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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Nov 1995 to 11 Sep 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
metabolism
Qualifier:
according to
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
1984
Qualifier:
according to
Guideline:
other: Pesticide Assessment Guidelines, Subdivision F, Paragraph 85-1, Metabolism Study Hazard Evaluation: Human and Domestic Animals, EPA
Version / remarks:
1984
Qualifier:
according to
Guideline:
other: Agricultural Chemicals Laws and Regulations, Japan (II) Testing Guidelines of Toxicology Studies, Society of Agricultural Chemical Industry
Version / remarks:
1985
Qualifier:
according to
Guideline:
other: Commission Directive 94/79/EC. Annex 1, Toxicological and Metabolism Studies No. L 354/18, 51
Version / remarks:
December 21, 1994
GLP compliance:
yes
Radiolabelling:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Tif:RAIf (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7-8 weeks (males), 9 weeks (females)
- Weight at study initiation: weights approximately 200-250 g (males and females)
- Housing: Closed all-glass metabolism cages; Open plexiglass metabolism cages; Animals which had been bile duct cannulated - non-restrictive metabolism cages
- Diet: Nafag No. 890 (Nafag, Gossau, Switzerland) ad libitum (except overnight prior to administration of the radiolabelled test material)
- Water: Tap water ad libitum; Animals which had been bile duct cannulated - tap water containing 5% glucose, 0.9% NaCl and 0.05% KCl ad libitum after surgery.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18-22 °C
- Humidity: 14 to 86%
- Air changes: Not reported
- Photoperiod: 12 hours light / 12 hours dark
Route of administration:
other: oral and intravenous
Vehicle:
other: Polyethylene glycol 200/ethanol 5/3 (v/v)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test material was dissolved in a mixture of polyethylene glycol 200/ethanol 5/3 (v/v).
Duration and frequency of treatment / exposure:
- oral administration: twice
- intravenous: once
Dose / conc.:
0.5 mg/kg bw/day
Remarks:
low dose
Dose / conc.:
100 mg/kg bw/day
Remarks:
high dose
No. of animals per sex per dose:
Group A1: 4 males / 5 females; Group B1, B2, C1, C2, D1 and D2: 5 males / 5 females; Group F1, F2 and F3: 12 males; Group F5, F6 and F7: 12 females; Group G1: 4 males; group G3: 5 males.
See Table 1 at "Any other information of materials and methods incl. tables" for specifications on the groups.
Details on study design:
In a metabolism study, the fate of the test substance was investigated in rats following oral and intravenous administration using [Thiazol-2-14C] and [Oxadiazin-4-14C] labelled test material. Single oral doses of the labelled test material were administered at two dose levels, the groups of rats used are shown in Table 1 in "Any other information on materials and methods incl. tables". One group received a single intravenous administration at the low dose level.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC
DOSING
The test material was dissolved at different concentrations in a mixture of polyethylene glycol 200/ethanol 5/3 (v/v):
Each animal received approximately 0.8 mL of the administration solution by oral gavage, except the animals of Groups G1, G3 (0.9 mL) and F2, F6 (1.0 mL). For the pre-treatment period the animals of Group C1 received the respective amount of non-radiolabelled test material dissolved in 0.7-0.8 mL of the administration solution. Group A1 were given an intravenous injection, into the tail vein, of approximately 0.3 mL of test material dissolved in physiological saline (0.9% NaCl).

STABILITY
The test substance was found to be stable in the radiolabelled solutions at the time of dosing (checked by TLC and HPLC). The test substance represented more than 95% of the radioactivity. For the repeated dosing experiment (Group C1, pre-treatment with non-radiolabelled test material) the stability of the test material in the administration vehicle was investigated over a period of 14 days prior to the first dosing, using radiolabelled material. After 14 days at room temperature the radiopurity determined by TLC was still above 96%.

BILE-DUCT CATHETERIZATION
The bile-duct was cannulated, under anaesthesia, with a catheter and fixed with a ligature. The catheter was positioned subcutaneously and exteriorised on the back of the animal, where the catheter was properly secured to allow unrestraint movement of the animal in the metabolism cage. After recovery the animals were observed for at least one hour. After this period those animals were selected showing an appropriate state of health and an average bile flow of at least 0.5 mL/h.

ANIMAL OBSERVATIONS
The animals were checked for appearance and behaviour during the experiment.

SAMPLE COLLECTION
- Urine, faeces and bile were individually and separately collected. The animals were killed by exsanguination after anaesthesia with carbon dioxide. Seven days after dosing with radiolabelled test material the animals were killed and tissues and organs including liver were taken.
- Urine, faeces, bile and liver specimens from male and female animals generated in that study were separately pooled and used for analysis in the present study.

STORAGE STABILITY
- This was investigated by comparison of the quantitative metabolite pattern after collection of the specimen, at the beginning of the analytical work, and at the end of the analytical period.
- Urine specimens were directly used for the quantitative determination by two dimensional TLC.

MEASUREMENT OF RADIOACTIVITY
Radioactivity in all specimens was measured by Liquid Scintillation Counting (LSC). Quantitative measurements of the radioactivity in aliquots of urine and other liquid specimens were generally carried out using scintillation mixture Irgasafe plus. The radioactivity in aliquots of faeces and other solid specimens was determined after combustion.

THIN LAYER CHROMATOGRAPHY (TLC)
Analytical and preparative TLC were performed on precoated plates of silica gel 0.25 mm thick. The plates were generally developed with chamber saturation. Non-radioactive spots were detected under UV light at 254 nm.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
HPLC was carried out on a Beckman HPLC system (system GOLD) consisting of two solvent-pumps, a personal computer (solvent programmer), an UV-detector (module 166), a Raytest radioactivity flow monitor RAMONA 5LS and a LINEAR three channel recorder, to record simultaneously both detector responses and the gradient profile. The UV-detector was operated at 254 nm.

SOLID PHASE EXTRACTION (SPE)
Solid phase extraction was carried out on a Bond Elut C18 cartridge (60 ml). The cartridge was sequentially conditioned with appropriate solvents dependent on the eluents.

FLASH CHROMATOGRAPHY
- Pre-purification of urine was carried out on a BIOTAGE 75, Flash-Top chromatography system equipped with a radioactivity flow monitor RAMONA-5, and a LINEAR three channel recorder. The flow was split for determination of radioactivity.
- The specimen was loaded on a BIOTAGE KP-C18-HS, 75S cartridge (300 g, 7.5 x 9.0 cm, 40 µm) equilibrated with ammonium formate buffer (0.01N, pH 4.6).

HIGH VOLTAGE ELECTROPHORESIS (HVE)
HVE was run for 30 minutes at 70 V/cm on a Camag high voltage electrophoresis apparatus using chromatography paper as carrier and the following buffer system: pH 7 aqueous acetic acid (3 mL/L)/pyridine/water (200/50/750 v/v). [14C]Glucose was used for correction of electroosmotic effects and fuchsin and methylene blue to check the homogeneity of the field on the carrier.

PREPARATION OF SPECIMENS
The specimens for the metabolite profiling and isolation of metabolites were generated in the toxicokinetic study (Müller & Stampf, 1996).

URINE
- Urine pools according to sex and animal group were prepared by mixing aliquots of the initially collected urine from each animal and time interval over the specified sampling time. Aliquots were directly used for TLC metabolite profiling in the analytical reference system.
- The urine pools U1/D1m and U1/D1f were combined to a composite pool, designated UA1. UA1, representing 92.9% of dose, was used for the isolation of metabolites. In addition, the urine pools U1/D2m and U1/D1f were combined to a composite pool designated UB1. UB1, representing 93.6% of dose, was used for co-chromatography experiments.

FAECES
- Faeces pools according to sex and animal group were prepared by mixing aliquots of the initially collected faeces from each animal and time interval over the specified sampling time. The faeces pools were extracted at room temperature twice with acetonitrile and once with methanol by shaking for about 60 minutes at about 200 rpm. Phase separation was obtained by sedimentation. The 3 extracts were combined. The volume of the combined extracts from each faeces pool was reduced on a rotatory evaporator and diluted with tetrahydrofurane to a volume of ca. 5 -15 ml for the low dose groups and ca. 40 - 60 ml for the high dose groups, respectively. Aliquots were directly used for TLC metabolite profiling in the analytical reference system.
- The faeces pool extracts from male and female animals from group D1 and D2 were combined to a composite pool designated FA1 and FB1, representing 2.8 and 2.2% of dose, respectively and were used for co-chromatography experiments.

BILE
Bile pools according to animal group were prepared by mixing 5% aliquots of the initially collected bile from each animal and time interval over the specified sampling time. Aliquots were directly used for TLC metabolite profiling in the analytical reference system.

LIVER:
Liver pools according to sex and animal group were prepared by mixing ca. 6 g aliquots of the initially collected liver from each animal at sacrifice. The liver pools were extracted at room temperature twice with acetonitrile and thrice with methanol by shaking for about 60 minutes at about 200 rpm. Phase separation was obtained by sedimentation. The 5 extracts were combined. The volume of the combined extracts from each liver pool was reduced on a rotatory evaporator to a volume <5 mL. Each pool was then diluted with water and extracted with ca. 8 mL chloroform by shaking for about 60 minutes at about 200 rpm. The organic phase was discarded. Aliquots of the aqueous phase were directly used for TLC metabolite profiling in various solvent systems.


METABOLITE CHARACTERISATION
ACIDIC TREATMENT
The metabolite (ca.1 µg test substance equivalent; 50 µL) was mixed with 100 µL of 2N HCl. The mixture was kept at 100°C for about 1 hour and at 60°C for about 21 hours in a heating block. The reaction was checked by TLC.

β- GLUCURONIDASE TREATMENT
An aliquot of the metabolite (about 1 µg test substance equivalent; 50 µL) was mixed with 2.1 mg glucuronidase (Type B-10 from bovine liver) in 100 µL acetate buffer (0.05 M, pH 4.9). The mixture was incubated in a shaking water bath at 37°C for 22 hours. The cleavage was checked by TLC.

SPECTROSCOPIC METHODS
A variety of spectroscopic instruments and techniques were used to identify the metabolites.

CALCULATIONS
All values assigned to extracts, fractions etc. of the metabolite profiling experiments are based on 100% recovery and are mean values of duplicate measurements. For all steps during the isolation procedure the recovery was corrected to 100%. The % of the dose assigned to fractions and metabolites refer to the amount physically available at this particular isolation step and did not necessarily correlate to the amounts originally present in the starting material as for the sake of an efficient isolation and purification procedure major amounts of a particular fraction may have been sacrificed.

Metabolites identified:
yes
Details on metabolites:
URINE METABOLITE PATTERN
- The chromatography revealed a complex metabolite pattern, consisting of up to 22 metabolite fractions. The metabolite fractions were designated Ul through U22. The pattern was dominated by fraction U12, corresponding to unchanged test material and accounting for 70 - 83% of dose, followed by metabolite fraction U16 and U17 amounting to 5 – 13% and 1 - 2% of dose, respectively. All the remaining fractions were at or below 1%.
- The pattern was essentially independent of sex, dose, pretreatment, and route of administration. Mainly one thiazole specific fraction, i.e. U20, was found accounting for less than 1% of dose. The urinary metabolite pattern of the bile-duct cannulated animals was qualitatively similar to the pattern of the corresponding urines from intact animals. The figures assigned to individual fractions were similar since renal excretion did not change significantly after bile-duct cannulation.

FAECES METABOLITE PATTERN
About 50 - 76% of the faeces radioactivity were extractable at room temperature independent of sex, dose, pretreatment and position of label. After bile-duct cannulation extractability was almost complete. The chromatography revealed a metabolite pattern consisting of up to 13 metabolite fractions. The metabolite fractions were designated F1 through F13. The pattern was similar, but less complex than those found for urine. The major fractions, i.e. F9 and F11, corresponded to the major urinary fractions U12 and U16, respectively, the former corresponding to unchanged test material. All the remaining fractions were below 1% of dose. The pattern was essentially independent of sex, dose, pretreatment, and route of administration. A minor thiazole specific fraction (i.e. F13) accounting for about 0.1% of dose was detected. It corresponded to the urinary fraction U20. The faecal metabolite pattern of the bile-duct cannulated animals (Group G1 and G3) was qualitatively similar to the pattern of the corresponding faeces from intact rats. The figures assigned to F9 were slightly increased after bile-duct cannulation. Nonetheless the qualitative similarity indicates microbial degradation from the gut flora.

BILE METABOLITE PATTERN
The bile pooled according to animal group was quantitatively analysed by two dimensional TLC. The TLC analysis revealed up to 5 metabolite fractions designated B1 through B5. The pattern was similar, but less complex than those found for urine. The major fractions, i.e. B3 and B5, corresponded to the major urinary fractions U12 and U16, respectively, the former corresponding to unchanged test material. As compared to urine and faeces a significant part of radioactivity remained at the origin upon TLC indicating a higher ratio of polar metabolites. The pattern was essentially independent of the position of label.

LIVER METABOLITE PATTERN
About 22 - 25% of the liver radioactivity was extractable at room temperature independent of sex and position of label The separately combined extracts were diluted with water, concentrated, and partitioned with chloroform. Almost no radioactivity was detected in the organic phase. The remaining aqueous phases were analysed by two-dimensional TLC. In both TLC-systems radioactivity remained almost completely at the origin, independent of the sex of the animals and the position of label.

FAECES METABOLITES
- The faeces pools FA1 and FB1 were compared with selected reference substances and/or authentic metabolites isolated from urine by co-chromatography. This revealed the presence of metabolite 1U, metabolite 2U, metabolite 3U, metabolite 14U, metabolite 18U, NOA407475 and NOA421275. See Table 3 in "Any other information on results incl. tables" for more information.
- Metabolite MU3 (acetic acid {amino-[(2-chloro-thiazol-5-ylmethyl)-amino]-methylene}-hydrazide) was identified as a minor metabolite corresponding to 0.009% of the total dose. Metabolite R6 was identified as N-acetyl-cysteine conjugate of the test substance corresponding to 0.09% of the total dose.

BILE METABOLITES
The bile pools B1/G1m and B1/G3m were compared with selected reference substances and/or authentic metabolites isolated from urine by co-chromatography. This revealed the presence of metabolite 1U, metabolite 2U, and metabolite 3U.

Table 1: Quantitative distribution of metabolite fractions in urine (% of dose)

Label

[Thiazol-2-14C] test material

Group

A1

single i.v.

B1

single p.o.

C1

multiple p.o.

D1

single p.o.

G1

Sex

male

female

male

female

male

female

male

female

male

Dose (mg/kg)

0.5

0.6

0.5

0.6

0.4

0.4

91

99

0.5

Metabolite fraction

 

 

 

 

 

 

 

U1-U11

3.0

3.4

4.1

3.4

4.9

3.9

3.4

2.3

3.9

U12

71.7

76.2

69.5

76.1

76.3

81.2

73.6

81.8

68.7

U13-U15

0.5

0.5

0.9

0.8

0.7

0.5

0.7

0.7

---

U16

7.1

7.5

10.8

6.6

10.2

5.5

12.2

6.9

5.1

U17-U22

1.6

1.7

2.6

1.4

2.1

1.3

3.0

1.3

0.9

Total

83.8

89.1

87.8

88.2

94.1

92.3

92.9

93.0

78.6

 

 

Label

[Oxadiazin-4-14C] test material

Group

B2

single p.o.

D2

single p.o.

G3

Sex

male

female

male

female

male

Dose (mg/kg)

0.4

0.5

101

104

0.5

Metabolite fraction

 

 

 

 

 

U1-U11

2.8

2.5

3.4

2.7

2.3

U12

74.2

82.4

72.3

82.6

76.2

U13-U15

1.0

0.8

1.2

0.9

0.6

U16

10.5

6.4

13.1

8.0

7.2

U17-U22

1.2

0.5

2.0

0.9

0.6

Total

89.6

92.5

92.0

95.2

86.8

Table 2: Quantitative distribution of metabolite fractions in faeces (% of dose)

Label

[Thiazol-2-14C] test material

Group

A1

single i.v.

B1

single p.o.

C1

multiple p.o.

D1

single p.o.

G1

Sex

male

female

male

female

male

female

male

female

male

Dose (mg/kg)

0.5

0.6

0.5

0.6

0.4

0.4

91

99

0.5

Metabolite fractionof extracts

 

 

 

 

 

 

 

F1-F8

0.9

0.5

1.3

1.0

1.7

0.9

1.7

1.1

0.8

F9

1.3

0.7

0.8

0.4

1.5

1.8

0.8

1.5

3.3

F10

---

---

---

---

---

---

0.1

0.1

---

F11

0.2

0.1

0.2

0.1

0.4

0.2

0.2

0.2

0.3

F12-F13

---

---

---

---

0.1

---

0.1

0.1

---

Sum extract

2.4

1.3

2.3

1.5

3.6

2.9

2.8

2.8

4.4

Non-extractable

2.1

1.2

2.1

1.1

2.5

0.9

2.0

1.4

0.4

Total

4.4

2.5

4.4

2.6

6.2

3.9

4.8

4.2

4.8

 

 

Label

[Oxadiazin-4-14C] test material

Group

B2

single p.o.

D2

single p.o.

G3

Sex

male

female

male

female

male

Dose (mg/kg)

0.4

0.5

101

104

0.5

Metabolite fraction of extracts

 

 

 

 

F1-F8

1.1

0.8

1.4

0.7

0.7

F9

0.7

0.6

0.9

0.8

2.1

F10

---

---

0.1

<0.1

---

F11

0.2

0.1

0.3

0.1

0.3

F12-F13

0.1

<0.1

0.1

<0.1

---

Sum extract

2.2

1.5

2.6

1.7

3.1

Non-extractable

2.0

1.2

2.6

1.7

0.5

Total

4.1

2.7

5.3

3.4

3.6

Table 3: Identified metabolites

Designation and chemical name

Designation and chemical name

IU3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-ylidene-N-nitroamine

14U2-methylsulfanyl-thiazole-5-carboxylic acid

2UN-(2-chloro-thiazol-5-yl-methyl)-N'-methyl-N"-nitro-guanidine

15U1-(2-chloro-thiazol-5-ylmethyl)-3-nitro-urea

3U1-(2-chloro-thiazol-5-ylmethyl)-3-methyl-urea

16U2-acetylamino-3-[5-(N'-methyl-N"-nitro-guanidinomethyl)-thiazol-2-ylsulfanyl]-propionic acid

4U(2-chloro-thiazol-5-ylmethyl)-urea

 

17U2-acetylamino-3-[5-(5-methyl-4-oxo-[1,3,5]oxadiazinan-3-ylmethyl)-thiazol-2-ylsulfanyl] propionic acid

5U2-acetylamino-3-(2-chloro-thiazol-5-ylmethanesulfinyl)-propionic acid

 

18UN-(2-chloro-thiazol-5-ylmethyl)-N'-nitro-guanidine

6U2-acetylamino-3-(2-chloro-thiazol-5-ylmethylsulfanyl)-propionic acid

 

19U3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-one

Conclusions:
The degradation of the test substance accounted for about 20 - 30% of dose. The metabolic pathways are independent of the route of administration, the dose level in the range of 0.5 and 100 mg/kg body weight, pretreatment, and the sex of the animals, within the limits of this study. Due to the rapid absorption and excretion, it is assumed that exposure time of the test substance to biotransformation enzymes is reduced resulting in excretion of high amounts of unchanged test substance.
Executive summary:

The toxicokinetic behaviour in male and female rats including bile-fistulated animals after single and multiple, intravenous and oral doses at two dose levels (0.5 and 100 mg/kg bw) was investigated earlier (Müller & Stampf, 1996). In continuation of that study, this present report is concerned with the identification of individual metabolites and the quantitative assessment of the metabolic pathways thus derived. The excreta generated in the toxicokinetic study were used to determine the quantitative metabolite pattern by two-dimensional TLC and to isolate the metabolites in pure form by chromatographic techniques applying LC, HPLC, and TLC in various modes.

The urinary pattern was complex but essentially independent of sex, dose, pretreatment with non-radiolabelled test material, and route of administration. Very minor label specific fractions were observed. The faecal pattern was similar, but less complex than that found for urine with some quantitative variations and independent of sex, dose, pretreatment, route of administration, and only slightly dependent on the position of label. The bile pattern was even less complex and independent of the position of label with some quantitative variations. All the excreta patterns were dominated by two major metabolite fractions accounting totally for 80 - 90% of dose.

 Ultimately 12 metabolites were isolated from urine of the high dosed male and female animals and identified by spectroscopic means (mainly1H-NMR and mass spectroscopy): 

Metabolite 1U

3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-ylidene-N-nitroamine

Metabolite 2U

N-(2-chloro-thiazol-5-ylmethyl)-N'-methyl-N"-nitro-guanidine

Metabolite 3U

1-(2-chloro-thiazol-5-ylmethyl)-3-methyl-urea

Metabolite 4U

(2-chloro-thiazol-5-ylmethyl)-urea

Metabolite 5U

2-acetylamino-3-(2-chloro-thiazol-5-ylmethanesulfinyl)-propionic acid

Metabolite 6U

2-acetylamino-3-(2-chloro-thiazol-5-ylmethylsulfanyl)-propionic acid

Metabolite 14U

2-methylsulfanyl-thiazole-5-carboxylic acid

Metabolite 15U

1-(2-chloro-thiazol-5-ylmethyl)-3-nitro-urea

Metabolite 16U

2-acetylamino-3-[5-(N'-methyl-N"-nitro-guanidinomethyl)-thiazol-2-ylsulfanyl]-propionic acid

Metabolite 17U

2-acetylamino-3-[5-(5-methyl-4-oxo-[1,3,5]oxadiazinan-3-ylmethyl)-thiazol-2-ylsulfanyl]-propionic acid

Metabolite 18U

N-(2-chloro-thiazol-5-ylmethyl)-N'-nitro-guanidine

Metabolite 19U

3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-one

Metabolite L14

2-oxo-propionic acid [3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-ylidene]-hydrazide

Metabolite MU3

acetic acid {amino-[(2-chloro-thiazol-5-ylmethyl)-amino]-methylene}-hydrazide

Metabolite R6

N-acetyl-cysteine conjugate of the test substance

 

In addition, methyl-urea, nitro-(3 -methyl-[1,2,3]-oxo-diazinon-4 -ylidene)-amine, [3 -(2 -chloro-thiazol-5 -ylmethyl)-[1,3,5]oxadiazinan-4 -ylidene]-nitro-amine, nitro-(3 -methyl-[1,3,5]-oxodiazinon-4 -ylidene)-amine, 3 -methyl-[1,3,5]oxadiazinan-4 -ylidene-amin, N-nitro-N'-methyl-guanidine, 3 -(2 -chlorothiazol-5 -ylmethyl)-5 -methyl-[1,3,5]oxadiazinan-4 -ylideneamin, (2 -chloro-thiazole-5 -carbonyl)-amino]-acetic acid, N-(2 -chloro-thiazol-5 -ylmethyl)-N'-methyl-guanidine, N-(2 -chloro-thiazol-5 -ylmethyl)-guanidine were identified as metabolites by co-chromatography with authentic reference substances. 

Totally about 70 - 80% of the dose was eliminated with the urine as unchanged test material. Metabolite 2U and 18U accounted totally for about 10% and 1% of dose, respectively, whereas all the other metabolites were below 1% of dose. Based on the structures of the metabolites the metabolic pathways of the test material were derived: cleavage of the oxadiazine ring to the corresponding nitroguanidine compound (major pathway); cleavage of the nitroguanidine group yielding a guanidine derivative (minor pathway); hydrolysis of the guanidine group to the corresponding urea (minor pathway); demethylation of the guanidine group (minor pathway); substitution of the chlorine of the thiazole ring by glutathione (minor pathway); cleavage between the thiazole and oxadiazine ring (minor pathway). Cleavage between the thiazole- and oxadiazine ring occurs to a very small extent. It is initiated either by attack of glutathione on the bridging methylene group or alternatively by oxidative dealkylation. The former gives rise to a thiazole-5-ylmethyl-glutathione derivative and a nitroguanidine compound whereas the latter produces ultimately the respective carboxylic acid derivative of thiazole and the corresponding cleavage counterpart. The glutathione derivatives are prone to further degradation of the glutathione moiety resulting in various sulfur-containing metabolites (e.g. mercapturates, sulfides, and sulfoxides). Both the thiazole and oxadiazine ring are susceptible to oxidative attack. The previous toxicokinetic study renders evidence that small but measurable amounts of radioactivity were exhaled after administration of either label, most probably as CO2. These minor pathways proceed rapidly to a lot of small molecules, which may, at least partially, enter the general metabolism. The various sulfur-containing metabolites and small metabolites from thiazole- and oxadiazine-ring degradation are probably the reason for the complex metabolite pattern detected in urine. The majority of metabolites were the result of more than one of the above mentioned transformations. The degradation resulted in metabolites which together with unchanged test material were eliminated very rapidly. The administered dose was almost completely absorbed, and was degraded partially and eliminated almost completely via urine. Excretion via bile and ultimately via faeces together with small amounts of unchanged test material escaping absorption was of very minor importance. Enterohepatic circulation was negligible.

The degradation of the test material accounted for about 20 - 30% of dose. The metabolic pathways are independent of the route of administration, the dose level in the range of 0.5 and 100 mg/kg body weight, pretreatment, and the sex of the animals, within the limits of this study. Due to the rapid absorption and excretion, it is assumed that exposure time of the test substance to biotransformation enzymes is reduced resulting in excretion of high amounts of unchanged test material.

Reference: Müller T and Stampf P, 1996. CGA293343:Absorption, distribution and excretion of [Thiazol-2-14C] and [Oxadiazin-4-14C] CGA293343 in the rat, Division Crop Protection, Ciba-Geigy Limited, Basle, Switzerland. Unpublished Report No. 11/96

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Sep 2001 to 01 Oct 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Version / remarks:
2000
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
[Oxadin-4-14C]
Species:
other: rat and human
Strain:
other: Hanlbm: WIST (SPF) (rats) and Caucasian donors (human)
Remarks:
male rats and male/female human
Sex:
male/female
Details on test animals and environmental conditions:
- RAT SKIN: Epidermal membranes were prepared from approximately 9 week old, male Hanlbm: WIST (SPF) rats.
- HUMAN SKIN: Abdominal cadaver skin from Caucasian donors (one male and one female) was obtained from the Institut für Pathologie, Kantonsspital Basel, Basel, Switzerland.
Type of coverage:
other: permeable tape (non-occluded)
Duration of exposure:
24 h
Doses:
- Nominal doses: 176.6 and 3450 µg of test material cm-2
- Actual doses: 20 g a.i./L and 350 g a.i./L
- Actual doses calculated as follows: not specified
- Dose volume: 6 µL
- Rationale for dose selection: not specified
Control animals:
no
Details on study design:
DOSE PREPARATION
The two doses were prepared to mimic the commercial 350 g/L formulation and the in use concentration of 20 g/L using the technical material, [14C]-labelled test material and formulation blank. The formulated test substance was checked for stability by TLC. The test material represented at least 98.4% and 97.9% of the radioactivity at the low and high dose levels, respectively.

APPLICATION OF DOSE:
- Those cells with skin membranes having acceptable Kp values in the integrity test were arranged on the manifolds and then a 6 µL aliquot of the formulated test substance was applied to the surface of each skin membrane. - - The donor chamber was covered with a permeable tape (non-occluded conditions). There were seven human skin samples and seven rat skin samples tested at each concentration.

REMOVAL OF TEST SUBSTANCE
Finally, the cells were washed with ethanol/water (1/:1, v/v) and the radioactivity in the cell wash was determined by LSC.

SAMPLE COLLECTION
- Duration of exposure and sampling: The skin was exposed to the test preparations for 24 hours during which time samples of receptor fluid were taken at suitable intervals (0-6 hours, every hour; 6-24 hours every 2 hours) to allow adequate characterisation of the absorption profile.
- Terminal procedures: Twenty four hours after application the skin membrane surface was rinsed with ethanol/water (1:1, v/v) and the radioactivity in the skin rinse was determined by LSC. The skin membrane was removed from the in-line cells and dissolved in tissue solubilizer prior to LSC.

SAMPLE PREPARATION
The two doses were prepared to mimic the commercial 350g/L formulation and the in use concentration of 20 g/L using the technical material, [14C]-labelled test material and formulation blank. The formulated test substance was checked for stability by TLC. The test material represented at least 98.4% and 97.9% of the radioactivity at the low and high dose levels, respectively.

ANALYSIS
All components of the test system (e.g. receptor fluid, skin wash, donor chamber) were analysed by LSC and the recovery determined.

OTHER:
- Definition of absorbed test material: The absorbed (systemically available) dose is considered to be the test material detected in the receptor fluid. Material removed from the surface of the epidermis by the washing procedure is regarded as unabsorbed.. In vivo, the majority of the dose in the epidermis, especially that in stratum corneum, would eventually be lost by desquamation.






Details on in vitro test system (if applicable):
SKIN PREPARATION
- Epidermal membranes were prepared from approximately 9 week old, male Hanlbm: WIST (SPF) rats. Abdominal cadaver skin from Caucasian donors (one male and one female) was obtained from the Institut für Pathologie, Kantonsspital Basel, Basel, Switzerland. The skin samples were wrapped in tinfoil and stored at -18°CC.
- Prior to use, the skin samples were removed from the freezer and allowed to thaw at room temperature. The subcutaneous fat was carefully removed from the full thickness skin and pieces of about 4 x 5 cm2 were stretched evenly over a cork block, with stratum corneum uppermost. Skin sections of about 200 µm thickness were cut off from the top using a dermatome.
- Pieces of skin membranes (approx. 1.8 x 1.8 cm2) were mounted in diffusion cells between the donor and receptor chamber so the stratum corneum was exposed to air and the basal part in contact with the receptor fluid. 0.9% NaCl was pumped through the receptor chamber for an equilibrium period of 0.5-1.0h.
- The integrity of the membranes was checked by applying tritiated water to the skin membrane surface under occluded conditions.
- The cumulative penetration was determined over 6 hours by collecting hourly fractions. The permeability coefficient (Kp) of each skin membrane was calculated for the 3-6 hours interval. Rat skin membranes with Kp > 3.5-10^-3 cm h-1 and human skin membranes with Kp > 2.5.10^-3 cm h-1 were excluded from the subsequent experiment.

PRINCIPLES OF ASSAY
- Diffusion cell: Diffusion of the test material into and across the skin to a receptor fluid was measured using glass diffusion cells in which the epidermis formed a horizontal membrane and provided an application area of 0.64cm2. Seven flow-through diffusion cells were used per aluminium manifold which was connected to a water bath to maintain the temperature of the skin membranes at 31 - 33°C. The receptor chambers were connected to a multi-channel peristaltic pump, with a pump speed of approximately 3 mL/h.
- Receptor fluid: The receptor fluid (50% ethanol in water) was chosen to ensure that the test substance would freely partition into this from the skin membrane and never reach a concentration that would limit its diffusion.
- Temperature: Throughout the experiment the receptor fluid was stirred and the epidermal membranes were maintained at a normal skin temperature of 32 ± 1°C in a water bath.


Signs and symptoms of toxicity:
no effects
Dermal irritation:
no effects
Absorption in different matrices:
RAT SKIN MEMBRANE
- After application of the test material at the low dose level, 0.82% of the applied dose penetrated through the rat skin membrane within 24 hours. At the high dose level the portion penetrating through the rat skin membrane within 24 hours accounted for 1.18% of the dose. The total amount which penetrated within this period accounted for 1.45 µg cm-2 at the low dose level and 40.8 µg cm-2 at the high dose level.
- The flux, which reflects the penetration rate under steady state conditions, was calculated to be 0.062 µg cm-2.h-1 at the low dose level and 1.18 µg cm-2.h-1at the high dose level. The time interval of steady state conditions varied at both dose levels between the individual cells from 1-24 hours and 6-24 hours. The 19-fold higher concentration of the undiluted formulation led to a corresponding 19-fold higher penetration rate as compared to the diluted formulation.

HUMAN SKIN MEMBRANE
- Within 24 hours, 0.19% of the applied low dose and 0.44% of the applied high dose penetrated through the human skin membrane, corresponding to 0.34 µg cm-2 and 15.0 µg cm-2, respectively. The calculated flux, under steady state conditions, accounted for 0.013 µg cm-2.h-1 and 0.20 µg cm-2.h-1 at the low and high dose levels, respectively. The time interval for steady state conditions was between 1-24 and 3-24 hours at the low dose level and between 1-24 and 12-24 hours at the high dose level. Again, the 19-fold higher concentration at the high dose level led to a corresponding 16-fold higher penetration rate (flux) as compared to the low dose level.

See Table 1 at "Any other information on results incl. tables" for further details.
Total recovery:
RAT SKIN MEMBRANE
The mean recovery was 97.2% of the applied radioactivity at the low dose level and 89.2% of the applied radioactivity at the high dose level.

HUMAN SKIN MEMBRANE
The mean recovery was 100.4% and 89.6% at the low and high dose level, respectively.

ANALYSIS OF SKIN RINSE
At both dose levels and in both species, at least 96.5% of the radioactivity was found as unchanged test material. Hence, it was concluded that at both dose levels and in both species the test substance remained stable for 24 hours on the skin membrane.

See Table 2 at "Any other information on results incl. tables" for further details.
Key result
Time point:
24 h
Dose:
176.6 µg cm^-2
Parameter:
other: Flux
Absorption:
0.062 other: µg cm^-2 h^-1
Remarks on result:
other: Rat skin membrane
Key result
Time point:
24 h
Dose:
3451 µg cm^-2
Parameter:
other: Flux
Absorption:
1.179 other: µg cm^-2 h^-1
Remarks on result:
other: Rat skin membrane
Key result
Time point:
24 h
Dose:
176.6 µg cm^-2
Parameter:
other: Flux
Absorption:
0.013 other: µg cm^-2 h^-1
Remarks on result:
other: Human skin membrane
Key result
Time point:
24 h
Dose:
3451 µg cm^-2
Parameter:
other: Flux
Absorption:
0.204 other: µg cm^-2 h^-1
Remarks on result:
other: Human skin membrane

Table 1: Summary of test material absorption through rat and human skin membranes

Test System

Rat skin membrane

Human skin membrane

Dose level

low

high

low

high

Applied dose (µg cm-2)

176.6

3451

176.6

3451

Applied volume (µL)

6

6

6

6

Application area (cm2)

0.64

0.64

0.64

0.64

Concentration (mg cm-3)

18.8

368.1

18.8

368.1

Penetration within:

µg cm-2

% of dose

µg cm-2

% of dose

µg cm-2

% of dose

µg cm-2

% of dose

6 hour

0.39

0.22

19.4

0.56

0.10

0.06

8.2

0.24

12 hour

0.78

0.44

28.8

0.83

0.17

0.10

12.8

0.37

24 hour

1.45

0.82

40.8

1.18

0.34

0.19

15.0

0.44

Flux (µg cm-2h-1)

0.062

1.179

0.013

0.204

Table 2: Summary of test material recovery (% of dose)

Test System

Rat skin membrane

Human skin membrane

Dose level

low

high

low

high

Applied dose (µg cm-2)

176.6

3451

176.6

3451

Perfusates

0-24 hour

0.82

1.18

0.19

0.44

Remaining dose

cell wash

0.19

0.43

0.08

2.42

 

skin rinse

95.71

75.19

99.92

69.00

 

skin membrane

0.50

12.40

0.23

17.73

 

subtotal

96.40

88.02

100.24

89.15

Recovery

97.22

89.20

100.24

89.59

Conclusions:
The test material penetrates through rat skin membrane faster and to a higher extent than through human skin membrane, at in-use concentrations as well as at the concentration of the undiluted formulation.
Executive summary:

The percutaneous penetration of the test material was determined in vitro using split-thickness skin membranes from rat and human skin. The skin membranes were set up in flow-through diffusion cells, the formulated [14C] radiolabelled test substance was applied onto the skin membranes and then the perfusates were collected at defined time intervals (0-6 hours at 1 hour intervals and 6-24 hours at 2 hour intervals). Two dose levels were used, i.e. a low dose level of nominal 0.2 mg test material / cm2 and a high dose level of nominal 3.5 mg test material/cm2. The low dose of 20 g a.i./L reflects concentrations recommended for use in the field, the high dose represents the undiluted formulation (350 g a.i./L). 24 hours after application, the skin membrane surface was rinsed with ethanol/water (1:1) and the radioactivity in the skin rinse was determined by LSC. The skin membrane was removed from the in-line cells and dissolved in tissue solubiliser prior to LSC. Finally, the cells were washed with ethanol/water (1:1) and the radioactivity in the cell wash determined.

During the 24 hours after application of formulated [Oxadiazin-4-14C] labelled test material, only 0.8% and 1.2% of the dose penetrated through rat split-thickness skin membranes at the low and high dose levels, respectively. The human split-thickness skin membranes showed a lower permeability of the test material. Within 24 hours after application, only 0.2% and 0.4% of the low and high doses, respectively, penetrated through human skin membranes.

A species difference in respect to the penetration of formulated test amterial was also reflected by the flux. The flux at steady state conditions was determined to be about 0.06 µg-cm-2 h-1 and 1.18 µg-cm-2 h-1 through rat skin membranes and about 0.01 µg.cm-2 h-1 and 0.20 µg.cm-2 h-1 through human skin membranes at the low and high dose level, respectively. This results in a human:rat ratio of the flux of about 1:4.8 at the low dose level and 1:5.8 at the high dose level.

The 19-fold higher concentration of the undiluted formulation led to a corresponding 19-fold and 16-fold higher penetration rate (flux) in rat and human skin membrane, respectively.

The radioactivity in the skin rinse at the end of exposure at both dose levels and for both species was analysed as unchanged test material. The experimental recoveries were between 89.2% and 100.4% of the applied dose. The test material penetrates through rat skin membrane faster and to a higher extent than through human skin membrane, at in-use concentrations as well as at the concentration of the undiluted formulation.

Description of key information

The degradation of the test material accounted for about 20 - 30% of dose. The metabolic pathways are independent of the route of administration, the dose level in the range of 0.5 and 100 mg/kg body weight, pretreatment, and the sex of the animals, within the limits of this study. Due to the rapid absorption and excretion, it is assumed that exposure time of the test material to biotransformation enzymes is reduced resulting in excretion of high amounts of unchanged test material, OECD 417 (Thanei, 1998)

The test material penetrates through rat skin membrane faster and to a higher extent than through human skin membrane, at in-use concentrations as well as at the concentration of the undiluted formulation; OECD 428 (Loffler, 2000)

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
50
Absorption rate - dermal (%):
10
Absorption rate - inhalation (%):
100

Additional information

OECD 417 - rat

The toxicokinetic behaviour in male and female rats including bile-fistulated animals after single and multiple, intravenous and oral doses at two dose levels (0.5 and 100 mg/kg bw) was investigated (Müller & Stampf, 1996). A further study (Thanei, 1998) was concerned with the identification of individual metabolites and the quantitative assessment of the metabolic pathways.

The excreta generated in the toxicokinetic study were used to determine the quantitative metabolite pattern by two-dimensional TLC and to isolate the metabolites in pure form by chromatographic techniques applying LC, HPLC, and TLC in various modes.

The urinary pattern was complex but essentially independent of sex, dose, pre-treatment with non-radiolabelled test material, and route of administration. Very minor label specific fractions were observed. The faecal pattern was similar, but less complex than that found for urine with some quantitative variations and independent of sex, dose, pre-treatment, route of administration, and only slightly dependent on the position of label. The bile pattern was even less complex and independent of the position of label with some quantitative variations. All the excreta patterns were dominated by two major metabolite fractions accounting totally for 80 - 90% of dose.

Ultimately 12 metabolites were isolated from urine of the high dosed male and female animals and identified by spectroscopic means (mainly1H-NMR and mass spectroscopy): 

Metabolite 1U

3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-ylidene-N-nitroamine

Metabolite 2U

N-(2-chloro-thiazol-5-ylmethyl)-N'-methyl-N"-nitro-guanidine

Metabolite 3U

1-(2-chloro-thiazol-5-ylmethyl)-3-methyl-urea

Metabolite 4U

(2-chloro-thiazol-5-ylmethyl)-urea

Metabolite 5U

2-acetylamino-3-(2-chloro-thiazol-5-ylmethanesulfinyl)-propionic acid

Metabolite 6U

2-acetylamino-3-(2-chloro-thiazol-5-ylmethylsulfanyl)-propionic acid

Metabolite 14U

2-methylsulfanyl-thiazole-5-carboxylic acid

Metabolite 15U

1-(2-chloro-thiazol-5-ylmethyl)-3-nitro-urea

Metabolite 16U

2-acetylamino-3-[5-(N'-methyl-N"-nitro-guanidinomethyl)-thiazol-2-ylsulfanyl]-propionic acid

Metabolite 17U

2-acetylamino-3-[5-(5-methyl-4-oxo-[1,3,5]oxadiazinan-3-ylmethyl)-thiazol-2-ylsulfanyl]-propionic acid

Metabolite 18U

N-(2-chloro-thiazol-5-ylmethyl)-N'-nitro-guanidine

Metabolite 19U

3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-one

Metabolite L14

2-oxo-propionic acid [3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-ylidene]-hydrazide

Metabolite MU3

acetic acid {amino-[(2-chloro-thiazol-5-ylmethyl)-amino]-methylene}-hydrazide

Metabolite R6

N-acetyl-cysteine conjugate of CGA293343

 

In addition, methyl-urea, nitro-(3 -methyl-[1,2,3]-oxo-diazinon-4 -ylidene)-amine, [3 -(2 -chloro-thiazol-5 -ylmethyl)-[1,3,5]oxadiazinan-4 -ylidene]-nitro-amine, nitro-(3 -methyl-[1,3,5]-oxodiazinon-4 -ylidene)-amine, 3 -methyl-[1,3,5]oxadiazinan-4 -ylidene-amin,N-nitro-N'-methyl-guanidine, 3 -(2 -chlorothiazol-5 -ylmethyl)-5 -methyl-[1,3,5]oxadiazinan-4 -ylideneamin, (2 -chloro-thiazole-5 -carbonyl)-amino]-acetic acid, N-(2 -chloro-thiazol-5 -ylmethyl)-N'-methyl-guanidine, N-(2 -chloro-thiazol-5 -ylmethyl)-guanidine were identified as metabolites by co-chromatography with authentic reference substances. 

Totally about 70 - 80% of the dose was eliminated with the urine as unchanged test material. Metabolite 2U and 18U accounted totally for about 10% and 1% of dose, respectively, whereas all the other metabolites were below 1% of dose. Based on the structures of the metabolites the metabolic pathways of the test material were derived: cleavage of the oxadiazine ring to the corresponding nitroguanidine compound (major pathway); cleavage of the nitroguanidine group yielding a guanidine derivative (minor pathway); hydrolysis of the guanidine group to the corresponding urea (minor pathway); demethylation of the guanidine group (minor pathway); substitution of the chlorine of the thiazole ring by glutathione (minor pathway); cleavage between the thiazole and oxadiazine ring (minor pathway). Cleavage between the thiazole- and oxadiazine ring occurs to a very small extent. It is initiated either by attack of glutathione on the bridging methylene group or alternatively by oxidative dealkylation. The former gives rise to a thiazole-5-ylmethyl-glutathione derivative and a nitroguanidine compound, whereas the latter produces ultimately the respective carboxylic acid derivative of thiazole and the corresponding cleavage counterpart. The glutathione derivatives are prone to further degradation of the glutathione moiety resulting in various sulfur-containing metabolites (e.g. mercapturates, sulfides, and sulfoxides). Both the thiazole and oxadiazine ring are susceptible to oxidative attack.

The first toxicokinetic study renders evidence that small but measurable amounts of radioactivity were exhaled after administration of either label, most probably as CO2. These minor pathways proceed rapidly to a lot of small molecules, which may, at least partially, enter the general metabolism. The various sulfur-containing metabolites and small metabolites from thiazole- and oxadiazine-ring degradation are probably the reason for the complex metabolite pattern detected in urine. The majority of metabolites were the result of more than one of the above mentioned transformations. The degradation resulted in metabolites which together with unchanged test material were eliminated very rapidly. The administered dose was almost completely absorbed, and was degraded partially and eliminated almost completely via urine. Excretion via bile and ultimately via faeces together with small amounts of unchanged test material escaping absorption was of very minor importance. Enterohepatic circulation was negligible.

The degradation of the test material accounted for about 20 - 30% of dose. The metabolic pathways are independent of the route of administration, the dose level in the range of 0.5 and 100 mg/kg body weight, pre-treatment, and the sex of the animals, within the limits of the study. Due to the rapid absorption and excretion, it is assumed that exposure time of CGA293343 to biotransformation enzymes is reduced resulting in excretion of high amounts of unchanged test material.

Reference: Müller T and Stampf P, 1996. CGA293343:Absorption, distribution and excretion of [Thiazol-2-14C] and [Oxadiazin-4-14C] CGA293343 in the rat, Division Crop Protection, Ciba-Geigy Limited, Basle, Switzerland. Unpublished Report No. 11/96

OECD 428 - rat and human skin membrane

The percutaneous penetration of the test material was determined in vitro using split-thickness skin membranes from rat and human skin (Loffler, 2002). The skin membranes were set up in flow-through diffusion cells, the formulated [14C] radiolabelled test substance was applied onto the skin membranes and then the perfusates were collected at defined time intervals (0-6 hours at 1 hour intervals and 6-24 hours at 2 hour intervals). Two dose levels were used, i.e. a low dose level of nominal 0.2 mg test material / cm2 and a high dose level of nominal 3.5 mg test material / cm2. The low dose of 20 g a.i./L reflects concentrations recommended for use in the field, the high dose represents the undiluted formulation (350 g a.i./L). 24 hours after application, the skin membrane surface was rinsed with ethanol/water (1:1) and the radioactivity in the skin rinse was determined by LSC. The skin membrane was removed from the in-line cells and dissolved in tissue solubiliser prior to LSC. Finally, the cells were washed with ethanol/water (1:1) and the radioactivity in the cell wash determined.

During the 24 hours after application of formulated [Oxadiazin-4-14C] labelled test material, only 0.8% and 1.2% of the dose penetrated through rat split-thickness skin membranes at the low and high dose levels, respectively. The human split-thickness skin membranes showed a lower permeability of the test material. Within 24 hours after application, only 0.2% and 0.4% of the low and high doses, respectively, penetrated through human skin membranes.

A species difference in respect to the penetration of formulated test material was also reflected by the flux. The flux at steady state conditions was determined to be about 0.06 µg-cm-2 h-1 and 1.18 µg-cm-2 h-1 through rat skin membranes and about 0.01 µg.cm-2 h-1and 0.20 µg.cm-2 h-1 through human skin membranes at the low and high dose level, respectively. This results in a human:rat ratio of the flux of about 1:4.8 at the low dose level and 1:5.8 at the high dose level.

The 19-fold higher concentration of the undiluted formulation led to a corresponding 19-fold and 16-fold higher penetration rate (flux) in rat and human skin membrane, respectively.

The radioactivity in the skin rinse at the end of exposure at both dose levels and for both species was analysed as unchanged test material. The experimental recoveries were between 89.2% and 100.4% of the applied dose. The test material penetrates through rat skin membrane faster and to a higher extent than through human skin membrane, at in-use concentrations as well as at the concentration of the undiluted formulation.

Conclusion absorption

The two studies presented above show that the substance is easily absorbed via the oral route and via the dermal route after oral exposure to rats and exposure to rat skin. Absorption by the human skin is estimated to be on average 5 times lower compared to absorption by the rat skin. Based on this information and the lack of information on inhalation absorption, the default absorption values from the REACH guidance (Chapter 8, R.8.4.2) are used for DNEL derivation, namely: 100% for inhalation and 50% for oral absorption. For dermal absorption, 10% is considered to be an absolute worst case based on the available information.