Pirimiphos-methyl

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

EU Method B.36 (Toxicokinetics)

Qualifier:

according to guideline

Guideline:

EPA OPP 85-1 (Metabolism and Pharmacokinetics)

GLP compliance:

yes

Radiolabelling:

yes

Species:

rat

Strain:

other: Alpk:APfSD

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Dose / conc.:

1 mg/kg bw (total dose)

Dose / conc.:

50 mg/kg bw (total dose)

Dose / conc.:

250 mg/kg bw (total dose)

Dose / conc.:

1 mg/kg bw/day

Remarks:

14 days non-radiolabelled test substance and 1 day radiolabelled test substance

The test substance is rapidly and extensively absorbed from the gastrointestinal tract following gavage administration of doses of 1, 50 or 250 mg/kg bw and repeated doses of 1 mg/kg bw/day (Table 2. the results at 50 mg/kg bw seem anomalous and are used for comparison with bile-duct cannulated rats only). Excretion is rapid, predominantly in the urine (>70%) at 48 hours, with approximately 50 % of the administered dose present in 12 hour urine samples. Some of the radiolabel present in faeces is due to biliary excretion. Overall the extent of absorption is considered to be >80% based on the
radioactivity found in the urine of animals dosed with a single dose of 1 or 250 mg/kg bw and the repeat dose at 1 mg/kg bw/d (>60%) plus the significant level of radioactivity found in the bile of animals dosed with a single dose of 50 mg/kg bw (17%). Variations in the levels of radiolabel in consecutive faecal samples indicated that test substance metabolites were subject to entero-hepatic circulation, which was more predominant in females and animals receiving repeated doses. This was consistent with the data from bile-duct cannulated animals, but could also involve deposition and release
in adipose tissue. Two days after dosing, less than 2% of the administered dose remained in the carcass and tissues, though abdominal fat had particularly high concentrations in females dosed with 250 mg/kg bw. Other than a marked increase in deposition in fat at 250 mg/kg bw, the results were essentially independent of dose level and duration. Indications of a sex difference in some aspects of the toxicokinetics of the test substance were consistent with metabolite data.

Table 2. Urine, faecal and tissue levels (means) in Alpk rats following administration of the test substance.

Dose	1 × 50 mg/kg bw (bile duct cannulated)		1 × 1 mg/kg bw		1 × 250 mg/kg bw		Repeat (14 + 1) × 1 mg/kg bw/d
	Male	female	male	female	male	female	Male	female
Urine 12h (% of dose)	33	28	59	46	53	50	54	57
Urine 12h - cannulated (% of dose)	22	7	NP	NP	NP	NP	NP	NP
Urine 48h non-cannulated (including terminal cage wash) (% of dose)	50	49	72	64	70	79	69	73
Urine 48h - cannulated (including terminal cage wash) (% of dose)	38	33	NP	NP	NP	NP	NP	NP
Faeces 12h (% of dose)	6	11	11	16	10	3	12	11
Faeces 12h - cannulated (% of dose)	2	<1	NP	NP	NP	NP	NP	NP
Faeces 48h non-cannulated (% of dose)	22	22	-	-	-	-	-	-
Faeces 48 h - cannulated (% of dose)	30	16	-	-	-	-	-	-
Total excretion 48 h (% of dose)	71	68	95	93	95	93	98	94
Total excretion 48 h - cannulated (% of dose)	85	69	NP	NP	NP	NP	NP	NP
Bile 6h (% of dose)	9	4	NP	NP	NP	NP	NP	NP
Bile 12h (% of dose)	14	11	NP	NP	NP	NP	NP	NP
Bile 48h (% of dose)	17	21	NP	NP	NP	NP	NP	NP
Tissue + carcass day 2 (% of dose)	NP	NP	0.3	1.3	0.7	1.9	0.8	1.0
Liver day 2 (mg equiv./g)	NP	NP	0.004	0.003	1.2	1.1	0.005	0.003
Kidney day 2  (mg equiv./g)	NP	NP	0.003	0.004	1.2	1.5	0.004	0.004
Abdominal fat day 4 (mg equiv/g)	NP	NP	0.004	0.009	5.6	56.0	0.007	0.011
Brain day 2 (mg equiv./g)	NP	NP	0.001	0.001	0.2	0.3	0.001	0.001
Blood day 2 (mg equiv./g)	NP	NP	0.002	0.002	1.1	1.1	0.002	0.003
Plasma day 2 (mg equiv./g)	NP	NP	0.001	0.001	1.1	0.7	0.001	0.001
Heart day 2 (mg equiv./g)	NP	NP	0.001	0.001	0.4	0.4	0.001	0.001

Pooled samples of urine, bile and faeces from the studies were examined for the presence of metabolites using a variety of chromatography techniques, mass spectroscopy (MS) and nuclear magnetic resonance spectroscopy (NMR). A total of 20 metabolites were detected, 13 of which were characterised. In most groups, >75% of the administered dose was characterised.Absorbed test substance is extensively metabolised, with no parent compound found in either urine or bile. Administration of a single dose or 15 doses of the test substance at 1 mg/kg bw produced a similar metabolic profile in both sexes with the main reactions being i) cleavage of the ester bond followed by glucuronidation or N-dethylation (M3, M11) or ii) O-demethylation (M6) followed by N-dethylation. Administration of 50 or 250 mg/kg bw indicated saturation in females of the pyrimidinyl esterase and the N-dethylation of M6. This difference in metabolism may be linked with the high levels of radiolabel in abdominal fat of females administered 250 mg/kg bw. Several metabolites retain functional groups consistent with cholinesterase inhibiting potential (M5). There was no direct evidence, in the samples analysed, of transformation to test substance oxon, though other oxons were detected in urine.

Table 3. Main metabolites in urine, faeces and bile (means; % of administered dose) in Alpk rats following administration of the test substance. 

Dose	1 × 50 mg/kg bw (bile duct cannulated)		1 × 1 mg/kg bw		1 × 250 mg/kg bw		Repeat (14 + 1) × 1 mg/kg bw/d
Metabolite#	Male	Female	Male	Female	Male	Female	Male	Female
Urine
parent	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
M1	1	5	3	4	7	9	3	6
M3	14	5	28	28	15	8	30	28
M11	1	<1	2	1	1	N.D.	1	1
De-ethyl M6	12	7	8	5	24	13	10	10
M6	1	11	N.D.	2	3	35	N.D.	2
M12	2	1	5	4	4	3	8	5
O-Glucuronide of M1	3	2	4	4	5	6	1	5
ethoxy-glucuronide of M12	<1	<1	3	1	1	-	2	2
Faeces
parent	29	15	13	15	4	3	15	5
M1	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.	1	1
M3	N.D.	N.D.	2	3	6	5	7	3
M11	N.D.	N.D.	6	5	5	2	5	3
O-Glucuronide of M1	N.D.	N.D.	N.D.	1	3	1	1	1
Bile
parent	N.D.	N.D.	-	-	-	-	-	-
M5	3	2	-	-	-	-	-	-
M1	1	1	-	-	-	-	-	-
M3	2	1	-	-	-	-	-	-
M11	<1	N.D.	-	-	-	-	-	-
De-ethyl M6	3	2	-	-	-	-	-	-
M6	<1	<1	-	-	-	-	-	-
M12	<1	N.D.	-	-	-	-	-	-
O-Glucuronide of M1	6	12	-	-	-	-	-	-

N.D. - Not Detected

Conclusions:

Excretion is rapid, predominantly in the urine (>70%) at 48 hours, with approximately 50 % of the administered dose present in 12 hour urine samples. Some of the radiolabel present in faeces is due to biliary excretion. Overall the extent of absorption is considered to be >80% based on the radioactivity found in the urine of animals dosed with a single dose of 1 or 250 mg/kg bw and the repeat dose at 1 mg/kg bw/d (>60%) plus the significant level of radioactivity found in the bile of animals dosed with a single dose of 50 mg/kg bw (17%). Variations in the levels of radiolabel in consecutive faecal samples indicated that the test substance metabolites were subject to entero-hepatic circulation, which was more predominant in females and animals receiving repeated doses. This was consistent with the data from bile-duct cannulated animals, but could also involve deposition and release in adipose tissue. Two days after dosing, less than 2% of the administered dose remained in the carcass and tissues, though abdominal fat had particularly high concentrations in females dosed with 250 mg/kg bw. Other than a marked increase in deposition in fat at 250 mg/kg bw, the results were essentially independent of dose level and duration. Indications of a sex difference in some aspects of the toxicokinetics of the test substance were consistent with metabolite data.
Absorbed test substance is extensively metabolised, with no parent compound found in either urine or bile. A total of 20 metabolites were detected, 13 of which were characterised. In most groups, >75% of the administered dose was characterised.

Executive summary:

The basic ADME of the test substance, following single and repeat dosing, was investigated in Alpk:APfSD rats in a series of studies performed between 1996 and 1998.All studies were performed according to GLP and the following Guidelines without significant deviations:- 87/302/EEC B.36 / OECD 417 (1984) / FIFRA § 85-1. All studies used unlabelled material of 99.6% purity and 2-14C-pyrimidyl labelled material of >98% purity and specific activity of 2.15 GBq/mMol. Doses were administered by gavage in corn oil. Urine samples were taken at 6, 12, 24, 36 and 48 hours after the 14 C dose, with faecal samples taken at 12, 24, 36 and 48 hours. Bile samples were taken on eight occasions between 2 and 48 hours after dosing. Animals were sacrificed at 48 hours (except 50 mg/kg bw non-cannulated animals - 72 hours) and samples of cage washes, blood and a range of tissues taken. Samples were analysed by liquid scintillation counting following appropriate processing. Autoradiography was performed on animals sacrificed 6 or 24 hours after a dose of 1 mg/kg bw.

The test substance is rapidly and extensively absorbed from the gastrointestinal tract following gavage administration of doses of 1, 50 or 250 mg/kg bw and repeated doses of 1 mg/kg bw/d (Table 2. the results at 50 mg/kg bw seem anomalous and are used for comparison with bile-duct cannulated rats only). Excretion is rapid, predominantly in the urine (>70%) at 48 hours, with approximately 50 % of the administered dose present in 12 hour urine samples. Some of the radiolabel present in faeces is due to biliary excretion. Overall the extent of absorption is considered to be >80% based on the radioactivity found in the urine of animals dosed with a single dose of 1 or 250 mg/kg bw and the repeat dose at 1 mg/kg bw/d (>60%) plus the significant level of radioactivity found in the bile of animals dosed with a single dose of 50 mg/kg bw (17%). Variations in the levels of radiolabel in consecutive faecal samples indicated that the test substance metabolites were subject to entero-hepatic circulation, which was more predominant in females and animals receiving repeated doses. This was consistent with the data from bile-duct cannulated animals, but could also involve deposition and release in adipose tissue. Two days after dosing, less than 2% of the administered dose remained in the carcass and tissues, though abdominal fat had particularly high concentrations in females dosed with 250 mg/kg bw. Other than a marked increase in deposition in fat at 250 mg/kg bw, the results were essentially independent of dose level and duration. Indications of a sex difference in some aspects of the toxicokinetics of the test substance were consistent with metabolite data.

Pooled samples of urine, bile and faeces from the studies were examined for the presence of metabolites using a variety of chromatography techniques, mass spectroscopy (MS) and nuclear magnetic resonance spectroscopy (NMR). A total of 20 metabolites were detected, 13 of which were characterised. In most groups, >75% of the administered dose was characterised.Absorbed test substance is extensively metabolised, with no parent compound found in either urine or bile. Administration of a single dose or 15 doses of the test substance at 1 mg/kg bw produced a similar metabolic profile in both sexes with the main reactions being i) cleavage of the ester bond followed by glucuronidation or N-dethylation (M3, M11) or ii) O-demethylation (M6) followed by N-dethylation. Administration of 50 or 250 mg/kg bw indicated saturation in females of the pyrimidinyl esterase and the N-dethylation of M6. This difference in metabolism may be linked with the high levels of radiolabel in abdominal fat of females administered 250 mg/kg bw. Several metabolites retain functional groups consistent with cholinesterase inhibiting potential (M5). There was no direct evidence, in the samples analysed, of transformation to test substance oxon, though other oxons were detected in urine.

Reference 2

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Sep 1996 to 06 Sep 1996

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)

GLP compliance:

yes

Radiolabelling:

no

Doses:

- Nominal doses: 503.7 g/L; 4.5 g/L
- Dose volume: 25.4 µL (10 µL/cm2, 2.54 cm2 application area)
- Rationale for dose selection: The two doses were prepared to mimic the commercial formulation

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human
- Preparative technique: Epidermal membranes were prepared from human whole skin by immersion in water at 60°C for 40-45 seconds and the epidermis teased off the dermis.
- Membrane integrity check: The integrity of the membranes was checked by measurement of the electrical resistance across the skin. Only those membranes with an acceptable resistance, thereby showing that they were intact, were used on the study.

PRINCIPLES OF ASSAY
- Diffusion cell: Measured using glass diffusion cells in which the epidermis formed a horizontal membrane and provided an application area of 2.54 cm2.
- Receptor fluid: The receptor chambers of the cells were filled with a recorded volume of receptor fluid (50% v/v ethanol in water) and placed in a water bath maintained at 30 ± 1°C. A pre-treatment sample (0 5mL) was taken from each receptor chamber for analysis by gas-liquid chromatography (GLC). An equal volume of fresh receptor fluid was added to each receptor chamber to replace the volume removed.
- Test temperature: Throughout the experiment the receptor fluid was stirred and the epidermal membranes were maintained at a normal skin temperature of 30 ± 1°C in a water bath.
- Occlusion: Unoccluded

Key result

Time point:

24 h

Dose:

4.5 g/L

Parameter:

percentage

Absorption:

8.46 %

Time point:

6 h

Dose:

4.5 g/L

Parameter:

percentage

Absorption:

1.9 %

Time point:

10 h

Dose:

4.5 g/L

Parameter:

percentage

Absorption:

3.4 %

Time point:

6 h

Dose:

503.7 g/L

Parameter:

percentage

Absorption:

0.11 %

Time point:

10 h

Dose:

503.7 g/L

Parameter:

percentage

Absorption:

0.22 %

Time point:

24 h

Dose:

503.7 g/L

Parameter:

percentage

Absorption:

0.56 %

Table 1. Summary of the test substance 500 g/L absorption through human epidermis

Application of Test Materials	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
Concentrate Formulation	0.5 - 54	1.17+0.097
(503.7 g a.i/L)			6	5.72	0.11
10 µL/cm^2(5037 µg ai/cm^2)			8	8.32	0.17
Unoccluded			10	10.9	0.22
Duration of exposure: 54h			24	28.4	0.56
n = 5
0.9:100 v/v aqueous spray diln	0.5 – 54	0.155 +0.028
(4.536 µg a.i/L)			6	0.883	1.9
10 µL/cm^2(45.36 µg ai/cm^2)			8	1.20	2.6
Unoccluded			10	1.53	3.4
Duration of exposure: 54h			24	3.84	8.5
n = 6

 

Conclusions:

The results obtained in this study demonstrate that the absorption of the test substance through human epidermis is slow when compared with the absorption rates of other penetrants measured using this in vitro technique (Dugard et al, 1984; Dugard and Scott, 1984). These data predict that the dermal absorption of the test substance from normal exposure to this 500g/L formulation would be low and the percentage of absorption would be 8.46 % in the worst case scenario.

Executive summary:

The absorption of the test substance from a nominal 500g/L formulation (actual content 504 g test substance/L) has been measured in vitro through human epidermis. The formulation was applied as the concentrate formulation and as a 0.9:100 v/v (4.54 g test substance/L) spray strength dilution of the formulation in water. The concentrate formulation and the spray strength dilution were applied to the epidermal membranes at a rate of 10 µL/cm2; all applications were left unoccluded throughout the entire exposure period. These applications were designed to simulate potential human dermal exposure to the formulation during normal use. 

Absorption of the test substance from both the concentrate and spray strength dilution maintained essentially constant rates over the entire exposure period (54 h). The absorption rate measured from the spray dilution (mean 0.155 µg/cm2/h) was approximately 8 times less than the rate obtained from the concentrate (1.172 µg/cm2/h). Mean amounts of the test substance absorbed during typical working day periods from the concentrate varied from 5.72 µg /cm2 (0.11% of dose) at 6 h to 10.9 µg/cm2 (0.22% of dose) at 10 h. This increased to 28.44 µg /cm2 (0.56% of dose) after 24 h exposure. The amounts absorbed from the spray dilution varied from 0.883 µg /cm2 (1.9% of dose) at 6 h to 1.53 µg/cm2 (3.4% of dose) at 10 h. This increased to 3.84 µg /cm2 (8.46% of dose) after 24 h exposure.

The results obtained in this study demonstrate that the absorption of the test substance through human epidermis is slow when compared with the absorption rates of other penetrants measured using this in vitro technique (Dugard et al, 1984; Dugard and Scott, 1984).  These data predict that the dermal absorption of the test substance from normal exposure to this 500g/L formulation would be low and the percentage of absorption would be 8.46 % in the worst case scenario.

Reference 3

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Sep 1996 to 24 Sep 1996

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)

GLP compliance:

yes

Radiolabelling:

no

Duration of exposure:

Duration of exposure and sampling: The skin was exposed to the test preparations for 30 hours. At recorded intervals, samples (0.5 mL) of the receptor fluid were taken for analysis by GLC. The volume of fluid in the receptor chamber was maintained by the addition of fresh receptor fluid (0.5 mL) to the chamber immediately after the removal of each sample.

Doses:

- Nominal doses: 503.7 g/L; 4.5 g/L
- Dose volume: 25.4 µL (10 µL/cm2, 2.54 cm2 application area)
- Rationale for dose selection: The two doses were prepared to mimic the commercial formulation

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Rat
- Preparative technique: Epidermal membranes were prepared from rat whole skin by chemical (sodium bromide) separation.
- Membrane integrity check: The integrity of the membranes was checked by measurement of the electrical resistance across the skin. Only those membranes with an acceptable resistance, thereby showing that they were intact, were used on the study.

PRINCIPLES OF ASSAY
- Diffusion cell: Measured using glass diffusion cells in which the epidermis formed a horizontal membrane and provided an application area of 2.54cm2.
- Receptor fluid: The receptor chambers of the cells were filled with a recorded volume of receptor fluid (50% v/v ethanol in water) and placed in a water bath maintained at 30 ± 1°C. A pre-treatment sample (0 5mL) was taken from each receptor chamber for analysis by gas-liquid chromatography (GLC). An equal volume of fresh receptor fluid was added to each receptor chamber to replace the volume removed.
- Test temperature: Throughout the experiment the receptor fluid was stirred and the epidermal membranes were maintained at a normal skin temperature of 30 ± 11°C in a water bath.
- Occlusion: Unoccluded

Key result

Time point:

24 h

Dose:

4.5 g/L

Parameter:

percentage

Absorption:

91 %

Time point:

6 h

Dose:

4.5 g/L

Parameter:

percentage

Absorption:

52.3 %

Time point:

8 h

Dose:

4.5 g/L

Parameter:

percentage

Absorption:

63.6 %

Time point:

10 h

Dose:

4.5 g/L

Parameter:

percentage

Absorption:

72.5 %

Time point:

6 h

Dose:

503.7 g/L

Parameter:

percentage

Absorption:

3.7 %

Time point:

8 h

Dose:

503.7 g/L

Parameter:

percentage

Absorption:

5.2 %

Time point:

10 h

Dose:

503.7 g/L

Parameter:

percentage

Absorption:

6.5 %

Time point:

24 h

Dose:

503.7 g/L

Parameter:

percentage

Absorption:

14 %

Table 1. Summary of the test substance 500 g/L Formulation absorption through rat epidermis

Application of Test Materials	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
Concentrate Formulation
(503.7 g a.i/L)			6	186	3.7
10 µL/cm^2(5037 µg ai/cm^2)	0.5 - 30	28.8+2.75	8	263	5.2
Unoccluded			10	327	6.5
Duration of exposure: 30h			24	706	14.0
n = 6
0.9:100 v/v aqueous spray diln
(4.5 g a.i/L)			6	23.7	52.3
10mL/cm^2(48.38 µg ai/cm^2)	0.5 – 4	4.79+0.227	8	28.8	63.6
Unoccluded			10	32.9	72.5
Duration of exposure: 30h	0.5 - 10	3.36+0.116	24	41.3	91.0
n = 12

Conclusions:

The results obtained in this study indicate that the absorption of the test substance from the 500 g/L formulation through rat epidermis would be considered slow.
The high percentage of the dose absorbed by 24 h from the spray strength dilution reflects the fact that only a very low dose (45.38 µg test substance/cm2) was applied.

Executive summary:

The absorption of the test substance from a nominal 500 g/L formulation, (actual content 504 g test substance/L) has been measured in vitro through rat epidermis. The formulation was applied as the concentrate formulation and as a 0.9:100 v/v (4.54 g test substance/L) spray strength dilution of the formulation in water. The concentrate formulation and the spray strength dilution were applied to the epidermal membranes at a rate of 10 µL/cm²; all applications were left unoccluded throughout the entire exposure period. These applications were designed to simulate potential human dermal exposure to the formulation during normal use.

 Absorption of the test substance from the concentrate formulation maintained an essentially constant rate (mean 28.8 µg/cm²/h) over the entire exposure period (30 h). During the working day periods, an average of between 3.7-6.5 % of the applied dose was absorbed. This increased to 14 % after 24 h exposure.

From the spray strength dilution, the fastest absorption rate was measured during the first 4 h of exposure (mean 4.79 µg/cm²/h), however the mean rate over a 10 h working day period was 3.36 µg/cm²/h. During the working day periods, an average of between 52-73% of the applied dose was absorbed. This increased to 91% after 24 h exposure. 

The results obtained in this study indicate that the absorption of the test substance from the 500 g/L formulation through rat epidermis would be considered slow.  The high percentage of the dose absorbed by 24h from the spray strength dilution reflects the fact that only a very low dose (45.38 µg test substance/cm²) was applied.

Description of key information

- Oral absorption: 70%; OECD 417, Brown 1997 a, b, c, d; Macpherson 1998

- Excretion: rapid, predominantly in the urine (>70%) at 48 hours; OECD 417, Brown 1997 a, b, c, d; Macpherson 1998

- Metabolism: extensively metabolised (20 metabolites detected); OECD 417, Brown 1997 a, b, c, d; Macpherson 1998

- Dermal absorption rate: 8.46 %; OECD 428, Ward 1996

- Bioaccumulation potential: No evidence of accumulation after repeated dosing. 

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

70

Absorption rate - dermal (%):

8.46

Absorption rate - inhalation (%):

100

Additional information

Oral absorption, excretion, metabolism and distribution; OECD 417, Brown 1997 a, b, c, d; Macpherson 1998

The basic ADME of the test substance, following single and repeat dosing, was investigated inAlpk:APfSD rats in a series of studies performed between 1996 and 1998. All studies were performed according to GLP and the following Guidelines without significant deviations: 87/302/EEC B.36 / OECD 417 (1984) / FIFRA § 85-1. All studies used unlabelled material and 2-14C-pyrimidyl labelled material and specific activity of 2.15 GBq/mMol. Doses were administered by gavage in corn oil. Urine samples were taken at 6, 12, 24, 36 and 48 hours after the 14C dose, with faecal samples taken at 12, 24, 36 and 48 hours. Bile samples were taken on eight occasions between 2 and 48 hours after dosing. Animals were sacrificed at 48 hours (except 50 mg/kg bw non-cannulated animals - 72 hours) and samples of cage washes, blood and a range of tissues taken. Samples were analysed by liquid scintillation counting following appropriate processing. Autoradiography was performed on animals sacrificed 6 or 24 hours after a dose of 1 mg/kg bw.

The test substance is rapidly and extensively absorbed from the gastrointestinal tract following gavage administration of doses of 1, 50 or 250 mg/kg bw and repeated doses of 1 mg/kg bw/day (the results at 50 mg/kg bw seem anomalous and are used for comparison with bile-duct cannulated rats only). Excretion is rapid, predominantly in the urine (>70%) at 48 hours, with approximately 50 % of the administered dose present in 12 hour urine samples. Some of the radiolabel present in faeces is due to biliary excretion. Overall, the extent of absorption is considered to be >80% based on the radioactivity found in the urine of animals dosed with a single dose of 1 or 250 mg/kg bw and the repeat dose at 1 mg/kg bw/day (>60%) plus the significant level of radioactivity found in the bile of animals dosed with a single dose of 50 mg/kg bw (17%). Variations in the levels of radiolabel in consecutive faecal samples indicated that the test substance metabolites were subject to entero-hepatic circulation, which was more predominant in females and animals receiving repeated doses. This was consistent with the data from bile-duct cannulated animals, but could also involve deposition and release in adipose tissue. Two days after dosing, less than 2% of the administered dose remained in the carcass and tissues, though abdominal fat had particularly high concentrations in females dosed with 250 mg/kg bw. Other than a marked increase in deposition in fat at 250 mg/kg bw, the results were essentially independent of dose level and duration. Indications of a sex difference in some aspects of the toxicokinetics of the test substance were consistent with metabolite data. Based on these findings and oral absorption of 80 % is used in the chemical safety assessment. No data on respiratory absorption is available, therefore, a default absorption value of 100% is assumed.

Pooled samples of urine, bile and faeces from the studies were examined for the presence of metabolites using a variety of chromatography techniques, mass spectroscopy (MS) and nuclear magnetic resonance spectroscopy (NMR). A total of 20 metabolites were detected, 13 of which were characterised. In most groups, >75% of the administered dose was characterised. Absorbed test substance is extensively metabolised, with no parent compound found in either urine or bile. Administration of a single dose or 15 doses of the test substance at 1 mg/kg bw produced a similar metabolic profile in both sexes with the main reactions being i) cleavage of the ester bond followed by glucuronidation or N-dethylation (M1, M2) or ii) O-demethylation (M3) followed by N-dethylation. Administration of 50 or 250 mg/kg bw indicated saturation in females of the pyrimidinyl esterase and the N-dethylation of M3. This difference in metabolism may be linked with the high levels of radiolabel in abdominal fat of females administered 250 mg/kg bw. Several metabolites retain functional groups consistent with cholinesterase inhibiting potential (M4, M5, M6, M7). There was no direct evidence, in the samples analysed, of transformation to test substance oxon, though other oxons were detected in urine (M7, M6).

Dermal absorption

The absorption of the test substance from a nominal 500g/L formulation (actual content 504 g test substance/L) has been measured in vitro through human epidermis. The formulation was applied as the concentrate formulation and as a 0.9:100 v/v (4.54 g test substance/L) spray strength dilution of the formulation in water. The concentrate formulation and the spray strength dilution were applied to the epidermal membranes at a rate of 10 µL/cm2; all applications were left unoccluded throughout the entire exposure period. These applications were designed to simulate potential human dermal exposure to the formulation during normal use. 

Absorption of the test substance from both the concentrate and spray strength dilution maintained essentially constant rates over the entire exposure period (54 h). The absorption rate measured from the spray dilution (mean 0.155 µg/cm2/h) was approximately 8 times less than the rate obtained from the concentrate (1.172 µg/cm2/h). Mean amounts of the test substance absorbed during typical working day periods from the concentrate varied from 5.72 µg /cm2 (0.11% of dose) at 6 h to 10.9 µg/cm2 (0.22% of dose) at 10 h. This increased to 28.44 µg /cm2 (0.56% of dose) after 24 h exposure. The amounts absorbed from the spray dilution varied from 0.883 µg /cm2 (1.9% of dose) at 6 h to 1.53 µg/cm2 (3.4% of dose) at 10 h. This increased to 3.84 µg /cm2 (8.46% of dose) after 24 h exposure.

The results obtained in this study demonstrate that the absorption of the test substance through human epidermis is slow when compared with the absorption rates of other penetrants measured using this in vitro technique (Dugard et al, 1984; Dugard and Scott, 1984).  These data predict that the dermal absorption of the test substance from normal exposure to this 500g/L formulation would be low and the percentage of absorption would be 8.46 % in the worst case scenario. Based on available information, the dermal absorption value used for the chemical safety assessment was conservatively set on 8.46%.