N,N'-diacetylhydrazine

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: - Study well documented, meets generally accepted scientific principles, acceptable for assessment - non-GLP

Data source

Reference

Reference Type:

publication

Title:

ACETYLHYDRAZINE AS AN INTERMEDIATE IN THE METABOLISM OF AROYLHYDRAZINES.

Author:

TURNBULL LB, YARD AS, MCKENNIS H Jr.

Year:

1962

Bibliographic source:

J Med Pharm Chem. 1962 Nov;91:1327-34.

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- rabbits applied with the test substances via ip injection; rats treated orally via gavage, dog iv injected with the test item
- rabbits treated with 1-acetyl-2-benzoylhydrazine (experiment I), benzhydrazide (II); rats: treated with C14 labelled (acetyl group) 1-acetyl-2-benzoylhydrazine (III + V), unlabelled 1,2-diacetylhydrazine (IV) and C14 labelled (acetyl groups) 1,2-diacetylhydrazine (VI); dog treated with acetylhydrazine (VII)
- rabbit urine (pooled for 30 h post dosing) analysed for 1,2-diacetylhydrazine content (I + II)
- rats kept in metabolic cages and urine, feces and exhaled air sampled up to 96 h (1,2 diacetylhydrazine) or 5 d (1-acetyl-2-benzoylhydrazine) post dosing and analysis for labelled carbon content (III + IV)
- rat urine (pooled for 24 h post dosing) analysed for labelled 1,2 diacetylhydrazine (V + VI)
- urine sampled for 2 h post dosing from the pentobarbital anethesized dog, analysis of the urine for the hydrazine content (VII)

GLP compliance:

no

Remarks:

study period predates implementation of GLP

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-diacetylhydrazine

EC Number:

221-576-1

EC Name:

N,N'-diacetylhydrazine

Cas Number:

3148-73-0

Molecular formula:

C4H8N2O2

IUPAC Name:

N'-acetylacetohydrazide

Details on test material:

- Name of test material (as cited in study report): 1,2-diacetylhydrazine
- Additionally used test items: 1-acetyl-2-benzoylhydrazine, benzhydrazide, acetylhydrazine fumarate
- Labelled substances: C14 labelled (acetyl group) 1-acetyl-2-benzoylhydrazine and C14 labelled (acetyl groups) 1,2-diacetylhydrazine

Radiolabelling:

yes

Remarks:

C14 labelling

Test animals

Species:

other: rat, rabbit and dog

Strain:

other: rat: Wistar, rabbit: New Zeeland White, dog: mongrel

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: not reported
- Sex: rat and dog: male; rabbit: female
- Age at study initiation: not reported
- Weight at study initiation:
• rabbits: 1.25 - 1.70 kg (experiment 1), 1.20 - 1.75 kg (II)
• rats: 127 ± 22.5 g (III), 152.3 ± 32.2 (IV), 416.7 ± 39.3 g (V), 428.3 ± 62.5 (VI)
• dog: 10.5 kg (VII)
- Fasting period before study: no
- Housing: rats (experiments III + IV): glass metabolism cages;
- Individual metabolism cages: rats: yes; rabbits and dog: not reported
- Diet (e.g. ad libitum): rats: ad libitum; rabbits and dog: not reported
- Water (e.g. ad libitum): rats: ad libitum; rabbits and dog: not reported
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): not reported

Administration / exposure

Route of administration:

other: rats: oral (gavage), rabbits: intraperitoneal, dog: intravenous

Vehicle:

other: in most experiments water, but not precisely reported for all experiments

Details on exposure:

- rabbit:
• 1-acetyl-2-benzoylhydrazine (experiment I): average dose 133.6 mg/kg bw, ip injection, no vehicle mentioned
• benzhydrazide (II): 1.09 g in total (all 8 animals), equivalent to an average dose of 92.4 mg/kg bw, ip injection, no vehicle mentioned
- rat:
• labelled 1-acetyl-2-benzoylhydrazine (III): 11.2 mg/animal (= 7.64 x 10^5 cpm), equivalent to an average dose of 88.0 mg/kg bw, oral gavage, dose applied dissolved in 2 mL of water
• labelled 1,2-diacetylhydrazine (IV): 8.73 mg/animal (= 6.92 x 10^5 cpm) equivalent to an average dose of 57.3 mg/kg bw oral gavage, no vehicle mentioned
• labelled 1-acetyl-2-benzoylhydrazine (V): 45 mg/animal ( at 7.64 x 10^7 cpm/mmole), equivalent to an average dose of 108.0 mg/kg bw, oral gavage, no vehicle mentioned
• unlabelled 1,2-diacetylhydrazine (VI): 50 mg/animal, equivalent to an average dose of 116.7 mg/kg bw, oral gavage, dose applied dissolved in 3 ml of water
- dog (VII): 2.69 g acetylhydrazine fumarate (per 10.5 g of bw)

Duration and frequency of treatment / exposure:

single treatment

Doses / concentrations

Remarks:

Doses / Concentrations:
see above under details on exposure

No. of animals per sex per dose / concentration:

- rabbit:
• 1-acetyl-2-benzoylhydrazine (experiment I): 4 females
• benzhydrazide (II):8 females
- rat:
• labelled 1-acetyl-2-benzoylhydrazine (III): 3 males
• labelled 1,2-diacetylhydrazine (IV): 3 males
• labelled 1-acetyl-2-benzoylhydrazine (V): 3 males
• unlabelled 1,2-diacetylhydrazine (VI): 3 males
- dog (VII): 1 male

Control animals:

no

Positive control reference chemical:

no

Details on study design:

- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): not reported

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES

- Tissues and body fluids sampled:
• rabbit:
- 1-acetyl-2-benzoylhydrazine (I): urine for 30 h post dosing
- benzhydrazide (II): urine for 30 h post dosing
• rat:
- labelled 1-acetyl-2-benzoylhydrazine (III): urine and faeces and exhaled CO2 for 5 d post dosing
- labelled 1,2-diacetylhydrazine (IV): urine and faeces and exhaled CO2 for 96 h post dosing
- labelled 1-acetyl-2-benzoylhydrazine (V): urine for 24 h
- unlabelled 1,2-diacetylhydrazine (VI): urine for 24 h
• dog (VII): urine for 2 h

- Time and frequency of sampling:
• rabbit:
- 1-acetyl-2-benzoylhydrazine (experiment I): single sampling after 30 h post dosing
- benzhydrazide (II): single sampling after 30 h post dosing
• rat:
- labelled 1-acetyl-2-benzoylhydrazine (III): daily for 5 d post dosing
- labelled 1,2-diacetylhydrazine (IV): 0- 24 h, 25 – 48 h, 49 – 72 h and 73 – 96 h post dosing
- labelled 1-acetyl-2-benzoylhydrazine (V): single sampling after 24 h post dosing
- unlabelled 1,2-diacetylhydrazine (VI): single sampling after 30 h post dosing
• dog (VII): single sampling after 2 h post dosing

- From how many animals: all animals in every experiment

- Method type(s) for identification:
• determination of 1,2-diacetylhydrazine and 1-acetyl-2-benzoylhydrazine in urine, all experiments (except experiment VII):
- urine filtering through Dowex (anion and cation exchanger matrices) followed by treatment with decolorizing carbon and concentration to dryness under reduced pressure
- solution in ethanol, paper chromatography on Whatman No. 1 paper,
- extraction of the relevant band from paper, recrystalisation and identification by melting point of mixture with respective control substance
• rat, labelled 1-acetyl-2-benzoylhydrazine (III) and labelled 1,2-diacetylhydrazine (IV):
- CO2: CO2 collected in bubble tower containing 10 % (w/w) KOH; radioactivity of aliquots determined on infinitely thick samples of barium carbonate following addition of barium chloride
- feces: feces separated from urine by collection on a screen; feces dried overnight at 100° and pulverized to a uniform powder for scintilation counting at infinite thickness against standards prepared by adding 1-acetyl-C14-2-benzoylhydrazine to control feces
- drying of urine samples over talc at 100° and similarly counted at infinite thickness
• dog, determination of hydrazine in urine (VII):
- treatment of urine with benzaldehyde,
- filtration of the formed crystalline substance from the urine and washing with hot water
- reextraction of wash water fractions with chloroform
- recrystalisation of the pooled substance and identification by melting point of mixture with control substance

- Limits of detection and quantification: not reported

Results and discussion

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

see below

Any other information on results incl. tables

Results:

- experiment I: 1-acetyl-2-benzoylhydrazine is metabolised to 1,2-diacetylhydrazine in the rabbit to a percentage of 17.8 mol%.

- experiment II: benzhydrazine is metabolised to 1,2-diacetylhydrazine in the rabbit to a percentage of 12 mol%.

- experiment III:

 • the total recovery of label is between 81.7 and 90.9 mol%.

 • 1-acetyl-2-benzoylhydrazine is metabolised to labelled CO2 in a percentage of 29.8 to 38.7 mol% in the rat during 5 days post dosing

 • 42.3 to 45.5 mol% of the radioactivity is found in the urine during 5 days post dosing

 • 6.4 to 7.6 mol% of the radioactivity is found in the feces during 5 days post dosing

 • see Table 1 for details

- experiment IV:

 • the total recovery of label is between 91.9 and 95.9 mol%.

 • 1,2-diacetylhydrazine is metabolised to labelled CO2 in a percentage of only 0.6 to 1.3 mol% in the rat during 96 h post dosing

 • 85.6 to 91.4 mol% of the radioactivity is found in the urine during 96 h post dosing

 • 3.9 to 5.3 mol% of the radioactivity is found in the feces during 96 h post dosing

 • see Table 2 for details

- experiment V: within 24 h 81 mol% of the orally applied labelled 1-acetyl-2-benzoylhydrazine was excreted as 1,2-diacetylhydrazine in rats.

- experiment VI: no results detailed in the report

- experiment VII: in the dog who is known from earlier experiments to be incompetent for the acetylation of acetylhydrazine to 1,2-diacetylhydrazine a significant amount of hydrazine was found in the urine sampled for 2 h post dosing with acetylhydrazine showing that hydrolysis from acetylhydrazine to hydrazine is possible in the mammalian organism.

- Table 1: 1-acetyl -C14-2-benzoyhydrazine in the male albino rat (a)

Day		Rat No. 1 (b), 102 g.				Rat No. 2, 135 g.				Rat No. 3, 145 g.
		Per cent of radioactivity recovered
		Urine	Respire- tory CO2	Feces	Total	Urine	Respire- tory CO2	Feces	Total	Urine	Respire- tory CO2	Feces	Total
1		44.3	33.9			41.8	32.1			43.8	27.1
2		0.9	3.1			0.2	1.7			0.2	1.4
3		0.2	0.9				1.0				0.7
4			0.5				0.3				0.4
5		0.1	0.3			0.3	0.3			0.3	0.2
Total		45.5	38.7	6.7	90.9	42.3	35.4	6.4	84.1	44.3	29.8	7.6	81.7

(a) All animals received 11.2 mg. of 1-acetyl-C14-2-benzoylhydrazine orally in 2 ml of water (7.64 x 10^5 cpm at infinite thickness).

(b) The urine from this animal was filtered through Celite and then concentrated to dryness. The residue was triturated with hot absolute alcohol. The ethanolic solution was diluted with water and was placed upon Dowex 50 (H+) which retained 41% of the activity in the solution. The activity was removed by elution with 2 M ammonium hydroxide. Upon paper chromatography radioactivity was found at the origin and distributed unevenly throughout the chromatogram. The fraction comprising the eluate and water washings showed upon paper chromatography two radioactive zones, Rf 0.53 and Rf 0.79, corresponding to 1,2-diacetylhydrazine and 1-acety1-2- benzoylhydrazine, respectively.

- Table 2: Metabolism Of C14 Labelled1,2-Diacetylhydrazine In The Male Albino Rat

Animal (a) and weight	% Radioactivity recovered
	0-24 h			25-48 h			49-72 h			73 -96 h
	Urine	Feces	CO2	Urine	Feces	CO2	Urine	Feces (c)	CO2	Urine	Feces (c)	CO2	Total
Rat No. 4 (b) (117 g.)	83.7	-	0.6	7.1	-	-	0.3	-	-	0.3	3.9	-	95.9
Rat No. 5 (180 g.)	88.4	-	0.7	1.4	-	0.6	0.1	6.4	-	-	-	-	97.6
Rat No: 6 (160 g.)	83.3	-	0.7	2.0	-	-	0.3	5.3	-	-	-	-	91.6

(a) All animals received a single oral dose of C14 labelled 1,2-diacetylhydrazine (8.73 mg., 6.93 x 10^5 cpm).

(b) The 24-hr. urine of this animal was filtered and concentrated to dryness under diminished pressure. The residue was triturated with hot alcohol. An aliquot of the filtrate showed upon paper chromatography only one radioactive zone, corresponding in Rf value to authentic 1,2-diacetylhydrazine.

(c) The determinations on the feces in all cases represent cumulative values.

Authors conclusion:

Man, rat and rabbit metabolise aroylhydrazines via 1-aroyl-2-acetyl-hydrazine and acetylhydrazine to 1,2-diacetylhydrazine.

 

Conclusion from the assessor:

- Comparison of experiment III and IV shows that significant amounts of CO2 are formed from 1-acetyl-2-benzoylhydrazine via acetylhydrazine, while hardly any CO2 is formed from 1,2-diacetylhydrazine (via acetylhydrazine). This indicates that 1,2-acetylhadrazine is rather stable and its hydrolysis to acetylhydrazine is rather slow as compare to the reacetylation to 1,2-diacetylhydrazine and the excretion of 1,2-diacetylhydrazine.

- 1,2 -diacetylhydrazine is excreted rather fast in the rat, therefore no potential for bioaccumulation is indicated.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results
Rats, rabbits and a dog were treated singly with 1-actely-2-benzoylhydrazine, benzhydrazide, acetyl hydrazine and 1,2-diacetylhydrazine via different routes, in some cases as C14 labbeled substances. 1-actely-2-benzoylhydrazine, 1,2-diacetylhydrazine and in the case of the dog hydrazine was quantified in urine via isolation and paper chromatography. In the case of the rats label was analysed in urine, feces and exhaled air. It was shown that man, rat and rabbit metabolise aroylhydrazines via 1-aroyl-2-acetyl-hydrazine and acetylhydrazine to 1,2-diacetylhydrazine.Significant amounts of CO2 are formed from 1-acetyl-2-benzoylhydrazine via acetylhydrazine, while hardly any CO2 is formed from 1,2-diacetylhydrazine (via acetylhydrazine). This indicates that 1,2-acetylhadrazine is rather stable and its hydrolysis to acetylhydrazine is rather slow as compare to the reacetylation to 1,2-diacetylhydrazine and the excretion of 1,2-diacetylhydrazine. 1,2 -diacetylhydrazine is excreted rather fast in the rat, therefore no potential for bioaccumulation is indicated.

Executive summary:

In this study that followed no guideline and predates the implementation of GLP the metabolism of aroylhydrazines should be analysed. Rabbits were applied with the test substances via ip injection; rats were treated orally via gavage, and a was dog iv injected with the test item. Rabbits were treated with 1-acetyl-2-benzoylhydrazine (experiment I), benzhydrazide (II). Rats were treated with C14 labelled (acetyl group) 1-acetyl-2-benzoylhydrazine (III + V), unlabelled 1,2-diacetylhydrazine (IV) and C14 labelled (acetyl groups) 1,2-diacetylhydrazine (VI). The dog was treated with acetylhydrazine (VII). In rabbits urine (pooled for 30 h post dosing) was analysed for 1,2-diacetylhydrazine content (I + II).

Rats were kept in metabolic cages and urine, feces and exhaled air sampled up to 96 h (1,2 diacetylhydrazine) or 5 d (1-acetyl-2-benzoylhydrazine) post dosing and analysis for labelled carbon content (III + IV). In another experiment rat urine (pooled for 24 h post dosing) was analysed for the content of labelled 1,2 diacetylhydrazine (V + VI). Finally urine was sampled for 2 h post dosing from the pentobarbital anethesized dog, and the urine was analysed for the hydrazine content (VII)

The following results were determined:

- experiment I: 1-acetyl-2-benzoylhydrazine is metabolised to 1,2-diacetylhydrazine in the rabbit to a percentage of 17.8 mol%.

- experiment II: benzhydrazine is metabolised to 1,2-diacetylhydrazine in the rabbit to a percentage of 12 mol%.

- experiment III:

 • the total recovery of label is between 81.7 and 90.9 mol%.

 • 1-acetyl-2-benzoylhydrazine is metabolised to labelled CO2 in a percentage of 29.8 to 38.7 mol% in the rat during 5 days post dosing

 • 42.3 to 45.5 mol% of the radioactivity is found in the urine during 5 days post dosing

 • 6.4 to 7.6 mol% of the radioactivity is found in the feces during 5 days post dosing

 • see Table 1 for details

- experiment IV:

 • the total recovery of label is between 91.9 and 95.9 mol%.

 • 1,2-diacetylhydrazine is metabolised to labelled CO2 in a percentage of only 0.6 to 1.3 mol% in the rat during 96 h post dosing

 • 85.6 to 91.4 mol% of the radioactivity is found in the urine during 96 h post dosing

 • 3.9 to 5.3 mol% of the radioactivity is found in the feces during 96 h post dosing

 • see Table 2 for details

- experiment V: within 24 h 81 mol% of the orally applied labelled 1-acetyl-2-benzoylhydrazine was excreted as 1,2-diacetylhydrazine in rats.

- experiment VI: no results detailed in the report

- experiment VII: in the dog who is known from earlier experiments to be incompetent for the acetylation of acetylhydrazine to 1,2-diacetylhydrazine a significant amount of hydrazine was found in the urine sampled for 2 h post dosing with acetylhydrazine showing that hydrolysis from acetylhydrazine to hydrazine is possible in the mammalian organism.

The authors concluded that man, rat and rabbit metabolise aroylhydrazines via 1-aroyl-2-acetyl-hydrazine and acetylhydrazine to 1,2-diacetylhydrazine.

The accessor of this endpoint study record concludes that significant amounts of CO2 are formed from 1-acetyl-2-benzoylhydrazine via acetylhydrazine, while hardly any CO2 is formed from 1,2-diacetylhydrazine (via acetylhydrazine). This indicates that 1,2-acetylhadrazine is rather stable and its hydrolysis to acetylhydrazine is rather slow as compare to the reacetylation to 1,2-diacetylhydrazine and the excretion of 1,2-diacetylhydrazine. 1,2 -diacetylhydrazine is excreted rather fast in the rat, therefore no potential for bioaccumulation is indicated.