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

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Apr 1993 to 29 Sep 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7600 (Dermal Penetration)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

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

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD BR VAF/Plus

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 50 - 61 days
- Weight at study initiation: 225 - 250 g
- Fasting period before study: yes
- Housing: Individually in Nalgene metabolism cages
- Individual metabolism cages: yes
- Diet: rodent chow ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Apr 1993 To: 29 Sep 1993

Administration / exposure

Type of coverage:

open

Vehicle:

other: Sentinel® 40 WG

Remarks:

water used as a dose vehicle in spray dilutions

Duration of exposure:

0.5, 1, 2, 4, 10 and 24 hours

Doses:

- Nominal doses: 0.15, 0.015 and 0.0025 mg a.i./cm2 skin
- Actual doses: 0.1670, 0.0182 and 0.0025 mg a.i./cm2 skin
- Actual doses calculated as follows: group means of the actual dose received for each rat at each dose level
- Dose volume: 9-10 µL/cm2 skin
- Rationale for dose selection: The doses were selected to give a range of concentrations in order to determine the effect of concentration on the absorption and elimination of the test substance in male rats following a single dermal dose

No. of animals per group:

24 rats per dose level, plus a control group of 6 rats

Control animals:

yes

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: Dose suspensions were prepared by dissolving the required amount of a mixture of unlabelled test substance and [14C]-test substance with an appropriate volume of the formulation base and water

APPLICATION OF DOSE: Using a syringe, 90-100 µL of the dose preparation was applied and spread evenly across the surface of the defined skin site (10 cm2). The syringe was weighed before and after dosing to determine the amount of dose given. After the dose had dried, a template was attached to the back of each animal and fitted with a non-occlusive cover to prevent loss of test compound from the application site. Control rats were given a dermal application of an aqueous suspension of the blank formulation

TEST SITE
- Preparation of test site: hair clipping 24 hours prior to dosing (an area of 40 cm2)
- Area of exposure: 10 cm2
- Type of cover / wrap if used: non-occlusive patch, 6.0 x 8.0 cm sheet made of cellulose and pectin based material

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: Sub-groups of 4 treated rats were skin washed and terminated at 0.5, 1, 2, 4, 10 and 24 hours after dosing. Animals were anaesthetised for the skin washing procedure. The application site washed 5 times with a mild soap solution (5% in water) using cotton-wool balls, and rinsed with water after each washing step.

SAMPLE COLLECTION
Sub-groups of 4 treated rats were anaesthetised and terminated by exsanguination at 0.5, 1, 2, 4, 10 and 24 hours after dosing. One control rat was terminated at each time point. The terminal blood sample, treated skin, template, cover and carcass was retained for analysis. Urine, faeces and an ethanol:water (1:1 v/v) cage wash were collected at the termination of each experiment. Urine and faeces were frozen upon excretion by collection over solid carbon dioxide.

SAMPLE PREPARATION
- Storage procedure: Urine, faeces and carcass samples were stored at approximately -10°C prior to analysis. Blood, covers, cotton wool balls from skin washes, skin and cage wash samples were refrigerated prior to analysis.
- Preparation details: see "Any other information on materials and methods incl. tables"

ANALYSIS
- Method type(s) for identification: liquid scintillation counting and HPLC for concentration and stability determination
- Liquid scintillation counting results (cpm) converted to dpm as follows: disintegration per minute (dpm) values were calculated using the appropriate instrument-stored quench correction data.
- Limits of detection and quantification: typical limit of detection (LOD) values are reported for each sample type as percent of dose.

Results and discussion

Signs and symptoms of toxicity:

no effects

Dermal irritation:

no effects

Absorption in different matrices:

- Non-occlusive cover: less than 4 % AD
- Skin wash: 74 - 97 % AD
- Skin test site: 2.08 ± 0.8 % AD and 1.22 ± 0.65 % AD for all mid and high dose animals, respectively
- Blood: Small amounts of radioactivity were found in the blood. The blood concentrations did not appear to increase with exposure time.
- Carcass: at high dose: 0.75 ± 0.32 % AD; at mid dose: from 0.59 % AD after 0.5 hour to 2.00 % AD after 10 hours of exposure, between 10 and 24 hours, residues in carcasses increased to 10.1 % AD
- Urine: less than 1 % AD
- Cage wash + cage wipe: less than 0.1 % AD; amounts in excess of 0.25 % AD were found occasionally (4/72 cage washes)
- Faeces: Very little radiolabel was excreted in the faeces by the end of 24 h. An average of less than 1% of the dose was found in feces at any exposure time and at any dose level. Faeces from one animal exposed to the low dose for 24 hours contained 2.15 %AD compared to an average of 0.08 % AD for the other three 24 hour animals

Total recovery:

- Total recovery: 85 - 114 %AD
- Recovery of applied dose acceptable: yes, mean recovery of radiolabel from each group of animals was greater than 92 %AD

Percutaneous absorption

Percutaneous absorption 1

Key result

Time point:

10 h

Dose:

0.0025 mg/cm2

Parameter:

percentage

Absorption:

16.01 %

Remarks on result:

other: Highest absorption percentage in the dose group

Remarks:

Sum of the absorbed dose and the dose retained in the skin

Percutaneous absorption 2

Time point:

24 h

Dose:

0.0182 mg/cm2

Parameter:

percentage

Absorption:

14.31 %

Remarks on result:

other: Highest absorption percentage in the dose group

Remarks:

Sum of the absorbed dose and the dose retained in the skin

Percutaneous absorption 3

Time point:

24 h

Dose:

0.167 mg/cm2

Parameter:

percentage

Absorption:

2.94 %

Remarks on result:

other: Highest absorption percentage in the dose group

Remarks:

Sum of the absorbed dose and the dose retained in the skin

Any other information on results incl. tables

Table 1. Mean distribution of [¹⁴C]-test substance residues following application of an aqueous suspension of a 40 WG formulation to rat skin at a dose level of 0.1670 mg/cm².

	Recovery (% of applied dose)
	High dose level: 0.1670 mg/cm^2
Termination time	0.5 hours	1 hours	2 hours	4 hours	10 hours	24 hours
Urine	0.00	0.00	0.00	0.00	0.00	0.04
Faeces	0.00	0.00	0.00	0.00	0.01	0.01
Cage wash	0.00	0.00	0.00	0.00	0.00	0.02
Carcass + blood	0.92	0.53	0.73	0.85	0.66	0.82
Absorbed dose	0.92	0.53	0.73	0.85	0.67	0.89
Treated skin	0.97	2.10	1.12	0.90	0.67	2.05
Skin wash	96.99	95.70	94.46	93.34	93.74	92.57
Template	1.67	0.08	1.23	0.87	0.52	1.73
Cover	0.00	0.00	0.00	0.00	0.01	0.03
Unabsorbed dose	98.67	95.78	95.69	94.21	94.27	94.32
Total recovered	100.55	98.41	97.54	95.95	95.61	97.27

 

Table 2. Mean distribution of [¹⁴C]-test substance residues following application of an aqueous suspension of a 40 WG formulation to rat skin at a dose level of 0.0182 mg/cm².

	Recovery (% of applied dose)
	Mid dose level: 0.0182 mg/cm^2
Termination time	0.5 hours	1 hours	2 hours	4 hours	10 hours	24 hours
Urine	0.00	0.00	0.00	0.01	0.12	1.19
Faeces	0.00	0.00	0.00	0.00	0.01	0.37
Cage wash	0.00	0.00	0.01	0.01	0.03	0.24
Carcass + blood	0.59	0.82	1.04	1.42	2.00	10.08
Absorbed dose	0.59	0.82	1.05	1.43	2.16	11.88
Treated skin	2.19	2.08	2.85	1.28	1.66	2.43
Skin wash	97.02	87.90	94.40	91.82	88.14	80.08
Template	1.00	2.30	0.83	1.97	1.46	1.58
Cover	0.00	0.00	0.00	0.07	0.04	0.48
Unabsorbed dose	98.02	90.21	95.23	93.85	89.64	82.14
Total recovered	100.80	93.11	99.12	96.56	93.45	96.45

Table 3. Mean distribution of [¹⁴C]-test substance residues following application of an aqueous suspension of a 40 WG formulation to rat skin at a dose level of 0.0025 mg/cm².

	Recovery (% of applied dose)
	Low dose level: 0.0025 mg/cm^2
Termination time	0.5 hours	1 hours	2 hours	4 hours	10 hours	24 hours
Urine	0.00	0.00	0.00	0.20	0.81	0.92
Faeces	0.00	0.00	0.00	0.01	0.02	0.60
Cage wash	0.01	0.01	0.01	0.41	0.09	0.23
Carcass + blood	0.86	2.74	4.48	10.30	9.90	5.95
Absorbed dose	0.87	2.75	4.50	10.92	10.81	7.69
Treated skin	3.28	3.98	6.06	4.91	5.20	8.91
Skin wash	84.99	90.81	82.16	76.47	76.05	74.48
Template	1.02	0.63	1.18	1.64	1.90	2.02
Cover	2.09	1.34	0.17	0.05	0.15	0.39
Unabsorbed dose	88.11	92.79	83.51	78.16	78.10	76.90
Total recovered	92.25	99.52	94.07	93.98	94.11	93.50

Applicant's summary and conclusion

Conclusions:

Absorption at the high dose level of 0.167 mg/cm2 was minimal, amounting to 2.94 % including the dose absorbed and the dose retained in skin after 24 hours. Absorption at the mid dose level of 0.0182 mg/cm2 was low, amounting to 14.31 % including the dose absorbed and the dose retained in skin after 24 hours. At the low dose level of 0.0025 mg/cm2, absorption increased from 4.15 % including the dose absorbed and the dose retained in skin after 0.5 hours to a plateau level of approximately 16 % including the dose retained in skin after 10 hours. At all three dose levels investigated, the majority of the applied test substance was readily washed from the application site skin after the exposure period. The absorbed dose was excreted mainly in urine and to a lesser extent in faeces.

Executive summary:

In a GLP compliant study the test substance in Sentinel 40WG formulation base was administered dermally to male Sprague Dawley rats at dose levels of approximately 0.0025,  0.015 and 0.15 mg/cm2, in dosing volumes of about 0.1 mL, applied to 10 cm2  of dorsal skin. The test material was left on the skin for 0.5, 1, 2, 4, 10 and 24 hours. Four rats from each dose group were used for each exposure period. The application site was protected by a template and covered by a non-occlusive patch. For all animals, the application site was washed with a 5 % soap/water solution at the end of the exposure period prior to termination. Non-occlusive patches, skin washes, templates, skin, cage washes, urine, feces, blood and carcasses were analyzed for radioactivity.   The test material was absorbed to some extent via the dermal route of exposure. Absorption was not proportional to dose level except for an exposure time of 0.5 hour when the average absorption at all dose levels was approximately 0.79 % of the doses. At a dose level of 0.0025 mg/cm2, absorption appeared to reach a maximum after 4 hours and plateau at 9.81 % of the applied dose. At a dose  level of 0.015 mg/cm2, absorption was minimal through 10 hours when the average absorption was 2.16 % of the dose; absorption increased to approximately 12 % after an exposure period of 24 hours. At a dose level of 0.15 mg/cm2, absorption reached a maximum of approximately  0.90 % at 30 minutes, which suggested that this dose level represented an infinite dose of the test substance. Greater than 92 % of the applied doses were recovered at each exposure period, washes. At least 80 % of the recovered radiolabel was found in skin. Templates and nonocclusive patches contained only small amounts of the doses. Radiolabel in skin was relatively constant at any dose level and, in general, the percent of the dose in skin appeared to decrease as dose level increased. Carcasses contained a minimum of 77 %, but usually greater than 90 % of the absorbed dose at each exposure period and at all dose levels. Less than 2 % of the dose was excreted by 24 hours at the two lower dose levels and less than 0.1 % was excreted at the highest dose level. The data from this dermal absorption study suggest that the test substance in Sentinel 40WG is not extensively absorbed at any dose level from  0.0025 to 0.15 mg/cm .  Maximum absorption appeared to be approximately 1 % to 12 % of the dose at dose levels of 0.0025 and 0.015 mg/cm2. Absorption at the high dose level of 0.167 mg/cm2 was amounting to 2.94 % including the dose retained in skin after 24 hours. Absorption at the mid dose level of 0.0182 mg/cm2 was low, amounting to 14.31 % including the dose absorbed and the doser etained in skin after 24 hours. At the low dose level of 0.0025 mg/cm2, absorption reached a plateau level of approximately 16 % including the dose absorbed and the dose retained in skin after 10 hours.