pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

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:

16 Oct 1991 to 15 Mar 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

toxicokinetics

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

Version / remarks:

Adopted 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Agricultural Chemicals Laws and Regulations, Japan (II) Testing Guidelines of Toxicology Studies Society of Agricultural Chemical Industry

Version / remarks:

Adopted 1985

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

Adopted April 1984

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Tif: RAI f (SPF)

Details on species / strain selection:

Laboratory rats were selected as standard for rodent species to comply with the corresponding toxicological studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: 175 to 214 g (for males at dosing), 189 to 204 g (for females at dosing).
- Housing: Different housing per group:
A1, B1, D1, D2, E1-E4, F2 & F4: Closed all-glass metabolism cages, suitable for the collection of expired air (cage system 1)
C1: Wire floor polycarbonate cages during 13 days of pre-treatment and in cage system 1 immediately after receiving the last dose of non-radiolabelled test substance.
F1 & F3: Open plexiglass metabolism cages (cage-system 2)
Control: Cage-System 2, except one animal each of Group K1 (control animals for Group A1), K2 (control animals for Group B1, C1, D1), K3 (control animals for Group D2), and all animals of Group K4 (control animals for Group E1 through E4), which were housed in Cage-System 1.
- Diet: libitum, certified standard diet (except the night before administration of the radiolabelled test substance).
- Water: ad libitum, throughout the study.
- Acclimation period: for all groups at least two days to the laboratory environment including one day to the metabolism cages under test conditions.
- Identification: The animals were identified by individual numbers marked on ear tags and on the cage. The animal numbers were allocated randomly.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40 to 80
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
16 Oct 1991 to 15 Mar 1993

Route of administration:

other: Oral gavage or intravenous administration (only for group A1)

Vehicle:

other: Oral gavage: 0.5% sodium carboxymethyl cellulose containing 0.4% Tween 80. Intravenous administration: physiological saline (0.9% NaCl in water)

Duration and frequency of treatment / exposure:

See for details 'any other information incl tables'.

Dose / conc.:

0.5 mg/kg bw/day

Remarks:

low dose, actual dose received: 0.49 to 0.61 mg/kg

Dose / conc.:

100 mg/kg bw/day

Remarks:

high dose, actual dose received: 91.8 to 109.6 mg/kg

No. of animals per sex per dose / concentration:

Groups A1, B1, C1, D1 & D2: 5 (both sexes)
Groups E1 - E4: 4 (only males)
Groups F1 - F4: 4 (only males)

Control animals:

yes, concurrent vehicle

Details on dosing and sampling:

TOXICOKINETIC STUDY
- Tissues and body fluids sampled: urine, faeces, expired air, cage washes, blood, plasma, tissues (bone, brain, fat (abdominal, heart, kidneys, liver , lungs, muscle (skeletal), spleen, gonads, uterus, carcass and total residues as % of total dose.
- Time and frequency of sampling:
[Groups A1, B1, C1, D1 & D2]
Urine and faeces: 0-24, 24-48 and 48-168 hours
Expired air 0-24, 24-48 and 48-72 hours
[Groups E1-E4]
Blood: 0.25, 0.5, 1, 2 and 4 hours (first two animals), 4, 8, 12, 24 and 48 hours (second two animals.
[Groups F1-F4]
Tissues:
F1: 15 min, 1.75, 3.25, 5.25 hours
F2: 4, 11, 17 and 21 hours
F3: 15 min, 50 min, 1.75 and 3.25 hours
F4: 4, 11, 17 and 21 hours

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: urine 0-24 hours, faeces 0-48 hours
- From how many animals: urine samples and faeces samples were pooled according to animal group and sex
- Method type for identification: HPLC-UV

DOSE ADMINISTRATION
For the oral administrations the test substance was dissolved (low dose level) or suspended (high dose level) in 0.5% sodium carboxymethyl cellulose containing 0.4% Tween 80. Two dose levels were used. Each animal received about 0.8 mL of the administration solution by stomach tube, except the animals of Group E3 (0.9 mL).
For the pre-treatment period the animals of Group C1 received the respective amount of non-radiolabelled test substance dissolved in 0.6 - 0.8 mL of the administration solution.
For the intravenous administration (Group A1) the test substance was dissolved in physiological saline (0.9% NaCl in water). Each animal received about 0.5 mL of the administration solution by syringe into the tail vein.

Type:

absorption

Results:

Radioactivity was rapidly and almost completely absorbed from the GI tract into the general circulation (84.7% of dose). Maximum concentrations in the blood were reached 15 min and 4 h after administration at the low and high dose level, respectively.

Type:

distribution

Results:

Seven days after administration, mean residue levels were below 0.038 ppm in all tissues. At high dose levels, residue concentrations in fat were 200 times higher than other tissues.

Type:

metabolism

Results:

HPLC analysis revealed complex urinary and faecal metabolite pattern, independent of the sex, pre-treatment, dose level. The cleavage of the bridge between the triazine and pyridine ring is of minor importance in the metabolism in the rat.

Type:

excretion

Results:

Within 24 hours totally 80 to 90% of the administered dose were excreted, mainly with urine.

Details on absorption:

An overview of absorption data of group A1, B1, C1, D1 & D2 can be found in Table 1 in ‘any other information on results incl. tables’.
Following oral administration in the first study, radioactivity was rapidly and almost completely absorbed from the gastrointestinal tract into the general circulation (range 57.4 to 84.7% of dose). Independent of the label, maximum concentrations in the blood were reached 15 minutes and 4 hours after administration at the low and high dose level, respectively. These maximum values accounted for about 0.3 ppm and 60 ppm, for the 0.5 and 100 mg/kg dose levels.
The renal elimination was used for the calculation of the bioavailability of the oral dose. This factor F can approximately be calculated by the ratio of the total urinary excretion after p.o. and i.v. administration. The bioavailability was determined to be 0.9 for both sexes, demonstrating also a high extent of absorption.

Details on distribution in tissues:

An overview of the tissues residues for groups B1, C1, D1 & D2 can be found in Table 2 in ‘any other information on results incl. tables’.
An overview of depletion kinetics in several tissues for groups F1, F2, F3 & F4 can be found in Table 3 in 'any other information on results incl. tables’.
Seven days after a single oral administration of [14C-triazine] labelled test substance at a low dose level, tissue residues were low. However, detectable residues were measured in all tissues. The mean values were below 0.025 ppm for all tissues except for heart with 0.038 ppm. At the 100 mg/kg dose level the tissue residues were only in fat accordingly higher, i.e. about 200 times. In brain, heart and muscle the residues were about 5 times, and in all other tissues and organs they were 20 to 50 times higher than at the low dose. This indicates a saturation of tissue binding sites and an unfettered distribution of radioactivity (most probably unchanged test substance) in the fat. This assumption is supported by the distribution of residual radioactivity in the pre-treated animals.
The experiments with [5-14C] pyridine labelled test substance revealed only slight differences compared to the triazine label. While the excretion pattern was independent of the label, and the urinary and faecal metabolite patterns were qualitatively similar, tissue residues were higher with the [5-14C] pyridine labelled compound. The total residual radioactivity accounted for about 3 - 4% of the dose compared to about 1% of the dose after administration of [6- 14C] triazine labelled test substance.
Seven days after administration only about 10 to 15% and 5% of the radioactivity covered in the whole blood were associated with the plasma in the animals of Group D1 and D2, respectively. This ratio was higher at earlier time points after dosage, i.e. about 60% at 4 hours after administration, decreasing to values of about 50% and 15% at 21 hours after dosing for Group D1 and D2, respectively.
The calculated half-life times (t½) for the depuration of the residual radioactivity from the tissues, assuming to follow (monophasic) first order kinetics, were in the range of 1 to 2 hours at the 0.5 mg/kg dose level (both labels). At the high dose level the half-life times were between 3 and 6 hours (except fat: 11 hours) and 2 to 11 hours for the triazine and pyridine label, respectively.

Details on excretion:

An overview of the excretion data for groups A1, B1, C1, D1 & D2 can be found in Table 4 in ‘any other information on results incl. tables’.
The principal route of excretion was via urine with a total of 56 to 80% of the dose. Excretion was rapid. Within 24 hours 52 - 74%, 10 - 37%, and 0.2 - 1.2% of the administered dose were detected in urine, faeces, and expired air, respectively. The excretion pattern was essentially independent of sex, route of administration and pre-treatment with non-radiolabelled test substance. At the high dose level, both sexes eliminated significantly more via kidneys than at the low dose level. The excretion data after intravenous and oral administration (Group A and B1) were on the other side only slightly different, confirming the complete absorption from the gastrointestinal tract.

Metabolites identified:

no

Remarks:

A quantitative distribution of the metabolite fractions is provided

Details on metabolites:

An overview of the quantitative distribution of the metabolite fraction in urine and faeces given as percent of dose for groups A1, B1, C1, D1 & D2 can be found in tables 5 and 6 in 'any other information on results incl, tables'.
URINE: The chromatography revealed a complex metabolite pattern, consisting of up to 14 metabolite fractions. The pattern were qualitatively similar for both sexes, both dose levels, and both labels. The most unpolar metabolite fraction, i.e. U14, accounted for 15 - 22% of the dose (high dose, both labels), 2% (low dose, i.v.), and less than 1 % (low dose, p.o.), cochromatographed with unchanged test substance.
FAECES: HPLC analysis of the extracts revealed up to 13 metabolite fractions. The pattern were qualitatively similar for all groups. Some fractions showed quantitative differences especially between both labels. One of the major metabolite fractions, i.e. F5, showed the same retention time as the urinary fraction U8. The metabolite fraction F11 cochromatographed with unchanged test substance and corresponded thus to the urinary fraction U14. The metabolite pattern revealed only slight differences between the labels indicating that the cleavage between the triazine and pyridine ring represents a minor metabolic pathway. But the resulting pyridine related metabolite(s) is (are) responsible for the observed significant higher tissue residues after administration the [14C-pyridine] labelled test substance.

Bioaccessibility (or Bioavailability) testing results:

not measured

Clinical signs: the appearance and the behaviour of the animals were observed at each sampling time. No test substance related or otherwise unusual behaviour of the animals were observed.

Dosing formulation stability: The stability of the test substance in the administration vehicle, at the time of dosing, was found to be stable. The test substance represented more than 95% of the radioactivity.

For the repeated dosing experiment (Group C1, pre-treatment with non-radiolabelled test substance) the stability of the test substance in the administration vehicle was investigated over a period of 19 days prior to the first dosing using radiolabelled material. After 19 days in the refrigerator the radiopurity determined by TLC was still above 96%.

Table 1. Absorption of radiolabelled test substance after intravenous and oral administration

Route of Adm.	intravenous		per oral
Label	[6-14C] triazine								[5-14C] pyridine
Group	A1		B1		C1		D1		D2
Sex	male	female	male	female	male	female	male	female	male	female
Dose[mg/kg]	0.54	0.52	0.52	0.53	0.51	0.55	97.8	93.8	99.4	99.6
Urine	65.6	69.9	56.3	62.1	56.8	64.2	72.5	78.3	76.9	80.3
Expired Air	0.3	0.6	0.2	0.4	0.2	0.5	1.2	1.4	0.4	0.6
Tissues	0.4	0.3	3.7	2.4	0.4	0.4	0.6	1.0	3.7	3.8
Sum	66.3	70.8	60.2	64.9	57.4	65.1	74.3	80.7	81.0	84.7

 

Table 2. Tissue residues in ppm test substance equivalents, 7 days after administration

Label	[6-14C]triazine						[5-14C]pyridine
Group	B1		C1		D1		D2
Sex	male	female	male	female	male	female	male	female
Dose (mg/kg) [mg/kg]	0.52	0.53	0.51	0.55	97.8	93.8	99.4	99.6
Blood	0.0043	0.0035	= LD	< LQ	0.153	0.183	0.962	1.125
Bone	0.0034	0.0026	0.0010	= LQ	0.131	0.127	0.679	0.656
Brain	0.0155	0.0110	< LQ	< LQ	0.078	0.096	3.190	3.600
Fat (abdominal)	0.0041	0.0039	0.0036	0.0035	1.088	0.833	0.813	0.671
Heart	0.0377	0.0275	< LQ	= LQ	0.112	0.141	7.461	8.648
Kidneys	0.0218	0.0171	0.0046	0.0041	0.912	0.864	4.094	4.666
Liver	0.0249	0.0188	0.0080	0.0073	0.985	0.969	3.750	3.940
Lungs	0.0077	0.0063	0.0011	0.0014	0.166	0.236	1.590	1.753
Muscle (skeletal)	0.0244	0.0171	0.0008	= LQ	0.151	0.119	5.057	5.240
Plasma	< LQ	< LQ	< LQ	= LQ	0.034	0.054	0.072	0.097
Spleen	0.0110	0.0087	< LQ	< LQ	0.138	0.183	1.692	1.970
Gonads	0.0069	0.0105	< LQ	< LQ	0.094	0.265	1.248	2.837
Uterus	---	0.0047	---	< LQ	---	0.179	---	1.551
Carcass	0.0141	0.0110	0.0012	0.0016	0.472	0.900	2.780	3.340
Total residues [% of dose]	3.7	2.4	0.4	0.4	0.6	1.0	3.7	3.8

Table 3. Depletion kinetics in half-life time (h)

Label	[6-14C]triazine		[5-14C]pyridine
Group	F1	F2	F3	F4
Dose (mg/kg)	0.53	99.7	0.57	105.3
Time interval (h)	¼-5¼	4- 21	¼-3¼	11- 21
Blood	1.2	3.1	0.9	3.4
Bone	1.9	3.6	1.2	6.0
Brain	1.4	3.3	1.2	8.8
Fat (abdominal)	1.5	10.9	1.7	10.6
Heart	1.1	2.9	1.0	6.6
Kidneys	1.4	4.4	1.4	7.8
Liver	1.5	5.5	2.3	10.8
Lungs	1.2	3.2	1.4	6.9
Muscle (skeletal)	1.5	2.9	1.1	4.8
Plasma	1.2	3.0	0.8	2.2
Spleen	1.3	3.3	1.4	7.7
Testes	1.8	2.8	1.9	4.3
Carcass	1.7	3.8	1.8	5.0

Table 4. Excretion after intravenous and oral administration [% of dose]

Label	[6-14C] triazine								[5-14C] pyridine
Administration	intravenously		orally						orally
Group	A1		B1		C1		D1		D2
Sex	male	female	male	female	male	female	male	female	male	female
Dose[mg/kg]	0.54	0.52	0.52	0.53	0.51	0.55	97.8	93.8	99.4	99.6
Urine 0 - 24h 24 -48h 48 -168h Subtotal	63.6 0.9 1.1 65.6	68.3 0.9 0.7 69.9	52.0 1.8 2.5 56.3	58.4 1.8 1.9 62.1	55.4 0.9 0.5 56.8	63.0 0.8 0.4 64.2	69.6 2.0 0.9 72.5	73.5 3.9 1.0 78.3	70.2 3.0 3.7 76.9	73.5 3.1 3.7 80.3
Faeces 0 - 24h 24 - 48h 48 -168h Subtotal	24.9 1.1 0.5 26.5	20.3 1.2 1.1 22.6	26.6 2.7 1.1 30.3	23.0 3.7 1.1 27.8	36.6 1.8 0.5 38.9	27.1 2.7 0.6 30.3	20.8 3.9 0.7 25.4	11.6 5.0 1.4 18.0	15.6 2.8 1.0 19.5	10.2 4.3 1.0 15.4
Expired Air	0.3	0.6	0.2	0.4	0.2	0.5	1.2	1.4	0.4	0.6
Cage Wash	0.2	0.7	0.5	0.4	0.3	0.4	0.3	0.4	0.3	0.3
Total Excretion	92.7	93.6	87.3	90.7	96.1	95.4	99.4	98.1	97.0	96.6

Table 5. Quantitative distribution of the metabolite fraction in urine given as percent of dose

Label	[6-14C]triazine								[5-14C]pyridine
Group	A1		B1		C1		D1		D2
Sex	male	female	male	female	male	female	male	female	male	female
Metabolite Fraction
U1	5.2	3.7	1.2	0.9	11.2	6.1	2.4	1.7	0.9	1.3
U2	9.0	7.2	1.7	1.3	5.2	4.9	5.1	3.6	1.8	1.7
U3	2.3	1.8	3.0	2.1	2.7	2.3	1.6	1.9	1.6	2.6
U4	1.5	2.4	1.1	0.7	2.5	2.8	1.7	3.0	1.3	1.1
U5	1.3	1.3	8.5	5.9	1.7	1.0	1.0	1.1	4.1	5.4
U6	3.1	2.5	6.1	5.0	5.7	4.4	2.0	1.2	1.5	1.7
U7	3.7	4.2	4.1	7.2	5.0	6.5	2.8	2.7	4.9	4.4
U8	5.4	8.8	5.3	11.2	6.3	12.6	4.0	5.0	4.4	3.6
U9	3.5	4.1	3.8	4.4	4.4	5.5	2.9	2.9	2.1	2.1
U10	3.1	2.7	2.8	3.1	2.4	3.0	3.1	4.0	3.8	2.6
U11	16.1	15.9	9.5	9.9	4.1	5.8	17.7	16.2	18.5	17.8
U12	1.1	1.3	0.7	0.8	1.2	1.8	3.4	2.4	4.1	3.2
U13	6.3	9.9	3.6	5.3	2.2	5.5	7.2	9.5	5.3	4.3
U14	2.1	2.6	0.6	0.7	0.6	0.8	14.5	18.3	16.9	21.7
Sum	63.6	68.3	52.0	58.4	55.4	63.0	69.6	73.5	70.2	73.5

 

Table 6. Quantitative distribution of the metabolite fraction in faeces given as percent of dose

Label	[6-14C]triazine								[5-14C]pyridine
Group	A1		B1		C1		D1		D2
Sex	male	female	male	female	male	female	male	female	male	female
Metabolite Fraction
0	5.3	4.7	2.3	2.2	6.2	6.6	3.9	2.5	1.1	0.9
F2	1.0	1.3	1.7	1.4	1.7	1.4	1.5	0.9	0.9	0.6
F3	1.6	1.1	2.2	1.3	2.1	2.0	1.9	1.3	0.3	0.3
F4	1.7	1.5	3.0	3.0	2.6	2.3	1.7	1.1	1.4	1.3
F5	4.9	3.1	6.0	5.7	8.3	4.7	2.5	1.3	2.4	1.1
F6	0.7	0.7	1.0	0.9	0.7	0.7	0.6	0.2	0.6	0.4
F7	0.3	0.3	1.5	1.9	0.7	0.3	0.5	0.3	1.1	1.6
F8	0.1	0.2	0.2	0.2	0.2	0.1	0.5	0.7	0.6	0.5
F9	0.8	1.4	0.8	0.9	0.7	0.7	1.5	1.4	1.9	1.7
F10	1.0	0.6	1.4	1.5	1.2	0.9	0.8	0.6	0.6	0.6
F11	0.3	0.2	0.3	0.5	0,3	0.1	0 7	0.4	1.1	1.1
F12	0.7	0.3	0.2	0.6	0.6	0.4	0.2	0.2	0.3	0.2
F13	0.7	0.5	1.4	1.3	1.2	1.2	0.9	0.6	0.7	0.6
Sum Eluat 2	19.0	16.0	21.9	21.3	26.7	21.5	17.1	11.5	13.1	10.8
Eluat 1 + 3	2.2	1.5	2.6	1.7	2.6	1.5	1.5	0.4	3.0	1.9
Extract 2	1.7	1.0	2.1	1.1	4.9	2.1	3.1	2.3	0.9	0.6
Nonextract.	3.2	3.1	2.7	2.5	4.2	4.6	3.1	2.3	1.5	1.1
Total	26.1	21.5	29.2	26.7	38.4	29.7	24.8	16.6	18.5	14.5

Conclusions:

- Absorption: Radioactivity was rapidly and almost completely absorbed from the GI tract into the general circulation (maximum, D2 mean 84.7). Maximum concentrations in the blood were reached 15 minutes and 4 hours after administration at the low and high dose level, respectively.
- Distribution: Seven days after administration, mean residue levels were below 0.038 ppm in all tissues. At high dose levels, residue concentrations in fat were 200 times higher than other tissues. This indicates a saturation of tissue binding sites and an unfettered distribution of radioactivity (most probably unchanged test substance) in the fat at high doses. This assumption is supported by the distribution of residual radioactivity in the pre-treated animals. This finding is not considered to be an indication for bioaccumulation based on the efficient metabolism and excretion
- Metabolism: HPLC analysis revealed complex urinary and faecal metabolite pattern, independent of the sex, pre-treatment, dose level. The cleavage of the bridge between the triazine and pyridine ring is of minor importance in the metabolism in the rat.
- Excretion: The principal route of excretion was via urine with a total of 56 to 80% of the dose. Excretion was rapid. Within 24 hours 52 - 74%, 10 - 37%, and 0.2 - 1.2% of the administered dose were detected in urine, faeces, and expired air, respectively

Executive summary:

In this GLP compliant ADME study performed according to OECD 417, single oral doses of the labelled test substance were administrated at two dose levels (low dose: 0.5 mg/kg; high dose: 100 mg/kg body weight) to several groups of male and female rats (strain: Tif: RAI f (SPF)). Group A1 received a single intravenous administration at the low dose level. Group B1, C1, D1 and D2 were exposed orally and urine, faeces and expired air were collected at different times. After 7 days the rats were sacrificed and tissues were analysed for radiolabelled content. Group E1 to E4 (all males) were designated for a timewise analysis of radiolabelled content in the blood. Groups F1 to F4 were designated for analysis for radiolabelled content in tissues at several time points.

No test substance related or otherwise unusual behaviour of the animals were observed. The orally administered test substance was fast and almost completely absorbed from the gastrointestinal tract into the general circulation (maximum, D2 mean 84.7% of dose, minimum, C1 mean 57.4% of dose). Independent of the label maximum concentrations in the blood were reached 15 minutes and 4 hours after administration at the low and high dose level, respectively. These maximum values accounted for about 0.3 ppm and 60 ppm, for the 0.5 and 100 mg/kg dose level. The residual radioactivity was determined in several tissues and organs of male rats at different time points after oral administration using both labels at both dose levels. The calculated half-life times (t½) for the depuration of the residual radioactivity from the tissues, assuming to follow first order kinetics, were in the range of 1 to 2 hours at the 0.5 mg/kg dose level (both labels) and between 3 and 6 hours (except fat: 11 hours) for the triazine label at the high dose level. For the high dose pyridine label the half-life times were in the range of 2 to 11 hours. Seven days after a single oral administration of 0.5 mg/kg, low but detectable residues were measured in all tissues and organs. The mean values were below 0.025 ppm test substance equivalents, except in the heart (0.038 ppm). Highest amounts besides the heart were found in liver (0.025 ppm), muscle (0.024 ppm, and kidneys (0.022 ppm).At the high dose level (Group D1, 100 mg/kg), the tissue residues were only in fat accordingly higher, i.e. about 200 times. This indicates a saturation of tissue binding sites and an unfettered distribution of radioactivity (most probably unchanged test substance) in the fat at high doses. This assumption is supported by the distribution of residual radioactivity in the pre-treated animals. This finding is not considered to be an indication for bioaccumulation based on the efficient metabolism and excretion. In brain, heart, and muscle the residues were about 5 times in all other tissues and organs 20 to 50 times higher. The highest residues were detected in fat (about 1 ppm test substance equivalents). These data indicate a saturation of tissue binding sites, except in fat, where an unhindered distribution of radioactivity (most probably unchanged test substance) was observed. The residual radioactivity determined in the pre-treated animals support this assumption. The principal route of excretion was urine (totally 56 to 80% of the dose). The absorbed amount was rapidly excreted. Within 24 hours 52 - 74%, 10 - 37%, and 0.2 - 1.2% of the administered dose were detected in urine, faeces, and expired air, respectively. The excretion pattern was essentially independent of the sex, the route of administration, and pre-treatment with non-radiolabelled test substance. Both sexes eliminated significantly more via the kidneys at the high dose level, i.e. 73 - 80% instead of 55 - 65% at the low dose level.

HPLC analysis revealed complex urinary and faecal metabolite pattern. The chromatograms were qualitatively similar for both sexes, both dose levels, the i.v. and p.o. administration, and the pre-treated animals. Some metabolite fractions showed quantitative differences between the sexes and the dose levels. The most unpolar urinary fraction, accounting for 15 - 18% and 1 - 2% of the dose at the high and low dose level, respectively, cochromatographed with unchanged test substance. In the faeces <1% of the administered dose were characterized as unchanged test substance.

The experiments with the [5-14C] pyridine labelled test substance revealed only slight differences compared to the triazine label. The excretion pattern was independent of the label. The urinary and faecal metabolite pattern were qualitatively similar for both labels but showed some quantitative differences.

In summary, the test substance was after oral administration to rats was fast and almost completely absorbed and rapidly eliminated, mainly with urine. The test substance was extensively metabolized and the metabolic pathways are independent of the sex, pre- treatment, and the dose level. The cleavage of the bridge between the triazine and pyridine ring is of minor importance in the metabolism of the test substance in the rat.

Reference 2

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jul 2011 to 28 Jul 2011

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD (Guidance Document No. 28). The Conduct of Skin Absorption studies

Version / remarks:

2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: European Commission Guidance Document on Dermal Absorption

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Dermal Technology Laboratory Ltd. Med IC4, Keele University Science and Business Park, Keele, Staffordshire, ST5 5NL, United Kingdom

Radiolabelling:

yes

Type of coverage:

open

Vehicle:

water

Duration of exposure:

24 hours

Doses:

- Test solutions: 1/2000 w/w aqueous spray strength dilution containing a nominal 0.25 g test substance/kg, a 1/500 w/w aqueous spray strength dilution containing a nominal 1 g test substance/kg and a formulation concentrate containing a nominal 500 g test substance/kg
- Nominal doses: 2.59, 10.5 and 1626 µg test substance per cm2
- Dose volume: 25.4 µL

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Type of skin: Human skin, abdomen, female, age 51 to 91 years
- Preparative technique: The skin samples were immersed in water at 60ºC for 40-45 seconds and the epidermis teased away from the dermis. Each membrane was given an identifying number and stored frozen, at approximately -20ºC, on aluminium foil until required for use.
- Membrane integrity check: yes, range 10.19 to 24.94 kΩ
- Storage conditions: stored for 50 to 56 weeks and one sample 192 weeks

PRINCIPLES OF ASSAY
Cells were selected such that each application was represented by six intact membranes from at least four different donors. The receptor chambers of the cells containing small magnetic stirrer bars were filled with a recorded volume of receptor fluid (50% v/v ethanol/water) and placed in a water bath maintained at a temperature of 32ºC ± 1ºC.
A pre-treatment sample (0.1 mL (aqueous dilutions) or 0.5 mL (slurry)) was taken from each receptor chamber for analysis by LSC. The volume reduction in the receptor chamber was immediately compensated by fresh receptor fluid.
The test substance formulations were applied to the skin surface by volume using a suitable positive displacement pipette. The dose was applied drop-wise over the skin to maximise coverage. The weight of dose applied was recorded.
Skin absorption for each application was followed for 24 hours. The exposed skin area was left unoccluded.
Samples (0.1 mL (aqueous dilutions) or 0.5 mL (slurry)) of receptor fluid were taken from the receptor chambers of this static cell system 1, 2, 3, 4, 6, 8, 10, 12, 16, 20 and 24 hours after application using an autosampler. The receptor fluid in the chambers was stirred continuously and the receptor volume was maintained by the replacement of a volume of fresh receptor fluid, equal to the sample volume, after each sample had been taken. The samples of receptor fluid were analysed by LSC.
After the final receptor fluid sample had been taken at the end of the exposure period, the remaining fluid in the receptor chamber was discarded. The receptor chamber was again refilled with fresh receptor fluid (approximately 5 mL), which was, afterwards, discarded.
The donor chamber was carefully removed and the underside (surface in contact with the membrane) wiped with one natural sponge pre-wetted with a dilute soap solution (3% Teepol in water) which was added to the wash sponges (below). The donor chambers were washed with methanol and the sample of the washing taken for analysis by LSC.
The epidermal surface of the skin was decontaminated by gently swabbing the application site with natural sponges pre-wetted with 3% Teepol in water, decontamination was assessed to be complete using a Geiger counter to measure the radioactivity on the skin/sponges, and with a further two sponges pre-wetted with water (one cell received three sponges) .The sponges were digested in a solubilising agent (Soluene 350) and a sample taken for analysis by LSC.
To assess penetration through human stratum corneum, successive layers of the stratum corneum were removed by the repeated application of adhesive tape to a maximum of 5 strips.
The surface of the skin was allowed to dry naturally. Strips of adhesive tape were sequentially pressed onto the skin surface and then carefully peeled off to remove layers of the stratum corneum. The adhesive strips were immersed individually in methanol to extract any test material. The extracts were sequentially numbered and analysed by LSC. The total number of tape strips was recorded.
The remaining epidermis was removed from the receptor chamber, digested in Soluene 350 and the whole digest analysed by LSC.

Absorption in different matrices:

The mean data for distribution in the test system are presented in Table 1 to 3 in terms of amount and percentage of the applied dose.
The absorption rates for each individual cell are given in Table 4.
Key result in 'percutaneous absorption', based on 1/2000 dilution of the formulation concentrate. Absorption = absorption in stratum corneum (tape strips 3-5) + remaining epidermis + absorbed = 2.425%
Undiluted concentrate: amounts of test substance absorbed at 8, 12 and 24 hours were 0.160, 0.234 and 0.410 μg/cm2 respectively. These respective amounts expressed as percentages of the applied dose equated to 0.010, 0.014 and 0.025%. Absorption flow was 0.015 µg/cm2/h during the 24 hour exposure period.
1/500 dilution: amounts of the substance absorbed at 8, 12 and 24 hours were 0.017, 0.025 and 0.051 μg/cm2 respectively. These respective amounts expressed as percentages of the applied dose equated to 0.157, 0.236 and 0.484%. Absorption flow was 0.002 µg/cm2/h during the 24 hour exposure period.
1/2000 dilution: amounts of the substance absorbed at 8, 12 and 24 hours were 0.012, 0.014 and 0.021 μg/cm2 respectively. These respective amounts expressed as percentages of the applied dose equated to 0.459, 0.557 and 0.801%. Absorption flow was 0.001 µg/cm2/h during the 24 hour exposure period.

Total recovery:

- Total recovery: Table 1 to 3 in ‘any other information on results incl. tables’.
- Recovery of applied dose acceptable: Yes
- Limit of detection (LOD): Table 5 to 7 in ‘any other information on results incl. tables’.
- Undiluted concentrate: Total recovery 108%, of which 107% was washed off at 24 hours.
- 1/500 dilution: Total recovery was 105%, of which 103% was washed off at 24 hours.
- 1/2000 dilution: Total recovery was 106%, of which 103% was washed off at 24 hours.

Key result

Time point:

24 h

Dose:

1/2000 dilution of formulation concentrate: 2.59 µg/cm2

Parameter:

percentage

Absorption:

2.451 %

Time point:

24 h

Dose:

1/500 dilution of formulation concentrate: 10.5 µg/cm2

Parameter:

percentage

Absorption:

1.2 %

Time point:

24 h

Dose:

33.3% w/w concentrate: 1626 µg/cm2

Parameter:

percentage

Absorption:

0.051 %

Table 1. 33.3% w/w aqueous slurry of the formulation concentrate: 1626 µg test substance per cm²

Test Compartment	µg test substance per cm²		% of applied dose
n=6	Mean	SEM	Mean	SEM
Donor chamber	0.185*	0.081	0.011*	0.005
Skin wash	1748	7.08	107	0.435
Stratum corneum(tape strips 1 & 2)	0.120*	0.031	0.007*	0.002
Stratum corneum(tape strips 3 – 5)	0.096*	0.046	0.006*	0.003
Remaining epidermis	0.321	0.074	0.020	0.005
Absorbed	0.410	0.064	0.025	0.004
Total recovered	1749	7.14	108	0.439

* < Limit of Quantitation Values

 

Table 2. 1/500 w/w aqueous dilution of the formulation concentrate: 10.5 µg test substance per cm²

Test Compartment	µg test substance per cm²		% of applied dose
n=6	Mean	SEM	Mean	SEM
Donor chamber	0.045	0.017	0.432	0.157
Skin wash	10.9	0.479	103	4.56
Stratum corneum(tape strips 1 & 2)	0.008	0.003	0.074	0.025
Stratum corneum(tape strips 3 – 5)	0.008	0.002	0.079	0.017
Remaining epidermis	0.066	0.013	0.630	0.126
Absorbed	0.051	0.019	0.484	0.177
Total recovered	11.1	0.463	105	4.41

* < Limit of Quantitation Values

 

Table 3. 1/2000 w/w aqueous dilution of the formulation concentrate: 2.59 µg test substance per cm²

Test Compartment	µg test substance per cm²		% of applied dose
n=6	Mean	SEM	Mean	SEM
Donor chamber	0.014	0.013	0.540	0.491
Skin wash	2.67	0.057	103	2.19
Stratum corneum(tape strips 1 & 2)	0.008	0.004	0.301	0.140
Stratum corneum(tape strips 3 – 5)	0.012	0.007	0.454	0.266
Remaining epidermis	0.030	0.022	1.17	0.861
Absorbed	0.021	0.006	0.801	0.218
Total recovered	2.75	0.043	106	1.65

* < Limit of Quantitation Values

Table 4. Mean absorption rates

Application of Test Materials and Actual Concentration of Dose Preparation	Mean Absorption Rates		Mean Amount and Percentage of Dose Absorbed
	Time period (h)	Absorption rate (µg/cm^2/h±SEM)	Time (h)	Amount (µg/cm^2)	Percentage Absorbed (%)
33.3% w/w aqueous slurry
(163 g/kg)	0-8	0.016 ± 0.004	6	0.136	0.008
10 µLcm^2(1626 µg/cm^2)	8-12	*0.024 ± 0.008	8	0.160	0.010
Unoccluded	12-24	0.014 ±0.004	12	0.234	0.014
Duration of exposure: 24h	0-24	0.015 ± 0.003	24	0.410	0.025
n = 6
1/500 w/w aqueous dilution
(1.05 g/kg)	0-8	0.002 ± 0.001	6	0.013	0.125
10 µL/cm^2(10.5 µg/cm^2)	8-12	0.002 ± 0.001	8	0.017	0.157
Unoccluded	12-24	0.002 ± 0.001	12	0.025	0.236
Duration of exposure: 24h	0-24	0.002 ± 0.001	24	0.051	0.484
n = 6
1/2000 w/w aqueous dilution
(0.259 g/kg)	0-8	0.001 ± 0.0003	6	0.010	0.403
10 µL/cm^2(2.59 µg/cm^2)	8-12	0.001 ± 0.0001	8	0.012	0.459
Unoccluded	12-24	0.001 ± 0.0001	12	0.014	0.557
Duration of exposure: 24h	0-24	0.001 ± 0.0001	24	0.021	0.801
n = 5

Table 6. Mean limit of quantitation values for 33.3% w/w Aqueous slurry of the formulation concentrate: 1626 µg test substance per cm2

Study compartment	DPM Value	µg/cm^2/h	µg/cm^2	% of applied dose
Absorption rate (0-8 hours)	-	0.015	-	0.001
Absorption rate (8-12 hours)	-	0.031	-	0.002
Absorption rate (12-24 hours)	-	0.010	-	0.0006
Absorption rate (0-24 hours)	-	0.005	-	0.0003
Receptor fluid	39.6	-	0.124	0.008
Donor chamber	16	-	0.204	0.013
Skin wash	17	-	0.542	0.033
Stratum corneum	18	-	0.574	0.035
Remaining epidermal membranes	24	-	0.008	0.0005

 

Table 6. Mean limit of quantitation values for 1/500 w/w Aqueous dilution of the formulation concentrate: 10.5 µg test substance per cm2

Study compartment	DPM Value	µg/cm^2/h	µg/cm^2	% of applied dose
Absorption rate (0-8 hours)	-	0.0007	-	0.006
Absorption rate (8-12 hours)	-	0.001	-	0.012
Absorption rate (12-24 hours)	-	0.0004	-	0.004
Absorption rate (0-24 hours)	-	0.0002	-	0.002
Receptor fluid	33.3	-	0.005	0.050
Donor chamber	28	-	0.004	0.037
Skin wash	20	-	0.007	0.066
Stratum corneum	25	-	0.009	0.083
Remaining epidermal membranes	19	-	0.001	0.010

 

Table 7. Mean limit of quantitation values for 1/2000 w/w Aqueous dilution of the formulation concentrate: 2.59 µg test substance per cm2

Study compartment	DPM Value	µg/cm^2/h	µg/cm^2	% of applied dose
Absorption rate (0-8 hours)	-	0.0007	-	0.026
Absorption rate (8-12 hours)	-	0.001	-	0.051
Absorption rate (12-24 hours)	-	0.0004	-	0.017
Absorption rate (0-24 hours)	-	0.0002	-	0.009
Receptor fluid	34.8	-	0.005	0.206
Donor chamber	26	-	0.004	0.140
Skin wash	17	-	0.006	0.229
Stratum corneum	32	-	0.011	0.432
Remaining epidermal membranes	18	-	0.001	0.039

 

Conclusions:

In this GLP compliant OECD 428 study, the penetration of the test substance through the human epidermis is generally very slow and the vast majority of the applied dose could be removed by gentle skin washing after 24 hours. The dermal absorption value obtained from the undiluted formulation concentrate (33.3% w/w), 0.051%. For 1/2000 aqueous dilution of the concentrate formulation, a dermal absoption of 2.425% was derived. This value is used as reference value for risk assessment, since it is considered to be conservative.

Executive summary:

The in vitro dermal absorption of the test substance concentrate formulation through human epidermal membranes was measured in an OECD 428 study under GLP. [14C]-test substance doses were applied as a nominal 33.3% w/w aqueous slurry of the formulation concentrate (500 g/kg) and as 1/500 w/w (nominally, 1 g/kg) and 1/2000 w/w (nominally, 0.25 g/kg) aqueous dilutions of the formulation concentrate. The doses were applied at 10 μL/cm²

and left unoccluded for an exposure period of 24 hours.The absorption process was followed by taking samples of the receptor fluid (50% v/v ethanol/water) at recorded intervals throughout the exposure period. The distribution of the test substance within the test system and a 24 hour absorption profile were determined, using liquid scintillation counting (LSC).

For the 33.3% w/w aqueous slurry of the formulation concentrate the mean recovery was 108% of the dose applied. The vast majority of the applied test substance (mean 107%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.015 µg/cm²/h, accounting for a 24-hour accumulating dose of 0.827 µg/cm² (0.051% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction. For the 1/500 w/w aqueous dilution of the formulation concentrate the mean recovery was 105% of the dose applied.The vast majority of the applied test substance (mean 103%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.002 µg/cm²/h, accounting for a 24-hour accumulating dose of 0.125 µg/cm² (1.193% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction. For the 1/2000 w/w aqueous dilution of the formulation concentrate the mean recovery was 106% of the dose applied.The vast majority of the applied test substance (mean 103%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.001 µg/cm²/h, accounting for a 24-hour accumulating dose of 0.063 µg/cm² (2.425% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction.

In conclusion, the penetration of the test substance through the human epidermis is generally very slow and the vast majority of the applied dose could be removed by gentle skin washing after 24 hours. The dermal absorption value obtained from the undiluted formulation concentrate (33.3% w/w), 0.051%. For 1/2000 aqueous dilution of the concentrate formulation, a dermal absoption of 2.425% was derived. The latter value is used as reference value for risk assessment, since it is considered to be conservative.

Description of key information

Basic toxicokinetics - Bissig 1993

- Absorption: Radioactivity was rapidly and almost completely absorbed from the GI tract into the general circulation (maximum, D2 mean 84.7%). Maximum concentrations in the blood were reached 15 minutes and 4 hours after administration at the low and high dose level, respectively.

- Distribution: Seven days after administration, mean residue levels were below 0.038 ppm in all tissues. At high dose levels, residue concentrations in fat were 200 times higher than other tissues.

- Metabolism: HPLC analysis revealed complex urinary and faecal metabolite pattern, independent of the sex, pre-treatment, dose level. The cleavage of the bridge between the triazine and pyridine ring is of minor importance in the metabolism in the rat.

- Excretion: The principal route of excretion was via urine with a total of 56 to 80% of the dose. Excretion was rapid. Within 24 hours 52 - 74%, 10 - 37%, and 0.2 - 1.2% of the administered dose were detected in urine, faeces, and expired air, respectively

- Bioaccumulation potential: At high dose levels, residue concentrations in fat were 200 times higher than other tissues. This indicates a saturation of tissue binding sites and an unfettered distribution of radioactivity (most probably unchanged test substance) in the fat at high doses. This assumption is supported by the distribution of residual radioactivity in the pre-treated animals. This finding is not considered to be an indication for bioaccumulation based on the efficient metabolism and excretion.

 

Dermal absorption - Johnson 2012

- the penetration of the test substance through the human epidermis is generally very slow and the vast majority of the applied dose could be removed by gentle skin washing after 24 hours. The dermal absorption value obtained from the undiluted formulation concentrate (33.3% w/w), 0.051%. For 1/2000 aqueous dilution of the concentrate formulation, a dermal absorption of 2.425% was derived. This value is used as reference value for risk assessment, since it is considered to be conservative and the most relevant for exposure conditions.

 

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 (%):

84.7

Absorption rate - dermal (%):

2.425

Absorption rate - inhalation (%):

100

Additional information

Basic toxicokinetics - Bissig 1993

In this GLP compliant ADME study performed according to OECD 417, single oral doses of the labelled test substance were administrated at two dose levels (low dose: 0.5 mg/kg; high dose: 100 mg/kg body weight) to several groups of male and female rats (strain: Tif: RAI f (SPF)). Group A1 received a single intravenous administration at the low dose level. Group B1, C1, D1 and D2 were exposed orally and urine, faeces and expired air were collected at different times. After 7 days the rats were sacrificed and tissues were analysed for radiolabelled content. Group E1 to E4 (all males) were designated for a timewise analysis of radiolabelled content in the blood. Groups F1 to F4 were designated for analysis for radiolabelled content in tissues at several time points.

No test substance related or otherwise unusual behaviour of the animals were observed. The orally administered test substance was fast and almost completely absorbed from the gastrointestinal tract into the general circulation (maximum, D2 mean 84.7% of dose, minimum, C1 mean 57.4% of dose). Independent of the label maximum concentrations in the blood were reached 15 minutes and 4 hours after administration at the low and high dose level, respectively. These maximum values accounted for about 0.3 ppm and 60 ppm, for the 0.5 and 100 mg/kg dose level. The residual radioactivity was determined in several tissues and organs of male rats at different time points after oral administration using both labels at both dose levels. The calculated half-life times (t½) for the depuration of the residual radioactivity from the tissues, assuming to follow first order kinetics, were in the range of 1 to 2 hours at the 0.5 mg/kg dose level (both labels) and between 3 and 6 hours (except fat: 11 hours) for the triazine label at the high dose level. For the high dose pyridine label the half-life times were in the range of 2 to 11 hours. Seven days after a single oral administration of 0.5 mg/kg, low but detectable residues were measured in all tissues and organs. The mean values were below 0.025 ppm test substance equivalents, except in the heart (0.038 ppm). Highest amounts besides the heart were found in liver (0.025 ppm), muscle (0.024 ppm, and kidneys (0.022 ppm).At the high dose level (Group D1, 100 mg/kg), the tissue residues were only in fat accordingly higher, i.e. about 200 times. This indicates a saturation of tissue binding sites and an unfettered distribution of radioactivity (most probably unchanged test substance) in the fat at high doses. This assumption is supported by the distribution of residual radioactivity in the pre-treated animals. This finding is not considered to be an indication for bioaccumulation based on the efficient metabolism and excretion. In brain, heart, and muscle the residues were about 5 times in all other tissues and organs 20 to 50 times higher. The highest residues were detected in fat (about 1 ppm test substance equivalents). These data indicate a saturation of tissue binding sites, except in fat, where an unhindered distribution of radioactivity (most probably unchanged test substance) was observed. The residual radioactivity determined in the pre-treated animals support this assumption. The principal route of excretion was urine (totally 56 to 80% of the dose). The absorbed amount was rapidly excreted. Within 24 hours 52 - 74%, 10 - 37%, and 0.2 - 1.2% of the administered dose were detected in urine, faeces, and expired air, respectively. The excretion pattern was essentially independent of the sex, the route of administration, and pre-treatment with non-radiolabelled test substance. Both sexes eliminated significantly more via the kidneys at the high dose level, i.e. 73 - 80% instead of 55 - 65% at the low dose level.

HPLC analysis revealed complex urinary and faecal metabolite pattern. The chromatograms were qualitatively similar for both sexes, both dose levels, the i.v. and p.o. administration, and the pre-treated animals. Some metabolite fractions showed quantitative differences between the sexes and the dose levels. The most unpolar urinary fraction, accounting for 15 - 18% and 1 - 2% of the dose at the high and low dose level, respectively, cochromatographed with unchanged test substance. In the faeces <1% of the administered dose were characterized as unchanged test substance.

The experiments with the [5-14C] pyridine labelled test substance revealed only slight differences compared to the triazine label. The excretion pattern was independent of the label. The urinary and faecal metabolite pattern were qualitatively similar for both labels but showed some quantitative differences.

In summary, the test substance was after oral administration to rats was fast and almost completely absorbed and rapidly eliminated, mainly with urine. The test substance was extensively metabolized and the metabolic pathways are independent of the sex, pre- treatment, and the dose level. The cleavage of the bridge between the triazine and pyridine ring is of minor importance in the metabolism of the test substance in the rat.

 

Dermal absorption - Johnson 2012

The in vitro dermal absorption of the test substance concentrate formulation through human epidermal membranes was measured in an OECD 428 study under GLP. [14C]-test substance doses were applied as a nominal 33.3% w/w aqueous slurry of the formulation concentrate (500 g/kg) and as 1/500 w/w (nominally, 1 g/kg) and 1/2000 w/w (nominally, 0.25 g/kg) aqueous dilutions of the formulation concentrate. The doses were applied at 10 μL/cm2

and left unoccluded for an exposure period of 24 hours. The absorption process was followed by taking samples of the receptor fluid (50% v/v ethanol/water) at recorded intervals throughout the exposure period. The distribution of the test substance within the test system and a 24 hour absorption profile were determined, using liquid scintillation counting (LSC).

For the 33.3% w/w aqueous slurry of the formulation concentrate the mean recovery was 108% of the dose applied. The vast majority of the applied test substance (mean 107%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.015 µg/cm2/h, accounting for a 24-hour accumulating dose of 0.827 µg/cm2(0.051% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction. For the 1/500 w/w aqueous dilution of the formulation concentrate the mean recovery was 105% of the dose applied. The vast majority of the applied test substance (mean 103%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.002 µg/cm2/h, accounting for a 24-hour accumulating dose of 0.125 µg/cm2(1.193% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction. For the 1/2000 w/w aqueous dilution of the formulation concentrate the mean recovery was 106% of the dose applied. The vast majority of the applied test substance (mean 103%) was washed off the skin at 24 hours. The mean absorption rate through the human epidermis was 0.001 µg/cm2/h, accounting for a 24-hour accumulating dose of 0.063 µg/cm2(2.425% of the dose applied) in the stratum corneum, remaining epidermis and systemic available fraction.

In conclusion, the penetration of the test substance through the human epidermis is generally very slow and the vast majority of the applied dose could be removed by gentle skin washing after 24 hours. The dermal absorption value obtained from the undiluted formulation concentrate (33.3% w/w),0.051%. For 1/2000 aqueous dilution of the concentrate formulation, a dermal absorption of 2.425% was derived. The latter value is used as reference value for risk assessment, since it is considered to be conservative.

Conclusions on adsorption and bioaccumulation

All available data was assessed and the studies representing the worst-case effects were included as key studies. The other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

Absorption of the test substance into the general circulation was rapidly and almost completely upon oral exposure (maximum absorption: 84.7% of dose). Maximum concentrations in the blood were reached 15 minutes and 4 hours after administration at the low and high dose level, respectively. The penetration of the test substance through the human epidermis is generally very slow and the vast majority of the applied dose could be removed by gentle skin washing after 24 hours. The dermal absorption value obtained from the undiluted formulation concentrate (33.3% w/w), 0.051%. For 1/2000 aqueous dilution of the concentrate formulation, a dermal absorption of 2.425% was derived. At high dose levels, residue concentrations in fat were 200 times higher than other tissues. This indicates a saturation of tissue binding sites and an unfettered distribution of radioactivity (most probably unchanged test substance) in the fat at high doses. This assumption is supported by the distribution of residual radioactivity in the pre-treated animals. This finding is not considered to be an indication for bioaccumulation based on the efficient metabolism and excretion.