparation and again after 90 minutes. Aliquots (about 1 g) were diluted with acetone to give approximate the standard concentrations and injected into the gas chromatograph. Standard solutions in the range of 10 to 100 µg/mL of test substance in acetone were prepared and used for the calibration of the gas chromatograph.

Gas chromatographic conditions:

Apparatus: Packard 428 gas chromatograph with FI-Detector and Shimazu CR3A Integrator
Column: OV-210 10 % on Gaschrom Q 80 - 100 mesh,1.5 m x 2 mm

Temperature: - Injector: 270 degrees centigrade,
- Oven: 230 degrees centigrade,
- Detector: 280 degrees centigrade

Carrier gas, Helium; 30 mL/min.
Injection volume, 3 µL

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear or observation of copulation plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

From day 6 through day 15 post coitum

Frequency of treatment:

Daily

Duration of test:

Until day 21 post coitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

6 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

12 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

24 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

48 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based upon the results of a dose-finding teratogenicity study in rats

Examinations

Maternal examinations:

CLINICAL OBSERVATIONS:
- Time schedule: Twice daily

BODY WEIGHT:
- Time schedule for examinations: Daily, from day 0 until day 21 post coitum

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, data were recorded on day 6, 11, 16 and 21 post coitum.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 21 post coitum
- Organs examined: All internal organs

Ovaries and uterine content:

The ovaries and uterine content was examined after termination:
Examinations included:
- Gravid uterus weight: Yes
- Position of foetuses in the uterus: Yes
- Number of corpora lutea: Yes

OTHER: Caesarean section
- The uteri of all females which were found at necropsy to be not pregnant were placed in an aqueous solution of ammonium sulphide to accentuate possible haemorrhagic areas of implantation sites. All tissues and organs except foetuses removed from animals killed at scheduled necropsy were discarded.

Blood sampling:

No data

Fetal examinations:

- External examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

OTHER: Procedure Wilson technique of the viscera and brain
- Slicing technique of Wilson (1965) for examination of the viscera and brain. One third of the number of live foetuses from each litter was fixed in a mixture of ethyl alcohol, formal and acetic acid. After evaluation the individual sections were preserved in a solution of ethyl alcohol and glycerine (one foetus per container). Descriptions of any abnormalities were recorded.

- The remaining foetuses (two thirds of the number of live foetuses) were placed in a solution of potassium hydroxide for clearing and stained with alizarin red. The skeletons were examined and all abnormalities were recorded. The specimens were preserved individually in plastic bags.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No sign or symptom was observed in any female of the vehicle control group and no test substance related finding was observed in any female of any dose group.

The following common observations were noted:
- Group 2 (6 mg/kg): Two animals, hairless areas on the fur and dullness of both eyes, respectively, were noted on day 6 post coitum.
- Group 3 (12 mg/kg): One animal, hairless areas were noted on day 13 post coitum. In female no. 64, a node (10 x 20 mm) in the left inguinal area was noted on day 19 post coitum
- Group 4 (24 mg/kg) : No sign or symptom was observed.
- Group 5 (48 mg/kg): One female, ruffled fur and whitish flux was noted on day 6 post coitum, and in another animal, hairless areas were noted on day 16 post coitum.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight and mean corrected body weight gain of the 6 and 12 mg/kg bw/day groups were not considered different from controls. There was a reduction in body weights (body weight loss) from day 6-7 and from day 7-8 of treatment in both the 24 and 48 mg/kg bw/day groups. Body weight gain recovered in the following days. There was an overall reduction in body weight gain for the period 6-11. Mean body weight gain was similar to controls for the remaining period at all dose levels. Mean body weights were statistically significant lower (p < 0.05) from day 12 of treatment at ≥ 24 mg/kg bw/day. This was due to the reduction in uterus weight (reduced foetus number) at these dose levels. In an additional evaluation of the body weight gain, a dose related and biologically significant body weight gain reduction of -18% versus control was observed at the 12 mg/kg bw/day dose level.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of groups 4 and 5 was significantly reduced (dose-related) during the treatment period in comparison to that of the vehicle control group. No significant or test substance related differences in food consumption were noted between the vehicle control group and group 2 (6 mg/kg) or 3 (12 mg/kg).

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related finding was noted in any female of any dose group and no abnormal finding was evident in any female of the vehicle control group.

The following incidental common findings were noted:
- Group 3 (12 mg /kg): In one animal, on the left side of the inguinal area, a dark­ red node (10 x 20 mm) was noted between the epidermis and the hypodermis.
- Group 5 (48 mg/kg): In one animal, the uterus was discoloured dark-red and contained reddish turbid fluid. This finding was considered to be the result of the nine dead embryos (5 embryonic and 4 foetal resorptions).

No abnormal finding was noted in groups 2 (6 mg/kg) and 4 (24 mg/kg).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

In group 4 (24 mg/kg) and 5 (48 mg/kg), a significantly increased postimplantation loss (dose-related) was noted

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

One animal in group 5 (48 mg/kg) had implantation sites only (resorption of all embryos).

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

The total losses consisted mainly of early embryonic resorptions, total resorptions and to a lower extent late resorptions, amounted to 22.2 % in group 4 (24 mg/kg) and 30.6 % in group 5 (48 mg/kg). A single female of the high dose group had resorptions only; inclusion of this female increased the percentage post implantation loss for this group to 34.2%.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

2 dead fetuses were found in group 4 (24 mg/kg) and 1 dead fetus in group 5 (48 mg/kg)

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

(=NOEL)

Effect level:

6 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of groups 1 (vehicle control), 2 (6 mg/kg) and 3 (12 mg/kg) were similar. No test substance related differences were evident.
In groups 4 (24 mg/kg) and 5 (48 mg/kg), the group mean body weights of foetuses were slightly reduced (8.3 %), in comparison with that of the vehicle control group and statistically significant. This finding was considered to be test substance related

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant differences between the sex ratios of foetuses of the dose groups and the vehicle control group were evident.

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External investigations revealed the presence of one runt in each of the 6, 12, 24 and 48 mg/kg groups. This finding was considered to be incidental and not related to treatment. No other malformed or anomalous foetus was found in the 6, 12, 24 mg/kg groups. At the highest dose one foetus was noted with a hydrocephalus and two foetuses had a palatoschisis (also reported as cleft palate), of which one was a runt.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal investigations revealed no treatment related malformations. The frequency of some relatively common findings appeared to be increased in a dose-related manner from 12 mg/kg bw/day, i.e., absent sternebrae and supranummery ribs. It was concluded by the authors that these increases were within the normal range of deviations and variations for animals of this strain. The stages of skeletal ossification in the control, 6 and 12 mg/kg groups were similar and reflected the normal range of variations for animals of this strain and age. In the 24 and 48 mg/kg groups, the incidence of incompletely ossified phalangeal nuclei and calcanea with still absent ossification was increased compared to that of the control. These findings may be the result of the reduced mean bodyweights of foetuses. In addition, the incidence of supernumerary ribs exceeded the historical control range at the two higher dose levels.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral investigations of foetuses by the Wilson technique revealed no abnormal findings in the control, 6 and 12 mg/kg groups. At 24 mg/kg a hydrocephalus internus was noted. At 48 mg/kg the previously mentioned hydrocephalus and the palatoschisis were confirmed. In addition, another hydrocephalus internus was noted in the high dose group.

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

(=NOEL)

Effect level:

12 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: cranium

other: skeletal: incomplete or absence of ossification (phalangeal nuclei and calcanea)

Description (incidence and severity):

At the highest dose one foetus was noted with a hydrocephalus and two foetuses had a palatoschisis (also reported as cleft palate), of which
one was a runt. A single incidence of hydrocephalus internus was also apparent at the 24 mg/kg bw/day dose level.
At 24 and 48 mg/kg bw/day incomplete ossification of phalangeal nuclei and
the absence of ossification in calcanea was observed.

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

24 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Table 1a. Differences in body weight gain of dams over the treatment period

Group (mg/k)	Days post-coitum
	0-6 gms (%)	6 – 11 gms (%)	11 – 16 gms (%)	6 – 16 gms (%)	16 – 21 gms (%)	6 – 21 gms (%)	Corrected body weight gain (%)*
1 (0)	18(+8.7)	17(+7.6)	27(+11.2)	44(+19.9)	48(+17.8)	92(+41)	9.5
2 (6)	18(+8.7)	16(+7.1)	26(+11)	42(+18.7)	51(+19)	93(+41)	8.4
3 (12)	17(+8.1)	14(+6.2)	25(+10.4)	39(+17.3)	53(+20)	92(+40)	7.2
4 (24)	18(+8.8)	12(+5.4)	23(+9.8)	35(+15.7)	43(+17)	78(+35)	9.3
5 (48)	17(+8.2)	11(+4.9)	22(+9.3)	33(+14.7)	42(+16)	75(+33)	9.5

Table 1b Reinterpretation of body weight gain data of dams over days 6 -11 compared to control group

Dose group	Body weight gain (Days 6 to 11)
6 mg/kg/day	-6%
12 mg/kg/day	-18%
24 mg/kg/day	-29%
48 mg/kg/day	-35%

Table 2. Summary of reproductive parameters.

Parameter		0 mg/kg	6 mg/kg	12 mg/kg	24 mg/kg	48 mg/kg
Number of females on study		25	25	25	25	25
Number of pregnant females (%)		24 (96)	22 (88)	23 (92)	25 (100)	23 (92)
Number of non-pregnant females		1	3	2	0	2
Number of females with 100 % intrauterine deaths		1	0	0	0	1
Number of females with live foetuses at necropsy		23	22	23	25	22
Number of corpora lutea: per group mean per dam		286 12.4	277 12.6	296 12.9	299 12.0	281 12.8
Number of implantations: per group mean per dam		261 11.3	259 11.8	279 12.1	279 12.1	255 11.6
Preimplantation loss: per group mean per dam		25 1.1	18 0.8	17 0.7	20 0.8	26 1.2
Live foetuses:	per group	248	248	271	217	177
	mean per dam	10.8	11.3	11.8	8.7*	8.0*
	% of implantations	95.0	95.8	97.1	77.8	69.4
	% males	49.2	46.8	48.3	45.2	48.9
Dead foetuses		0	0	0	2	1
Early resorptions:	per group	12	10	8	56	65
	mean per dam	0.5	0.5	0.3	2.2	3.0
	% of implantations	4.6	3.9	2.9	20.1	25.5
Late resorptions:	per group	1	1	0	4	12
	mean per dam	0.0	0.0	0.0	0.2	0.5
	% of implantations	0.4	0.4	0.0	1.4	4.7
Total resorptions:	per group	13	11	8	60	77
	mean per dam	0.6	0.5	0.3	2.4	3.5
	% of implantations	5.0	4.2	2.9	21.5*	30.2*
Post implantation losses:	per group	13	11	8	60	78
	mean per dam	0.6	0.5	0.3	2.5	3.5
	% of implantations	5.0	4.2	2.9	22.2	30.6
Mean weight of live foetuses:		4.8	4.8	4.7	4.4*	4.4*

*) P ≤ 0.05

Table 3. Summary of foetal findings.

Parameter	0 mg/kg	6 mg/kg	12 mg/kg	24 mg/kg	48 mg/kg
External examinations: Number of foetuses examined No. of malformations (No. litters affected)	248 0 (0)	248 1 (1)	271 1 (1)	217 1 (1)	177 3 (3)
Visceral examinations: Number of foetuses examined No. of malformations (No. litters affected)	78 0 (0)	79 0 (0)	91 0 (0)	71 1 (1)	60 3 (3)
Total number of palatoschisis (litter)					2 (2)
Total number of hydrocephalus (litter)				1 (1)	2 (2)
Skeletal examinations: Number of foetuses examined No. of anomalies (No. litters affected)	170 5 (4)	169 4 (4)	180 10 (7)	146 14 (8)	117 6 (6)
Supernumerary ribs (no.foetuses(%))	7 (2.05)	19(5.6)	31(8.6)	59(20)	65(27.8)

Applicant's summary and conclusion

Conclusions:

The maternal NOAEL in this study was 6 mg/kg based on body weight gain reduction during early treatment at 24 mg/kg. The developmental NOAEL was 12 mg/kg based on reduced foetal body weight, increased post implantation loss and increased incidence foetal malformations at 24 and 48 mg/kg.

Executive summary:

A developmental toxicity study in rats was performed in accordance with OECD TG 414 and GLP principles, to determine the potential of the test substance to induce structural and/or other anomalies in the foetus which may arise from the exposure of the mother during pregnancy. The test substance was administered orally by intubation once daily to mated female Wistar rats (outbred, SPF-quality) from day 6 through day 15 post coitum. Animals were treated at dose levels of 0, 6, 12, 24 and 48 mg/kg bw by oral gavage at a constant dosing volume of 10 ml/kg bw. The vehicle that was used in this study was CMC. Each group consisted of 25 mated female rats. On day 21 post coitum, all females were sacrificed and the foetuses removed by caesarean section. The investigations of females/dams and foetuses were performed in accordance with international recommendations. All parameters recorded were evaluated and reported.

Results showed that no death occurred and no substance related signs or symptoms were observed. At necropsy, no test substance related findings were evident in any female of any group. The oral administration of the test substance caused loss of body weight in the dams of group 5 (48 mg/kg), from the first to second and from the second to third day of treatment. Thereafter the body weight gain was similar to that of the vehicle control group. The reason that the body weights remained below that of the vehicle control group was as noted as in group 4 (24 mg/kg), primarily the consequence of the lower number of foetuses per dam. A dose related and biologically significant body weight gain reduction of -18% versus control was observed at the 12 mg/kg bw/day dose level.The mean food consumption of groups 4 and 5 was significantly reduced (dose-related) during the treatment period in comparison to that of the vehicle control group. No significant or test substance related differences in body weight gain and food consumption were noted between the vehicle control group and group 2 (6 mg/kg) or 3 (12 mg/kg). The evaluations of the reproduction data resulted in dose related significantly increased post implantation losses of 22.2 % in group 4 (24 mg/kg) and 34.2 % in group 5 (48 mg/kg). In the vehicle control group and in groups 2 (6 mg/kg) and 3 (12 mg/kg) losses of 5.0 %, 4.2 % and 2.9 % were noted, respectively. In the foetuses, slight but significantly reduced mean body weights of 8.3 % were noted in groups 4 and 5 when compared to that of the vehicle control group. During the external investigations of foetuses, three foetuses of different litters/dams with anomalies were found in group 5 (48 mg/kg). One foetus had hydrocephalus and two foetuses had palatoschisis (one of which was a runt). These findings may be test substance related. The presence of one runt in groups 2, 3 and 4, respectively, was considered to be incidental.

Besides the mentioned foetus with hydrocephalus in group 5, one foetus in group 4 with hydrocephalus internus was found during visceral investigations by Wilson Technique. This single finding was considered to be incidental because of isolated appearance of foetuses with hydrocephalus internus in the historical background data of this rat strain employed. The skeletal investigations of foetuses for anomalies resulted in similar findings of nonspecific conventional variations in all groups. The comparison of the stage of skeletal development yields an increased incidence of incompletely or still absent ossifications of phalangeal nuclei and calcanea in the foetuses of group 4 (24 mg/kg) and group 5 (48 mg/kg). This result corresponded to the reduced body weights of foetuses.

In conclusion, mean body weight loss was seen in treated dams at the beginning of the treatment period (days 6-8) from 24 mg/kg bw/day. A dose related and biologically significant body weight gain reduction of -18% versus control was observed at the 12 mg/kg bw/day dose level.Mean bodyweight gain recovered thereafter. Mean food consumption was reduced in the 24 and 48 mg/kg bw/day from days 6 -11 and days 11- 16. Embryo/foetal toxicity was evident at 24 and 48 mg/kg from the following observations: decreased total number of live foetuses per dam, increased numbers of early and late resorptions, decreased foetal bodyweight and incomplete ossification of phalangeal nuclei and the absence of ossification in calcanea. At the highest dose one foetus was noted with a hydrocephalus and two foetuses had a palatoschisis (also reported as cleft palate), of which one was a runt. A single incidence of hydrocephalus internus was also apparent at the 24 mg/kg bw/day dose level. Given the probably treatment-related incidence in the 48 mg/kg bw/day, relationship to treatment is possible. The maternal NOAEL in this study was 6 mg/kg based on bodyweight gain reduction during early treatment at 12 mg/kg. The developmental NOAEL was 12 mg/kg based on reduced foetal bodyweight, increased post implantation loss and increased incidence foetal malformations at 24 and 48 mg/kg.