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Reference
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with generally accepted scientific principles, with incomplete reporting on methodological deficiencies, which do not affect the quality of the relevant results.
Objective of study:
metabolism
Principles of method if other than guideline:
C14-labelled 3,5-dimethylpyrazole was orally administered to rats in order to characterise the metabolism of the compound.
GLP compliance:
no
Radiolabelling:
yes
Remarks:
C14
Species:
rat
Strain:
other: CD
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River.
- Glucose-primed rats were used as described in Dulin (1962).
- Weight at study initiation: 140g
- Fasting period before study: 16 hours
- Housing: In cages designed to collect and separate the animals excrement.
Route of administration:
oral: gavage
Vehicle:
other: 0.25% methylcellulose solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Two samples of the test material were prepared in volumetric flasks.
Remarks:
Doses / Concentrations:
25 mg/kg, (1.19 x 10^7 dpm/rat), samples were within 1.4% of each other.
Control animals:
not specified
Positive control reference chemical:
no
Details on study design:
The metabolism, of the test material was investigated by orally exposing rats and anayising their urine in order to identify any metabolites.

Animals received one of three pre-treatments:
- Fasted rats received only the test material.
- Rats received corn oil (0.2 ml/rat) immediately prior to dosing with the test material.
- Rats received SKF-525A (40 mg/kg), injected intraperitoneally, 30 minutes prior to treatment with the test material.

Both the corn oil and SKF-525A, when administered at the stated dose levels, inhibits the hypoglycemic activity of the test material by up to 200 mg/kg in the gastrointestinal tract.

SKF-525A = β-diethylaminoethyl diphenylpropylacetate hydrochloride
Details on dosing and sampling:
Hypoglycemic Activity Sampling:
- Tissues and body fluids sampled: urine.
- Time and frequency of sampling: Up to 2 hours post administration.
- Method type(s) for identification: Liquid scintillation counting, radiochromoatogram and both paper and thin-layer chromatography.

Radioactive Samples:
- Apparatus: Liquid scintillation spectrometer, Packard Tri-Carb, Models 314EX2A abd 314X.
- Counts were performed in duplicate.
- The procedure followed that set out in Herberg (1960).
Details on absorption:
The test material is absorbed completely from the gatroinestinal tract.
Details on excretion:
Essentially the entire dose (95%) is found in the urine within 24 hours, where 60% is excreted during the first 3 hours.
Metabolites identified:
yes
Details on metabolites:
It is completely converted (>99%) to 4 metabolites, 2 of which together with their conjugated forms account for about 98% of the oral dose.

The metabolites were identified as follows along with their average excreted percentages; 5-methylpyrazole-3-carboxylic acid (13.1 ± 3.8), conjugated 5-methylpyrazole-3-carboxylic acid (13.6 ± 4.4), conjugated 4-hydroxy-3,5-dimethylpyrazole (70.8 ± 5.5), the identity of the 4th is unknown (1.3 ± 1.9).

Distribution of metabolites of 3,5-dimethylpyrazole-C14

In the urine of fasted rats. Both paper and thin-layer chromatography showed essentially no unchanged 3 , 5-dimethylpyrazole in the urine. The Rf values of the metabolites are presented in table 1 . Based on the radioactive intensity of the 4 zones, in 5 fasted rats

the average percentages excreted (±S.D.) as metabolites designated as A, B, C, and D were 13.1 ± 3.8, 13.6 ± 4.4, 70.8 ± 5.5, and

1.3 ± 1.9, respectively (table 2) . Three of the fasted rats did not excrete a detectable amount of metabolite D.

Table 1: Paper Chromatographic Rf valuesa

Compoundb

System

BAW

BPW

KCl

Metabolite A

0.76

0.37

0.69

Metabolite B

0.72

0.32

0.77

Hydrolysis product of metabolite B

0.76

0.37

 

Metabolite C

0.45

0.49

0.85

Hydrolysis product of metabolite C

0.61

0.77

 

Metabolite D

0.36

0.09

NRc

3,5-Dimethylpyrazole

0.80

0.82

0.79

5-Methylpyrazole-3-carboxylic acid

0.76

0.37

0.69

4-Hydroxy-3,5-dimethylpyrazole

0.61

0.77

 

a The Rf values were generally reproducible to within 0.05 Rf unit from one chromatogram to the next, but were reproducible to within 0.01 Rf unit when replicate parallel measurements were made on the same chromatogram. The latter procedure was always used when comparing unknowns with standards.

b A, 5-methylpyrazole-3-carboxylic acid; B, conjugated 5-methylpyrazole-3-carboxylic acid; C, conjugated 4-hydroxy-3 , 5-dimethylpyrazole; D, unknown.

c NR = not resolved.

Table 2: Excretion and distribution of metabolites from rats treated with 3,5 -dimethylpyrazole

Pretreatment

Percent of C14in

0 - 24 hr Urine

Percent of C14in 0 - 24 hr Urine as Metabolte:

A

B

C

D

Fasted

89.1a

7.4

18.7

70.7

3.5

Fasted

95.3b

13.3

7.9

78.8

0.0

Fasted

93.1

 

 

 

 

Fasted

101.3

14.4

17.5

65.0

3.0

Fasted

94.1c

12.2

11.6

72.9

0.0

Fasted

97.2c

18.0

12.4

66.6

0.0

Mean ± S.D.

95.0 ± 4.1

13.1 ± 3.8

13.6 ± 4.4

70.8 ± 5.5

1.3 ± 1.9

 

Corn Oil

83.1

27.0

13.2

59.0

0.8

Corn Oil

21.8

10.6

10.2

75.6

2.7

Corn Oil

52.2

13.6

11.1

72.9

2.5

Mean ± S.D.

52.3 ± 30.6

17.1 ± 8.7

11.5 ± 1.4

69.2 ± 8.7

2.0 ± 1.0

 

SKF-525A

83.5

12.8

9.7

72.5

3.5

SKF-525A

62.5

 

 

 

 

Mean ± S.D.

73.0 ± 14.8

 

 

 

 

aAn additional 4.8% was recovered in the 24- to 168-hour samples.

bAn additional 1.8% was recovered in the 24- to 168-hour samples.

cAn additional 0.3% was recovered in the 24- to 48-hour sample.

The relative areas of the radioactive peaks in the BPW and BAW systems were virtually identical, strongly suggesting that the peaks were resolved completely (for example, integration of the 4 radioactive peaks resulting from the 24-hr urine collection of 1 rat gave relative areas of 7.9, 18.9, 70.1, and 3.4 using the BAW system and 6.8, 18.4, 71.3, and 3.6 using the BPW system).

Isolation and identification of metabolites

3,5-Dimethylpyrazole is extracted quantitatively from urine into chloroform at pH 8.7; the fact that essentially no radioactivity (<0.5%) is extracted under these conditions supports the theory that very little 3,5 -dimethylpyrazole is excreted intact. Extraction data are more reliable than radiochromatographic evidence because 3,5-dimethylpyrazole sublimes rather easily. At pH 1.5, metabolites A and B were essentially quantitatively extracted into the butanol, while the very polar metabolite C remained in the urine.

Metabolite A is 5-methylpyrazole-3-carboxylic acid, the active metabolite. This was established from a comparison of Rf values in 2 paper and 2 thin-layer chromatographic systems with authentic material. This metabolite was isolated previously in crystalline form and characterized after the administration of unlabeled 3,5-dimethylpyrazole upon acid hydrolysis. The total radioactivity in the hydrolyzed sample is unchanged, but the radioactive peak corresponding to metabolite A increases and metabolite B disappears. The extraction behavior of metabolite B suggests that it is an acidic compound. Metabolite B is unaffected by Ketodase or Glusulase hydrolysis, suggesting that it is not a sulfate or glucuronide conjugate. These data and the fact that the polarity of metabolite B is very similar to 5-methylpyrazole-3-carboxylic acid in all of the thin-layer and paper chromatographic systems used suggest that metabolite B is an acid. The behavior of metabolite B suggests that it is conjugated with glycine, but so far this has not been demonstrated definitively.

Metabolite C is acid-hydrolyzable, indicating that it is also a conjugate. Attempts at isolating the conjugated material were unsuccessful because of its salt-like nature; e.g. even leaching of lyophilized urine with ethanol gave poor recovery of C14. The highly polar nature of this conjugated metabolite suggests a sulfate or glucuronide conjugate, but it was resistant to both Glusulase and Ketodase hydrolysis.

Because of the difficulty encountered in the extraction of the conjugated form of metabolite C, it was decided to attempt the isolation of its hydrolysis product. Urine from 10 rats (350 ml), treated with 25 mg of unlabeled 3,5-dimethylpyrazole twice daily for 3 days,

was collected, reduced in volume by lyophilization, and combined with the urine of a rat treated with labeled 3,5-dimethylpyrazole. The urine was adjusted to pH 0 with concentrated HCl and heated under reflux for 5 hours. During hydrolysis, the pH increased to 2.2. The urine was filtered, adjusted to pH 9, and filtered again (urinary precipitates were checked for radioactivity before discarding) . The alkaline urine was then extracted with two 2-liter portions of ethyl acetate. The ethyl acetate solution was reduced in volume, and material which precipitated was filtered (nonradioactive). The ethyl acetate solution was evaporated to dryness and the residue, which contained 54% of the original C14, was dissolved in ethanol, applied to a silica gel column, and eluted with CHCl3:

CH3OH (5:1). Tubes 30 to 50 (10 ml in each tube) contained the radioactivity corresponding to the hydrolysis product of metabolite C (and possibly metabolite D also). This material was applied to another silica gel column and was subjected to a gradient elution with

CHCl3,: CH3OH, 99:1 (tubes 1-124), 97:3 tube) contained the radioactivity corresponding Tubes 290 to 355 contained the radioactivity as a single peak. Evaporation of the radioactive fractions yielded crystals, which were recrystallized twice from ethanol yielding 343 mg of colorless needles, m.p. 176 °C 179 °C. The ultraviolet spectrum has a single peak, λ EtOH max = 232

max 4900) . The nuclear magnetic resonance spectrum of this material in D2O solution exhibited only singlet absorption peaks

at 2.17 ppm (methyl protons) and 4.72 ppm (exchangeable protons), having areas of 6 and 2, respectively. The absorption peak

representing the vinyl hydrogen at the 4-position, present in the spectrum of 3,5-dimethylpyrazole, is lacking, having been replaced with a group with 1 exchangeable proton. The nuclear magnetic resonance data, in conjunction with the equivalent weight in glacial acetic acid and elemental analysis, can only fit the structure attatched below.

Calculated for C5H8N2O : C, 53.55; H, 7.13; N, 24.99 ; equivalent weight, 112. Found : C, 53.77; H, 728; N, 25.12; equivalent weight, 117. Sachs and Rohmer (1902) reported a melting point of 173.5 °C for the proposed structure. The infrared spectrum (KBr pellet) support the proposed structure, e.g., bands (cm-1) at 3400, 3250, 3130, and 2580 (NH/OH); 3000 and 2920 (CH); 1615, 1535, and 1485 (C=C/N=C); 1415 and 1385 (CH3); 1300, 1235, 1175, and 1050 (CO/CN).

Metabolite D, which is formed to the extent of about 1% of the dose, has not been isolated; this metabolite was not detected in the urine of some of the rats examined. Metabolite D is unaffected by enzymatic or acid hydrolysis.

Rate of absorption, excretion, and metabolism

The absorption of orally administered 3,5-dimethylpyrazole is essentially complete in fasted rats; 95 ± 4% of the dose is found in the urine within 24 hours (table 2).

Sequential urine collections were obtained from 2 fasted rats at 1, 2, 3, 4, 6, 8, and 24 hours after administration of 3,5-dimethylpyrazole. Semilogarithmic plots of C14 excreted vs. time give an estimation of the excretion half-life. In the 2 fasted rats an excretion half-life of 2.4 hours was obtained; i.e., an average of about 60% of the C14 is excreted in the urine during the first 3 hours. The estimated half-life of C14 excretion in rats treated with SKF-525A is 4.4 hours. The results of these excretion half-life studies are complicated by the fact that the rats were not catheterized. Each of the corn oil-treated rats voided only once during the first 8 hours; consequently, estimation of excretion half-life was impossible in this case.

The sequential urine collections from a fasted rat were chromatographed in an attempt to determine the approximate rat of excretion of each metabolite (table 3) . The percentage of the excreted C14 as metabolite C in the early urine collections was found to be greater than in later ones. A semiogarithmic plot of percentage of each metabolite to be excreted vs. time gives an estimate of the excretion half-life of each metabolite. The half-life values obtained by this procedure for 1 fasted rat were 2.4, 2.0, 1.6, and 1.7 hours for metabolites A, B, C, and D, respectively; i.e., these times were required for 50% of the total of each of these metabolites to appear in the urine.

Effect of hypoglycemic blocking agents on metabolism

The effect of corn oil and SKF-525A on the metabolism of 3,5-dimethylpyrazole was studied, since these agents have been shown to inhibit the hypoglycemic activity of this compound. Specifically, it was of interest to determine whether these agents prevented the formation the active metabolite, 5-methylpyrazole-3-carboxylic acid (metabolite A). These agents, even at 10 times the doses used in these studies, inhibit the hypoglycemic activity of 3,5-dimethylpyrazole. Consequently, in order for effects on metabolism by these agents to explain inhibition of activity, a pronounced alteration of metabolism would be required. For example, metabolite A is active in the fasted rat at less than 1 mg/kg.

Table 3: Time course of metabolite excretion pattern of the fasted rat

Collection Period (hr)

Percent of Dose Excreted

Percent Excreted during Each Collection Period as Metabolite:

A

B

C

D

0 - 1

5.8

5.6

6.3

88.1

0.0

1 - 2

22.7

8.5

14.1

77.4

2.9

2 - 3

25.8

15.2

18.2

62.9

3.9

3 - 4

12.4

14.2

19.0

63.3

3.5

4 - 6

24.7

20.0

21.2

55.9

2.8

6 - 8

4.4

21.3

21.4

55.2

2.1

0 - 8

95.8

14.4

17.5

65.0

3.0

None of these agents significantly alters the pattern of metabolites excreted in 24 hours (table 2) . Since the inhibiting effects of these agents were demonstrated 2 hours after administration, the distribution of metabolites in the early urine collections was also studied.

Early metabolism, as evidenced by excretion, was also not affected significantly. The inhibition of hypoglycemic activity by these agents apparently cannot be explained by their effect on the metabolism of 3,5-dimethylpyrazole. This conclusion is supported by the recent finding that the hypoglycemic activity of the active metabolite, 5-methylpyrazole-3-carboxylic acid, is also inhibited by these agents.

Although not statistically significant, reduction of the C14 excreted in 24 hours was observed in the rats pretreated with SKF-525A (table 2) . The decreased excretion from the corn oil-treated rats compared to the fasted rats is significant : .1 > P > .05 by a modified t test (Snedecor, 1956) . This may reflect trapping of the pyrazole in the gut by unabsorbed corn oil ; the trapping in the gut may also account for the greater variability of C14 excretion from the corn oil-treated rats. This decreased urinary excretion in the corn oil treated rat, which probably reflects decreased absorption, is not sufficient to account for the inhibition of hypoglycemic activity by this agent.

Hypoglycemic activity of the metabolites

The hypoglycemic potency of 5-methylpyrazole-3-carboxylic acid is sufficient to account for all of the hypoglycemic activity found in the urine of 3 , 5-dimethylpyrazole-treated rats. The interesting effects of this compound on in vivo and in vitro carbohydrate and lipid metabolism have been described in detail (Gerritsen and Dulin, 1965).

A sample of 4-hydroxy-3,5-dimethylpyrazole, the hydrolysis product of metabolite C, was tested for hypoglycemic activity and was found to have insignificant hypoglycemic activity compared with 5-methylpyrazole-3-carboxylic acid. Urine from which 5-methylpyrazole-3-carboxylic acid has been completely extracted is inactive when tested for hypoglycemic activity; consequently, metabolite C, which does not extract under these conditions, must also be inactive.

Metabolites B and D have not been isolated in crystalline form, but extracts containing these metabolites were inactive in lowering blood glucose.

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
The fate of the test material when administered orally was shown to have no potential for bioaccumulation. The test material was determined to be completely absorbed in the gastrointestinal tract, converted into four metabolites and almost entirely excreted via the urine within 24 hours.
Executive summary:

In a non-GLP compliant study which was performed according to sound scientific principles, the potential for bioaccumulation of the test material was investigated in rats. Test animals received an oral dose of 25 mg/kg, via oral gavage. Their urine was analysed to identify the fate of the radiolabelled test material. The test material was determined to be completely absorbed in the gastrointestinal tract, converted into four metabolites and almost entirely excreted via the urine within 24 hours. Therefore it is considered that there is no potential for bioaccumulation after oral exposure.

3,5-Dimethylpyrazole-C14 is absorbed completely from the gastrointestinal tract of fasted rats when administered as an aqueous suspension. Essentially all of the oral dose (95%) is found in the urine within 24 hours. It is converted completely (>99%) to 4 metabolites. Two metabolites, which together with their conjugated forms account for about 98% of the oral dose of 3,5-dimethylpyrazole, have been isolated in crystalline form and their structures determined. In 5 normal, fasted rats the average percentages excreted (±S.D.) as 5-methylpyrazole-3-carboxylic acid and its conjugate form were 13.1 ± 3.8 and 13.6 ± 4.4, respectively. The major metabolite, which was excreted to the extent of 70.8 ± 5.5% in fasted rats, has been unequivocally identified as conjugated 4-hydroxy-3, 5-dimethylpyrazole; it has been isolated from hydrolyzed rat urine in its unconjugated form. An unknown metabolite, which is formed to the extent of 1.3 ± 1.9% of the dose, was not detected in the urine of some of the rats studied.

From the urinary excretion data, the half-life for the appearance of C14 in the urine of the rat was determined to be about 2.4 hours; i.e., about 60% of the C14 is excreted during the first 3 hours.

The fate of 3, 5-dimethylpyrazole in fasted rats compared to rats treated with corn oil and SKF-525A indicates that the inhibition of hypoglycemic activity by these agents probably cannot be explained by their effect on the absorption and/or metabolism of 3, 5-dimethylpyrazole.

Description of key information

Key study; Smith (1965), non GLP study performed to sound scientific principles, Klimisch 2, no potential for bioaccumulation, four metabolites identified and main excretory route was urine.

Key value for chemical safety assessment

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

Additional information

In a non-GLP compliant study (Smith, 1965) which was performed according to sound scientific principles, the potential for bioaccumulation of the test material was investigated in rats. Test animals received an oral dose of 25 mg/kg, via oral gavage. Their urine was analysed to identify the fate of the radiolabelled test material. The test material was determined to be completely absorbed in the gastrointestinal tract, converted into four metabolites and almost entirely excreted via the urine within 24 hours. Therefore it is considered that there is no potential for bioaccumulation after oral exposure.

3,5-Dimethylpyrazole-C14 is absorbed completely from the gastrointestinal tract of fasted rats when administered as an aqueous suspension. Essentially all of the oral dose (95 %) is found in the urine within 24 hours. It is converted completely (>99 %) to 4 metabolites. Two metabolites, which together with their conjugated forms account for about 98 % of the oral dose of 3,5-dimethylpyrazole, have been isolated in crystalline form and their structures determined. In 5 normal, fasted rats the average percentages excreted (± S.D.) as 5-methylpyrazole-3-carboxylic acid and its conjugate form were 13.1 ± 3.8 and 13.6 ± 4.4,

respectively. The major metabolite, which was excreted to the extent of 70.8 ± 5.5 % in fasted rats, has been unequivocally identified

as conjugated 4-hydroxy-3, 5-dimethylpyrazole; it has been isolated from hydrolyzed rat urine in its unconjugated form. An unknown metabolite, which is formed to the extent of 1.3 ± 1.9 % of the dose, was not detected in the urine of some of the rats studied.

From the urinary excretion data, the half-life for the appearance of C14 in the urine of the rat was determined to be about 2.4 hours;

i.e., about 60 % of the C14 is excreted during the first 3 hours.

Toxicokinetic Assessment of the Substance 3,5-Dimethylpyrazole

1.0       Introduction

Physico-chemical properties, the results of a metabolism study and acute and repeat dose toxicity studies for 3,5-dimethylpyrazole have been used to determine a toxicokinetic profile.

2.0       Physicochemical properties

The substance 3,5-dimethylpyrazole (CAS No. 67-51-6) is a white powder and has the molecular formula C5H8N2 with a molecular weight of 96.1 g/mol. It is very water soluble (28.9 g/L as 20 °C) with a log Pow value of 2.1 at 35 °C. 3,5-Dimethylpyrazole contains a 1,2-pyrazole group and is therefore weakly basic in nature, with a pH in aqueous solution of 7.5. It is hydrolytically stable at pH 4, 7 and 9, with a half-life of > 1 year at 25 °C. It has a vapour pressure of 0.37 Pa at 20 °C and a particle size of 269 µm (MMAD, mass median aerodynamic diameter).

3.0       Absorption

3.1       Oral absorption

The low molecular weight, high water solubility and log P value of 3,5-dimethylpyrazole will favour absorption from the gastrointestinal tract.

The metabolism of 3,5-dimethylpyrazole has been studied in the rat (Smith, Forist, Gerritsen, 1965). A single oral dose of 14C-labelled 3,5-dimethylpyrazole was administered by oral gavage to fasted male rats at 25 mg/kg in 0.25 % methyl cellulose. A mean of 95 % of the administered dose (n=6) was recovered in the urine within 24 hours, indicating that oral absorption of 3,5-dimethylpyrazole is complete.

Acute and repeat oral toxicity studies have been conducted which show clear evidence of systemic toxicity, and therefore of oral absorption, consistent with the findings of the metabolism study.

In an acute oral toxicity study (Parcell, 1994a), there were signs of systemic toxicity following administration of single oral doses of 3,5-dimethylpyrazole in 1 % aqueous methyl cellulose to fasted rats at 1260-2000 mg/kg for females and 2000 mg/kg for males. From group sizes of 5 animals, two female rats dosed at 1600 mg/kg and two males and four females dosed at 2000 mg/kg died during the study. Of the surviving animals, slightly low bodyweight gains were recorded for one female at 1260 mg/kg and three males and one female at 2000 mg/kg. For the rats that died, macroscopic examination revealed changes in the kidneys, spleen and alimentary tract. The surviving animals showed no macroscopic abnormalities at post mortem. Clinical signs were observed in rats at all dose levels, with complete recovery in the surviving rats by day 3 (1260 mg/kg), day 4 (1600 mg/kg) or day 5 (2000 mg/kg) after dosing.

In a second acute oral toxicity study (Wallace, 1976a), 3,5-dimethylpyrazole was administered to rats in propylene glycol. No symptoms of toxicity were observed at 500 mg/kg, however clinical signs were observed at doses of 1000 mg/kg and above, and all animals died at doses of 4000 mg/kg and above.

In the repeat oral dose study (OECD 422 - Zmarowski, 2012), rats were dosed at 20, 60 and 200 mg/kg/day by oral gavage in propylene glycol. Male rats were dosed for 29-31 days, and female rats for 45-56 days. At the top dose level of 200 mg/kg/day, treatment-related effects were seen on bodyweight, food and water consumption, functional observations, clinical pathology, macroscopic findings and microscopic findings in the thymus, liver, spleen, testes and epididymides. At 60 mg/kg/day, females showed increased water consumption and males showed liver effects.

For risk assessment purposes, 3,5-dimethylpyrazole is considered to be completely absorbed (100 %) following oral administration.

3.2       Dermal absorption

3,5-Dimethylpyrazole is a solid, and therefore will need to dissolve into the surface moisture of the skin before it can be absorbed. Whilst the low molecular weight and high water solubility in combination with the moderate log Pow value (log Pow 2.1) would favour partitioning of any dissolved material into the stratum corneum, the presence of heterocyclic ammonium ions are likely to cause binding to skin components and slow dermal uptake.

Following a single 24-hour occluded application at 2000 mg/kg to male and female rat skin, where 3,5-dimethylpyrazole was applied moistened with water, there were no signs of systemic toxicity or skin irritation and no treatment- related abnormalities were found at examination post mortem. All animals showed an overall weight gain during the study (Hutchinson, 2002a).

Following a 4-hour, semi-occluded application of 500 mg of 3,5-dimethylpyrazole to rabbit skin, applied with a small volume (0.5 mL) of water, there were no signs of skin irritation (Parcell, 1994). In a second study, no signs of skin irritation were seen following application of 500 mg of 3,5-dimethylpyrazole to clipped or abraded rabbit skin for 4 hours under an occlusive dressing (Wallace, 1976b). Similarly, no evidence of skin sensitisation was seen in studies in the guinea pig (Denton, 1994; Hutchinson, 2002c).

There was no evidence of any dermal penetration of 3,5-dimethylpyrazole in the rat, rabbit or guinea pig. As evidence of systemic toxicity was seen in the oral toxicity studies, the lack of findings in the dermal studies lead to the conclusion that dermal absorption of 3,5-dimethylpyrazole is low.

On this basis, a dermal absorption of 100 % in humans is considered appropriate for 3,5-dimethylpyrazole, and this default value will be used for risk assessment purposes as worst case.

3.3       Inhalation

3,5-Dimethylpyrazole has a low vapour pressure (0.37 Pa at 20 °C) and therefore inhalation of vapour is not anticipated.

3,5-Dimethylpyrazole is a powder with particles with a mass mean aerodynamic diameter of 269 µm (MMAD) (Livingston, 2012). Based upon the particle size distribution determined by laser diffraction, 0.26 % of the material is capable of becoming airborne, (particle size ≤100 µm) and potentially inhalable. 3,5-Dimethylpyrazole does not contain particles of a respirable size (≤15 µm) that would be capable of penetrating lower into the respiratory tract that the nasopharyngeal region, and therefore there will be no absorption via the inhalation route. Any particles deposited in the nasopharyngeal region would pass rapidly into the gastrointestinal tract.

In the absence of any experimental data on toxicity following inhalation exposure, and considering that the particle size distribution indicates that any potentially inhaled substance will be likely to be swallowed, the same extent of oral absorption (100 %) will be used for inhalation for risk assessment purposes.

4.0       Distribution

Any 3,5-dimethylpyrazole that is absorbed will be distributed via the blood to the liver and other organs and tissues. Due to its high water solubility and moderate log Pow, it would not be expected to accumulate in fatty tissues. The changes in organ weights and findings in the thymus, liver, spleen, testes and epididymides in the OECD 422 study in rat, along with changes to haematological and clinical chemistry parameters, provide evidence that the substance is widely distributed (Zmarowski, 2012).

5.0       Metabolism

The metabolism of 3,5-dimethylpyrazole has been studied in the rat (Smith, Forist, Gerritsen, 1965). Following administration of a single oral dose of 14C-labelled 3,5-dimethylpyrazole at 25 mg/kg to fasted male rats, the majority of the dose was recovered in the urine within 24 hours. Metabolism was extensive, with less than 0.5 % of the radioactivity recovered in urine being unmetabolised 3,5-dimethylpyrazole. Four metabolites of 3,5-dimethylpyrazole were detected in urine. The major metabolite was the conjugate of 4-hydroxy-3,5-dimethylpyrazole (71 % of dose), and two further metabolites were identified as 5-methylpyrazole-3-carboxylic acid (13 % of dose) and its conjugate (14 % of dose). The minor metabolite (1 % of dose) was not identified.

The metabolism of 3,5-dimethylpyrazole therefore proceeds principally via hydroxylation of the pyrazole ring followed by conjugation. A less significant pathway involves oxidation of the 3-methyl group followed by conjugation.

6.0       Elimination

3,5-Dimethylpyrazole is of low molecular weight and is very water soluble, therefore it is expected that elimination of unchanged material will be via the urine. As metabolism generally results in metabolites of higher polarity, any metabolites would also be expected to be eliminated in the urine.

This has been demonstrated in the metabolism study conducted in the rat (Smith, Forist, Gerritsen, 1965) which showed that the majority (>95 %) of an orally administered dose was recovered in the urine within 24 hours, principally as two metabolites and their conjugates.

7.0       Conclusion

A metabolism study has shown that absorption of 3,5-dimethylpyrazole is complete following oral administration. 3,5-dimethylpyrazole is extensively metabolised, and quickly excreted via the urine, principally as two metabolites and their conjugates. The toxicity studies provide further evidence that 3,5-dimethylpyrazole is well absorbed by the oral route and that the absorbed material is widely distributed. For risk assessment purposes, 3,5-dimethylpyrazole is considered to be completely absorbed (100 %) following oral administration.

Based on the physical-chemical properties of 3,5-dimethylpyrazole, no significant absorption by the dermal and inhalation routes is expected.

Although no evidence of dermal absorption was seen in the acute dermal toxicity, skin irritation or skin sensitisation studies, based on estimation of mammalian dermal absorption in accordance with principles adopted within the EFSA guidance on estimating dermal absorption of pesticide active substances (EFSA, 2012), a dermal absorption value of 25 % will be used for risk assessment purposes.

In the absence of any experimental data on toxicity following inhalation exposure, and considering that particle size distribution indicates that any potentially inhaled substance will be likely to be swallowed, the same extent of oral absorption (100 %) will be used for risk assessment purposes.