Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jun 2007 to 20 Sep 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

distribution

excretion

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

Apr 1984

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)

Version / remarks:

Aug 1998

Qualifier:

according to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Version / remarks:

Dec 1994

Qualifier:

according to guideline

Guideline:

other: JMAFF, 12 Nohsan No 8147.

Version / remarks:

Nov 2000

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Remarks:

Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 - 7 weeks (high dose), 13 weeks (low dose)
- Weight at study initiation: Low dose: 217 - 224 g (males), 184 - 199 g (females); High dose: 157 - 170 g (females), 126 - 143 g (females)
- Housing: All animals were group housed in stock cages during the initial acclimatisation period. On study animals were housed individually in all glass metabolism cages.
- Diet: ad libitum
- Water: Mains water, supplied by a plastic bottle, ad libitum
- Acclimation period: 7 days (low) 1 day (high)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 40 – 70
- Air changes pe hour: 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 Jun 2007 To: 20 Sep 2007

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

DIET PREPARATION
- Both dose preparations were formulated as homogenous suspensions and comprised unlabelled and [14C]-test substance in the dose vehicle. For more details see Table 1 in 'Any other information on materials and methods incl. tables'.
- Low dose level: An appropriate amount (14.53 mg) of the [14C]-test substance was accurately weighed into a 5 mL volumetric flask. An appropriate volume (371 μL, equivalent to 4.061 mg) of 10.95 mg/mL test substance stock solution (prepared by dissolving 109.5 mg test substance into 10 mL acetonitrile) was accurately dispensed into the 5 mL volumetric flask. The flask was made up to volume with acetonitrile and gently shaken until the compound was dissolved. Aliquots were removed to determine the radio-diluted specific activity. The solution was transferred with washings of acetonitrile to a mortar board and gently evaporated to dryness under a steady stream of nitrogen. Once dried, the content of the mortar board was lightly wetted with corn oil and ground into a fine paste with a pestle. The paste was then transferred to a pre-weighed glass container. Additional corn oil was added to the mortar board again and ground with the pestle before transferring into the glass container. This process was repeated several times before the contents of the glass jar were then up to the final dose volume to achieve the required target concentration.
- High dose level: The high dose was prepared under another study An appropriate amount (1.818 mg) of test substance was accurately weighed into a 10 mL volumetric flask. An appropriate amount (19.4 mg) of [14C]-test substance was accurately weighed in a glass vial, dissolved in acetonitrile and transferred (with washings), to the 10 mL volumetric flask. The flask was made up to volume with acetonitrile, and gently shaken until the compound was dissolved. Aliquots were removed to determine the radio-diluted specific activity. The high dose formulation was prepared as detailed above using the same method as the low dose formulation.

Duration and frequency of treatment / exposure:

Single dose

Dose / conc.:

1 mg/kg diet

Remarks:

Group 1. Low dose excretion and tissue distribution

Dose / conc.:

75 mg/kg diet

Remarks:

Group 2. High dose excretion and tissue distribution

No. of animals per sex per dose / concentration:

4

Control animals:

no

Details on study design:

- Dose selection rationale: A dose level of 1 mg/kg was selected to represent a no-effect dose following a single oral administration. A dose level of 75 mg/kg was selected, following preliminary dose range finding work, to represent a dose that caused a mild and transient toxicological or pharmacological effect following single oral administration. Each rat was given a single oral dose of nominally 1 mg or 75 mg [14C]- test substance /kg and housed individually in a metabolism cage. Urine, faeces and cage washes were collected at intervals over 7 days after which time the rats were terminated. Selected tissues were removed and together with the residual carcass were analysed for residual radioactivity.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY
- Dose administration: The formulations were administered by gastric gavage at a target dose volume of 5 mL/kg to achieve target dose levels of 1 mg/kg and 75 mg/kg for low and high dose levels, respectively. For both dose levels, the animals received a target radioactive dose of 5 MBq/kg. Each animal was accurately weighed on the day of dosing. The syringes were weighed prior to and following each dosing. The actual dose received by each animal was determined with reference to the radioactive concentration, the weight of dose administered and the calculated specific activity of the dose formulation.
- Tissues and body fluids sampled: Urine and faeces were collected individually and separately by collection over solid carbon dioxide (up to 48 h post dose). Urine and faeces were frozen immediately upon collection. At the end of each faeces collection period, cage wash samples were collected (water).
- Time and frequency of sampling: Urine was collected at intervals of 12, 24, 48, 72, 96, 120, 144 and 168 hours after dosing. Faeces and cage wash were collected at intervals of 24, 48, 72, 96, 120, 144 and 168 hours after dosing.
To investigate pharmacokinetics, terminal blood samples were collected via the vena cava at 168 hours after dosing.
- Other: Animals were terminated by overexposure to anaesthetic vapour followed by cervical dislocation. Each terminal blood sample was divided between two heparinised tubes, one of which was centrifuged to separate plasma. The following tissues were taken for radioactivity analysis: adrenals, bone mineral, brain, renal fat, heart, kidneys, liver, lungs, muscle, ovaries, pancreas, spleen, testes (males), thymus, thyroid, uterus (females), gastrointestinal tract plus contents and residual carcasses. All samples were analysed for radioactivity by liquid scintillation counting either directly or following sample oxidation.

METABOLITE CHARACTERISATION STUDIES
Metabolite characterisation was undertaken in a separate study.

ANALYTICAL METHOD
- Sample oxidation: Duplicate samples (where possible) were oxidised in a Packard Tri-Carb 307 Automatic Sample Oxidiser. The [14C]-carbon dioxide generated was absorbed and mixed with scintillant, prior to analysis by liquid scintillation counting. The efficiency of oxidation of test samples relative to [14C]-standard oxidation efficiencies, which was determined at regular intervals during each series of oxidations. Combustion of standards showed that recovery efficiencies were all greater than 97 %.
- Liquid scintillation counting: All samples prepared in scintillation fluid were subjected to liquid scintillation counting for 5 minutes, together with representative blank samples, using a Packard TR 2100 Liquid Scintillation Analyser with automatic quench correction by an external method. Where possible, samples were analysed in duplicate and allowed to heat and light stabilise prior to analysis. Prior to calculation of each result, a background count rate was determined and subtracted from each sample count rate.

Statistics:

Not applicable.

Preliminary studies:

Not applicable.

Details on distribution in tissues:

Seven days after dosing the low dose level rats, the liver and kidney were the only two tissues with detectable radioactive residues. Seven days after dosing the high dose level rats, radioactive residues were only detectable in the liver (males and females) and kidney (males only). These findings were consistent with the rapid and extensive excretion observed in both sexes. Tissue distribution was similar in both sexes. In mean blood, plasma and other tissues the total radioactivity was below the limit of reliable detection.

Details on excretion:

Excretion of the test substance was fairly rapid, with the majority of the administered radioactivity excreted by 48 hours post dose (89.8 to 102.4% of applied dose). Rapid excretion with main route via faeces (77.3 to 83.0 %) and also (13.3 to 27.1 %) via urine. The routes and rates of excretion were similar for males and females for both low and high dose level animals, although urinary excretion appeared to be slightly greater in females.

Metabolites identified:

no

ANALYSIS OF RADIOLABELLED TEST SUBSTANCE

The HPLC and TLC radiochemical purities of [14C]- test substance as determined prior to the preparation of the dose formulation were 99.7 % and 100.0 % respectively. These purities were considered acceptable and the radiolabelled test substance was used without repurification. The mean radiochemical purities of [14C]-test substance in the low dose level formulation, as determined in pre-dose and post-dose aliquots, were 100.0 %, as determined by both HPLC and TLC. These data indicate that the test material was stable during dose preparation and for the duration of the dosing procedure. The mean radiochemical purities of [14C]-test substance in the high dose level formulation, as determined in pre-dose and post-dose aliquots, was >99.8% and >100.0 %, as determined by HPLC and TLC, respectively. These data indicate that the test material was stable during dose preparation and for the duration of the dosing procedure.

 

ACHIEVED DOSES

The mean achieved concentration of test substance for the low dose formulation, based upon the radioactive analysis, was 0.207 mg/g and 1.013 MBq/g of dose solution. The mean achieved concentration for the high dose formulation was 17.531 mg/g and 1.104 MBq/mg of dose solution. The achieved concentrations for low and high dose levels (taking the specific gravity of corn oil (0.9 g/mL) into account) were within 7 % and 1 % of the test substance target concentrations, respectively. The homogeneity of [C14]-test substance in the low and high dose level preparations was satisfactory. The radiochemical stability of test substance in the dose preparation was also shown to be satisfactory for at least 14 days which covered the period of use in the present study

 

CLINICAL OBSERVATIONS

No clinical observations were observed in either dose group during the study.

RECOVERY OF RADIOACTIVITY

The total mean percentage recoveries of administered radioactivity including excreta, tissues and residual carcasses following oral gavage dosing at 1 mg/kg were 106.0 % for males and 106.4 % for females. The total mean percentage recoveries of administered radioactivity including excreta, tissues and residual carcasses following oral gavage dosing at 75 mg/kg were 95.7 % for males and 100.5 % for females.  

DETAILS ON DISTRIBUTION

- Single low dose: Seven days after dosing, mean blood and plasma concentrations of total radioactivity were below the limit of reliable detection. The highest mean concentration was noted in the liver with values of 0.010 μg equiv/g and 0.005 μg equiv/g in males and females respectively. The kidney also had detectable levels, with mean values of 0.003 μg equiv/g and 0.002 μg equiv/g for males and females respectively. All other tissues had measurements below the limit of reliable detection.

- Single high dose: Seven days after dosing, mean blood and plasma concentrations of total radioactivity were below the limit of detection. The highest mean concentrations were found in the liver with values of 0.586 μg equiv/g and 0.284 μg equiv/g in males and females respectively. In males, the kidney also had detectable levels of total radioactivity with a mean concentration of 0.121μg equiv/g. In females, mean concentrations in the kidney were below the limit of reliable detection. All other concentrations in males and females were below the limit of reliable detection at this terminal time point.

DETAILS ON EXCRETION

- Single low dose: Following a single oral dose of 1 mg [C14]-test substance/kg, the major route of elimination was via the faeces in both males and females, with means of 83.0 and 77.3% of the administered radioactivity recovered by seven days post dose, respectively. Urinary excretion accounted for means of 19.6 and 27.1 % of the administered dose in both males and females, respectively, by the end of the sampling period. Excretion was rapid with the majority of the administered radioactivity being excreted in the first 48 h post dose (mean of ca 99.1  % and 102.4 % in males and females, respectively). There was no significant radioactivity remaining in the carcass or gastrointestinal tract, indicating excretion was complete by 168 hours post dose.

- Single high dose: Following a single oral dose of 75 mg [C14]-test substance /kg, the major route of elimination was via the faeces in both males and females, with means of 79.4 and 78.5 % of the administered radioactivity recovered by seven days, respectively. Urinary excretion accounted for means of 13.3 and 17.5 % of the administered dose in both males and females, respectively, by the end of the sampling period. Excretion was fairly rapid with the majority of the administered radioactivity being excreted in the first 48 hours post dose (mean of ca 89.8 and 92.2 % in males and females, respectively). There was no significant radioactivity remaining in the carcass or gastrointestinal tract, indicating excretion was complete by 168 hours post dose.

Table 3 Distribution of radioactivity tissues/organs 168 hours after administration of a single oral dose of [14C]-test substance to rats

Tissue/organ	Group mean tissue residues (µg equivalents of test substance/g )
	Group 1		Group 2
	1 mg/kg		75 mg/kg
	Male (n=4)	Female (n=4)	Male (n=4)	Female (n=4)
Adrenals	<0.002	<0.002	<0.147	<0.079
Bone Mineral	<0.001	<0.001	<0.013	<0.003
Brain	<0.001	<0.001	<0.008	<0.003
Fat- renal	<0.001	0.005	<0.022	<0.218
Heart	<0.001	<0.001	<0.023	<0.00001 -B
Kidneys	0.003	0.002	0.121	<0.057
Liver	0.010	0.005	0.586	0.284
Lungs	<0.001	<0.001	<0.035	<0.005
Muscle	<0.001	<0.001	<0.025	<0.027
Ovaries	NA	<0.002	NA	<0.071
Pancreas	<0.001	<0.001	<0.042	<0.042
Plasma	<0.001	<0.001	<0.007	<0.010
Residual Carcass-A	<0.0001 -B	<0.001	<0.088	<0.053
Spleen	<0.001	<0.001	<0.032	<0.010
Testes	<0.001	NA	<0.012	NA
Thymus	<0.001	<0.001	<0.011	<0.00001 -B
Thyroid	<0.003	<0.002	<0.087	<0.073
Uterus	NA	<0.001	NA	<0.002
Whole Blood	<0.002	<0.001	<0.058	<0.054

A = Value includes residual carcass and blood.

B = The number of significant figures has been raised (to a maximum of 5 decimal places) where appropriate when the value of detection is below the LOD.

Table 4 Recovery of radioactivity in tissues and excreta after administration of a single oral dose of [14C]-test substance to rats

		Group mean excretion data (percentage of radioactive dose recovered)
		Group 1 1mg/kg		Group 2 75 mg/kg
		Male (n=4)	Female (n=4)	Male (n=4)	Female (n=4)
Urine	0 - 12 h 12 - 24 h	14.7 3.2	20.5 4.7	7.5 3.5	7.8 6.8
	24 - 48 h	1.2	1.5	1.7	2.4
	48 - 72 h	0.3	0.3	0.3	0.3
	72 - 96 h	0.1	0.1	0.1	0.1
	96 - 120 h	0.0	0.0	0.1	0.1
	120 - 144 h	0.0	0.0	0.0	0.0
	144 - 168h	0.0	0.0	0.0	0.0
	Subtotal	19.6	27.1	13.3	17.5
Faeces	0 - 24 h	66.8	61.2	58.6	50.4
	24 - 48 h	13.2	14.5	18.4	24.8
	48 - 72 h	2.1	1.3	1.7	2.8
	72 - 96 h	0.7	0.2	0.4	0.3
	96 - 120 h	0.2	0.1	0.2	0.1
	120 - 144 h	0.1	0	0.1	0.1
	144 - 168h	0	0	0.1	0
	Subtotal	83	77.3	79.4	78.5
Cage wash		3.3	1.9	2.9	4.4
GI tract + contents		0.0	0.0	0.0	0.0
Tissues + carcass		<0.1	<0.1	0.1	<0.1
Total Recovery		106.0	106.4	95.7	100.5

Conclusions:

Following single oral administration of 1 or 75 mg [14C]-test substance to rats, elimination was rapid, with the majority of the administered radioactivity excreted by 48 h post dose. The major route of elimination was via the faeces, with urinary elimination also an important route of excretion. The routes and rates of excretion were similar for males and females for both low and high dose level animals, although urinary excretion appeared to be slightly greater in females. Seven days after dosing the low dose level rats the liver and kidney were the only two tissues with detectable radioactive residues. Seven days after dosing the high dose level rats radioactive residues were only detectable in the liver (males and females) and kidney (males only). These findings were consistent with the rapid and extensive excretion observed in both sexes. Tissue distribution was similar in both sexes.

Executive summary:

In an OECD TG 417 study in compliance with GLP, eight males and 8 female rats were given a single oral dose of 1 or 75 mg [14C]-test substance/kg to investigate the excretion of radioactivity over seven days. After this period, the rats were killed and residual radioactivity was measured in blood and plasma, selected tissues and the remaining carcasses.

Following a single oral dose of 1 mg [14C]-test substance//kg, the major route of elimination was via the faeces in both males and females, with means of 83.0 and 77.3 % of the administered radioactivity recovered by seven days post dose, respectively. Urinary excretion accounted for means of 19.6 and 27.1 % of the administered dose in both males and females, respectively, by the end of the sampling period. Excretion was fairly rapid with the majority of the administered radioactivity being excreted in the first 48 hours post dose (mean of ca 99.2 and 102.4 % in males and females, respectively). The total mean percentage recoveries of administered radioactivity including excreta, tissues and residual carcasses following oral gavage dosing at 1 mg/kg were 106.0 % for males and 106.4 % for females. Following a single oral dose of 75 mg [14C]-test substance//kg, the major route of elimination was via the faeces in both males and females, with means of 79.4 and 78.5 % of the administered radioactivity recovered by seven days post dose, respectively. Urinary excretion accounted for means of 13.3 and 17.5 % of the administered dose in both males and females, respectively, by the end of the sampling period. Excretion was fairly rapid with the majority of the administered radioactivity being excreted in the first 48 hours post dose (mean of ca 89.8 and 92.2 % in males and females, respectively). The total mean percentage recoveries of administered radioactivity including excreta, tissues and residual carcasses following oral gavage dosing at 75 mg/kg were 95.7 % for males and 100.5 % for females. Seven days after dosing the low dose level rats, radioactive residues in the majority of tissues were very low and the tissue distribution of radioactivity was similar for both sexes. The liver and kidney were the only two tissues with detectable residues, which were just over the limit of reliable detection.

Seven days after dosing the high dose level rats, radioactive residues were below the limit of reliable detection in all tissues apart from the liver and kidney, and tissue distribution was similar in both sexes. The liver contained mean concentrations of 0.586 and 0.284 μg equiv/g, in males and females respectively. The kidney contained detectable levels in males (0.121 μg equiv/g), but was below the limit of reliable detection in females.

Following single oral administration of 1 mg [14C]-test substance/kg or 75 mg [14C]-test substance/kg to rats, elimination was rapid with the majority of the administered radioactivity excreted by 48 hours post dose. The major route of elimination was via the faeces, with urinary elimination also an important route of excretion. The routes and rates of excretion were similar for males and females and for both the low and high dose level animals, although urinary excretion appeared to be slightly greater in females. Seven days after dosing the low dose level the liver and kidney were the only two tissues with detectable radioactive residues. Seven days after dosing the high dose level radioactive residues were only detectable in the liver (males and females) and kidney (males only).

Hence, the findings were consistent with the rapid and extensive excretion via faeces and urine observed in both sexes. Tissue distribution was similar in both sexes.

Reference 2

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Mar 2008 to 18 Apr 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

2004

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

[pyrazolyl-5-14C]

Species:

other: Human skin membrane

Type of coverage:

open

Vehicle:

water

Doses:

Concentrate (125 g/L) and two dilutions (nominally 1/83 and 1/1250)

Time point:

24 h

Concentrate / Dilution:

concentrate

Absorption:

2 %

Remarks on result:

other: dermal absorption = (stratum corneum + remaining epidermis + receptor fluid) + k * SD

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

1/83 v/v

Absorption:

6.1 %

Remarks on result:

other: dermal absorption = (stratum corneum + remaining epidermis + receptor fluid) + k * SD

Key result

Time point:

24 h

Concentrate / Dilution:

dilution

Dose:

1/1250 v/v

Absorption:

19.13 %

Remarks on result:

other: dermal absorption = (stratum corneum + remaining epidermis + receptor fluid) + k * SD

TEST SUBSTANCE ABSORPTION RATE

- Formulation concentrate: The absorption rate between 0 - 8 hours was 0.037 µg/cm2/h, after which it increased to 0.066 µg/cm2/h between 8 - 16 hours. Fastest absorption occurred between 16 - 24 hours, when test substance was absorbed at a rate of 0.085 µg/cm2/h. Between 0 - 24 hours the mean absorption rate was 0.061 µg/cm2/h.

-1/83 v/v aqueous spray dilution;The absorption rate between 0 - 6 hours the absorption rate was 0.008 µg/cm2/h, after which it increased to 0.016 µg/cm2/h between 6 - 12 hours. Fastest absorption occurred between 12- 24 hours, when test substance was absorbed at a rate of 0.018 µg/cm2/h. Between 0 - 24 hours the mean absorption rate was 0.015 µg/cm2/h.

-/1250 v/v aqueous spray dilution:test substance absorption was essentially linear throughout the entire 24 hour exposure period. Between 0-24 hours the mean absorption rate was 0.003 µg/cm2/h.

 

MASS BALANCE AND TEST SUBSTANCE DISTRIBUTION

-Formulation concentrate: Mean recovery of the applied test material was 104 %. The majority of the applied dose, 97.5 % was removed by gentle skin washing 24 hours after application. The proportion of the applied dose present in receptor fluid following the total 24 hour exposure was 0.122 %. This percentage equated to 1.48 µg/cm2. A total of 1.43 % of the applied dose remained in the epidermal membrane following a 24 hour skin washing procedure. Of this total, 0.261 % was present in the outer layers of the stratum corneum.

- 1/83 v/v aqueous spray dilution: Mean recovery of the applied test material was 101 %. Skin washing 24 hours after application removed 95.5 % of the applied dose. The proportion of the applied dose present in receptor fluid following the total 24 hour exposure was 2.66 %. In terms of actual amounts this percentage equated to 0.360 μg/cm2. A total of 1.19 % of the applied dose remained in the epidermal membrane following a 24 hour skin washing procedure. Of this total, 0.470 % was present in the outer layers of the stratum corneum.

- 1/1250 v/v aqueous spray dilution: Mean recovery of test material was 104 % of the applied dose. Skin washing 24 hours after application removed 94.2 % of the applied dose. The proportion of the applied dose present in receptor fluid following the total 24 hour exposure was 6.19 %. In terms of actual amounts this percentage equated to 0.063 μg/cm2. A total of 2.90 % of the applied dose remained in the epidermal membrane following a 24 hour skin washing procedure. Of this total, 0.761 % was present in the outer layers of the stratum corneum.

Table 1 Summary of Test substance Absorption through Human Epidermis

Application of Test Materials	Mean Absorption Rates		Mean Amount and Percentage of Dose Absorbed
	Time period (h)	Absorption rate (mg/cm^2/h ±SEM)	Time (h)	Amount (mg/cm^2)	Percentage absorbed
Formulation concentrate (122 g substance / L)	0-8	0.037 ± 0.007	6	0.191	0.016
10 mg/cm2 (1218μg/cm2)	8-16	0.066 ± 0.013	8	0.283	0.023
Unoccluded	16-24	*0.085 ± 0.018	10	0.387	0.032
Duration of exposure: 24 h	0-24	0.061 ± 0.012	24	1.48	0.122
n = 6			LOQ	0.038	0.003
1/83 v/v aqueous spray dilution (1.35 g substance / L)	0-6	0.008 ± 0.001	6	0.047	0.346
10 mg/cm^2(13.5 mg ai/cm^2) Unoccluded	6-12 12-24	0.016 ± 0.004 *0.018 ± 0.005	8 10	0.075 0.107	0.558 0.792
Duration of exposure: 24 h	0-24	0.015 ± 0.004	24	0.360	2.66
n = 6			LOQ	0.0004	0.003
1/1250 v/v aqueous spray dilution.
(0.10 g substance / L)			6	0.015	1.43
10 mg/cm^2(1.02 mg ai/cm^2)	0-24	*0.003 ± 0.001	8	0.020	1.95
Unoccluded			10	0.025	2.48
Duration of exposure: 24 h			24	0.063	6.19
n = 6			LOQ	0.0002	0.024

*Fastest rate of absorption over the 24h time course.

Table 2 Summary of Test Substance Distribution in the Test System after 24 Hours

Formulation concentrate
Test Compartment n = 5	μg test substance per cm²		% of applied dose
	Mean	SEM	Mean	SEM
Donor chamber	65.2	8.54	5.36	0.701
Skin wash	1187	18.5	97.5	1.522
Stratum corneum	3.18	0.356	0.261	0.029
Remaining epidermis	14.2	2.64	1.17	0.216
*Absorbed	1.48	0.298	0.122	0.024
Total recovered	1272	21.0	104	1.72
1/83 v/v dilution
Test Compartment n = 6	μg test substance per cm²		% of applied dose
	Mean	SEM	Mean	SEM
Donor chamber	0.212	0.145	1.57	1.07
Skin wash	12.9	0.288	95.5	2.13
Stratum corneum	0.064	0.021	0.470	0.156
Remaining epidermis	0.098	0.034	0.723	0.254
*Absorbed	0.360	0.096	2.66	0.708
Total recovered	13.7	0.370	101	2.73
1/1250 v/v dilution
Test Compartment n = 6	μg test substance per cm²		% of applied dose
	Mean	SEM	Mean	SEM
Donor chamber	0.004	0.002	0.358	0.176
Skin wash	0.959	0.052	94.2	5.12
Stratum corneum	0.008	0.002	0.761	0.185
Remaining epidermis	0.022	0.005	2.14	0.488
*Absorbed	0.063	0.026	6.19	2.55
Total recovered	1.06	0.026	104	2.55

Stratum corneum = amount in tape strips; Remaining epidermis = epidermal tissue remaining after tape stripping;

Absorbed = amount in receptor fluid

Table 3 Calculations human skin membrane (Concentrate) - Individual Distribution of test substance from the Formulation Concentrate in the Test System

Test Compartment	Percentage of Dose Recovered (%): concentrate
	Cell 50	Cell 51	Cell 52	Cell 53	Cell 54	Cell 56
Donor chamber	3.73	7.40	6.43	6.69	3.25	4.63
Skin wash	91.6	101	94.5	98.8	101	98.0
Stratum corneum	0.18	0.17	0.334	0.308	0.263	0.313
Remaining epidermis	1.64	1.71	1.48	0.363	0.836	0.981
Absorbed	0.126	0.068	0.041	0.179	0.121	0.195
Total recovered	97.3	110	103	106	106	104

Stratum corneum = amount in tape strips; Remaining epidermis = epidermal tissue remaining after tape stripping;

Absorbed = amount in receptor fluid

Absorption mean % = (1.95 + 1.95 + 1.86 + 0.85 + 1.22 + 1.49)/6 = 1.55 %

Std = 0.45

Multiplication factor (k) = 1

Dermal absorption% = 1.55 + (0.45*1) = 2.00 %

Table 4 Calculations human skin membrane (1/83 v/v Spray) - Individual Distribution of test substance from the 1/83 v/v Spray Strength Dilution in the Test System

Test Compartment	Percentage of Dose Recovered (%): 1/83 v/v Spray
	Cell 90a	Cell 91a	Cell 92a	Cell 93a	Cell 94a	Cell 95a
Donor chamber	6.71	0.065	0.739	1.87	0.005	0.004
Skin wash	89.6	91.2	96.1	102	101	92.8
Stratum corneum	1.10	0.181	0.478	0.722	0.165	0.173
Remaining epidermis	1.53	0.192	0.594	1.48	0.310	0.235
Absorbed	4.26	1.40	3.37	4.90	1.17	0.885
Total recovered	103	93.1	101	111*	103	94.1

Stratum corneum = amount in tape strips; Remaining epidermis = epidermal tissue remaining after tape stripping;

Absorbed = amount in receptor fluid

Absorption mean % = (6.89 + 1.77 + 4.44 + 1.65 + 1.29)/5 = 3.21 %

Std = 2.41

Multiplication factor (k) = 1.2

Dermal absorption% = 3.21 + (2.41*1.2) = 6.10 %

*(Cell 93a were excluded in the calculation of the mean absorption due to high recovery).

Table 5 Calculations human skin membrane (1/1250 v/v Spray) - Individual Distribution of test substance from the 1/1250 v/v Spray Strength Dilution in the Test System

Test Compartment	Percentage of Dose Recovered (%): 1/1250 v/v Spray
	Cell 35b	Cell 36b	Cell 37b	Cell 42b	Cell 44b	Cell 46b
Donor chamber	<0.001	<0.001	0.898	0.385	<0.001	0.866
Skin wash	104	106	84.8	101	94.8	74.1
Stratum corneum	0.112	0.559	0.816	0.892	0.691	1.50
Remaining epidermis	0.863	1.52	4.21	1.65	1.77	2.82
Absorbed	1.48	2.20	4.50	1.88	9.82	17.3
Total recovered	107	111*	95.2	105	107	96.5

Stratum corneum = amount in tape strips; Remaining epidermis = epidermal tissue remaining after tape stripping;

Absorbed = amount in receptor fluid

Absorption mean % = (2.46 + 9.53 + 4.42 + 12.28 + 21.62)/5 = 10.06 %

Std = 7.56

Multiplication factor (k) = 1.2

Dermal absorption% = 10.06 + (7.56*1.2) = 19.13 %

* (Cell 36b were excluded in the calculation of the mean absorption due to high recovery).

Conclusions:

Based on this in vitro dermal absorption study in human epidermis, a dermal absorption value of 2.00% was found for the concentrate and 19.13% for the highest dilution.

Executive summary:

In an OECD TG 428 study in compliance with GLP the absorption and distribution of test substance from an EC formulation was measured in vitro through human epidermis. The doses were applied as the formulation concentrate (nominally 125 g / L) and as two aqueous spray dilutions (1/83 v/v and 1/1250 v/v) of the formulation in water. The doses were applied to the epidermal membranes at a rate of 10 μL/cm2 and the weight recorded. The cells were left unoccluded for an exposure period of 24 hours. The absorption process was monitored using [14C]-labelled test substance, which was added to a blank formulation and water as appropriate, prior to application. The distribution of test substance within the test system was investigated and a 24 hour absorption profile was determined. The samples were analysed by LSC.

The absorption rate of the concentrate between 0 - 8 hours was 0.037 µg/cm2/h, after which it increased to 0.066 µg/cm2/h between 8 - 16 hours. Fastest absorption occurred between 16 - 24 hours, when test substance was absorbed at a rate of 0.085 µg/cm2/h. Between 0 - 24 hours the mean absorption rate was 0.061 µg/cm2/h. For the 1/83 v/v aqueous spray dilution, The absorption rate between 0 - 6 hours the absorption rate was 0.008 µg/cm2/h, after which it increased to 0.016 µg/cm2/h between 6 - 12 hours. Fastest absorption occurred between 12- 24 hours, when test substance was absorbed at a rate of 0.018 µg/cm2/h. Between 0 - 24 hours the mean absorption rate was 0.015 µg/cm2/h. For the 1/1250 v/v aqueous spray dilution, test substance absorption was essentially linear throughout the entire 24 hour exposure period. Between 0-24 hours the mean absorption rate was 0.003 µg/cm2/h.

Absorption was calculated according the Efsa guidance on dermal absorption (2017). Following the test conditions, dermal absorption is considered as the sum of the concentration in the Stratum corneum, remaining epidermis and receptor fluid. Cells with recovery rates below 90% or 110% were excluded from the calculation. To include an uncertainty factor on variance a multiplication factor based on the number of cells was multiplied with the standard deviation of the average dermal absorption observed. This results in the following dermal absorption values: 2.00, 6.10 and 19.13% for the concentrate, 1/83 dilution and 1/1250 dilution, respectively.

Based on this in vitro dermal absorption study in human epidermis, a dermal absorption value of 2.00% was found for the concentrate and 19.13% for the highest dilution.

Description of key information

- Oral absorption: approximately 85 % based on calculated radioactivity eliminated in urine and bile, and that in the residual carcass, OECD TG 417, Shaw 2008b.

- Metabolism: the substance undergoes extensive metabolism in the rat via hydroxylation, followed by further oxidation resulting in multiple hydroxyl moieties with subsequent formation of glucuronic acid or sulphate conjugates, OECD TG 417, Green 2014.

- Excretion: Rapid excretion predominantly via faeces and to a lesser extent to urine, with the urinary excretion being slightly more relevant in females, OECD TG 417, Shaw 2008a.

- Dermal absorption: 19.1 % (diluted product), in vitro absorption study in human skin, OECD TG 427, Davies 2008b.

- Inhalation absorption: Based on the lack of information on inhalation absorption, the default absorption value from the REACH guidance (Chapter 8, R.8.4.2) is used for DNEL derivation, namely: 100%.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

85

Absorption rate - dermal (%):

19.1

Absorption rate - inhalation (%):

100

Additional information

Absorption: Oral absorption of 63 – 73 % of applied dose in bile cannulated rats is considered to under-estimate absorption, as the majority of radioactivity in faeces of bile duct cannulated animals was shown to be unchanged parent (11 – 27 % of applied dose), representing unabsorbed material whereas the faeces of non-bile cannulated rats contained unchanged parent in only minor quantities (0.4 - 1.4 % of applied dose). Assuming that unchanged parent in faeces is representative of the unabsorbed dose, oral absorption is greater in non-bile cannulated animals, possibly because of the influence of bile on the absorption of test substance. Estimating oral absorption from the amount of unchanged parent present in the faeces from intact animals gives values > 85 % of applied dose (Shaw 2008b).

Dermal route: Studies demonstrated the amount of the test substance absorbed through human skin membrane and rat skin membrane over 24 hour with a concentrate and two dilutions (1/83 v/v and 1/1250 v/v). The recalculated values for sum of % Stratum corneum + % Remaining epidermis + % Absorbed+ k(n=6 for Formulation Concentrate and n=5 for both 1/83 and 1/1250 v/v Spray) x SD were 2.00 , 6.10 and 19.1 % for human skin membrane. No conclusion can be made for the in vivo rat skin based on the obtained data due to the limited number of remaining cells and high inconsistent fluctuations (Read 2008 and Davies 2008a) according to the dermal absorption EFSA guidance (2017).

Distribution: At low dose level radiolabelled dosing in rats, the liver and kidney were the only two tissues with detectable residues. Seven days after dosing the high dose level rats radioactive residues were only detectable in the liver (males and females) and kidney (males only). These findings were consistent with the rapid and extensive excretion observed in both sexes. Tissue distribution was similar in both sexes. Mean blood and plasma concentrations were below the limit of reliable detection at 48 hours post dose in both dose groups.

Metabolism: The test substance was extensively metabolized giving rise to up to 25 metabolite types (including conjugates) with potential for multiple isomers in most groups. The biotransformation was postulated to proceed by hydroxylation in the bicyclo-isopropyl region, followed by further oxidation to form the carboxylic acid and/or to give rise to multiple hydroxyl moieties with subsequent formation of glucuronic acid or sulphate conjugates. This oxidative pathway also occurred following N-demethylation of test substance giving rise to a corresponding set of oxidative N-desmethyl metabolites and their conjugates. No cleavage of the molecule was observed. In general, unconjugated metabolites were eliminated in urine and conjugated metabolites eliminated in bile (Green, 2014).

Excretion: Excretion of the test substance was fairly rapid, with the majority of the administered radioactivity excreted by 48 hours post dose (89.8 to 102.4% of applied dose). Rapid excretion with main route via faeces (77.3 to 83.0 %) and also (13.3 to 27.1 %) via urine. The majority of the dose excreted via faeces derives from excretion via bile accounting for 55 – 58 % and 48 - 57 % of applied dose in males and females, respectively, by 48 hours after dosing. The routes and rates of excretion were similar for males and females for both low and high dose level animals, although urinary excretion appeared to be slightly greater in females (Shaw 2008a).