N,N'-diacetylhydrazine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Unsuitable test system for assessing on developmental toxicity, however acceptable, sufficiently documented publication which meets basic scientific principles; data of the original publication (Schultz et al 1988) double-checked and used for further QSAR development (Mekenyan et al 1996)

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Structure-Activity Relationships for Osteolathyrism. I. Effects of Altering the Semicarbazide Structure

Author:

Schultz TW, Ranney TS, Riggin GW, Cajina-Quezada M

Year:

1988

Bibliographic source:

Transactions of the American Microscopical Society 107(2):113-126

Reference 2

Reference Type:

publication

Title:

'Dynamic' QSAR for semicarbazide-induced mortality in frog embryos

Author:

Mekenyan OG, Schultz TW, Veith GD, Kamenska V

Year:

1996

Bibliographic source:

PMID: 8854223 J Appl Toxicol 16(4):355-63

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: ASTM E1439-98: Standard Guide for Conducting the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) (10 June 1998)

Principles of method if other than guideline:

“The Frog Embryo Teratogenesis Assay - Xenopus (FETAX) is a 96-hour whole-embryo developmental toxicity test that uses early-stage embryos of the South African clawed frog (Xenopus laevis) to measure the effects of chemicals on mortality, malformation, and growth inhibition. It was proposed as a screening assay to identify potential human teratogens and developmental toxicants” (NTP, Department of Health and Human Sciences of the U.S.A., NICEATM-ICCVAM, Test Method Evaluations, Developmental Toxicity as of update from 11/07/2011).
The 96-h toxicity (log LC50) and 96-h teratogenicity (log EC50) endpoints are determined and the the teratogenic index (LC50/EC50) is calculated to show the possible relationship of teratogenic risk and acute toxicity.

GLP compliance:

not specified

Limit test:

no

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 the publication): Sym-diacetyl hydrazine
- Purity: The test item was taken as delivered and not further purified
- Supplier: Aldrich Chemical Company, Milwaukee, Wisconsin, U.S.A.

Test animals

Species:

other: Aquatic frog embryos (Xenopus laevis)

Strain:

not specified

Details on test animals or test system and environmental conditions:

TEST ORGANISM
- Common name: African clawed frog
- Age at study initiation: Early to mid-blastula embryos
TEST SYSTEM
- Test vessel: 60 ∙ 15 mm dishes
- Material, fill volume: Pyrex glass, 10 mL
TEST MEDIUM / WATER PARAMETERS
- FETAX solution (625 mg NaCl, 96 mg NaHCO3, 30 mg KCl, 15 mg CaCl2, 60 mg CaSO4∙2H2O and 75 mg MgSO4 per litre of distilled water)
OTHER TEST CONDITIONS
- Test water temperature: 23±0.5 °C
- Adjustment of pH: The stock solutions were adjusted to pH 7.2
- Photoperiod: 12 h light / 12 h dark

Administration / exposure

Route of administration:

other: via aqueous medium

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Embryos were obtained from adult pairs injected with human chorionic gonadotropin to induce ovulation and amplexus.

Duration of treatment / exposure:

96 h

Frequency of treatment:

Contiously during the whole test duration

Duration of test:

96 h

No. of animals per sex per dose:

25 embryos of unknown sex in any treatment and control, with a minimum of 3 replicates were tested for each concentration series

Control animals:

yes, concurrent no treatment

Details on study design:

Semicarbazide hydrochloride (CAS 563-41-7) was used as reference substance (positive control).

Examinations

Maternal examinations:

No maternal examinations were performed in accordance with the FETAX protocol.

Fetal examinations:

The mortality, number and type of malformations, stage of development and length were recorded after 96 hours. To this end histological and electron microscopical examinations were conducted on representative embryos with emphasis on the axial skeleton to identify osteolathyrism, a collagen cross-linking deficiency, which is the specific effect of the positive control substance.

Statistics:

Probit analysis and 95 % CL calculation by SAS software (Statistical Analytics System) was employed to determine the LC50 and the EC50

Indices:

Based on mortality and malformation data obtained over a range of dose levels, the 50 % lethal concentration (LC50) and the 50 % effective concentration for malformations (EC50) of exposed embryos are calculated. These two point estimates are used to calculate the teratogenic index (TI), which is equal to the LC50 divided by the EC50 (NICEATM 2000).
- NICEATM (2000) Background Review Document Frog Embryo Teratogenesis Assay – Xenopus (FETAX). Prepared by The National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). Edited by National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC 27709, U.S.A. 273 p

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:not examined

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: at high doses

Details on embryotoxic / teratogenic effects:
The test item was able to induce the connective tissue defect osteolathyrism (Mekenyan et al 1996).

Effect levels (fetuses)

1

Dose descriptor:

other: TI (i.e. the Teratogenic Index calculated as LC50/EC50)

Effect level:

1.5 other: dimensioless

Based on:

test mat.

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

conc. level: LC50

Effect level:

18.96 other: g/L

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

3

Dose descriptor:

conc. level: EC50

Effect level:

12.72 other: g/L

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Results with reference substance (positive control)

96 h LC50 57.15 mmol/L (95 % CL 46.65-73.03), i.e. 2.86 times more toxic than the test item on molar basis

96 h EC50 0.05 mmol/L (95 % CL 0.03-0.064), i.e. 2190 times more morphologically effective (teratogenicity, malformations) than the test item on molar basis

TI (= LC50/EC50) 1143, i.e. 762 times higher (on molar basis) than the one caluculated for the test item

Applicant's summary and conclusion

Conclusions:

Osteolathyrism at levels close to lethality, no indication for relevant teratogenic risk, test system not reliable.

Executive summary:

Schultz et al (1988) investigated the test item Sym-diacetyl hydrazine (CAS 3148-73-0), which represents the submission item, for lethal and morphological effects (teratogenicity, malformations) to embryos of the African clawed frog (Xenopus laevis). The non-GLP study was followed the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) protocol as published in ASTM E1439-98. However the experiments are considered “reliable with restrictions” (Klimisch 2) with regard to the determination of a 50 % effect level of lethal and sublethal effects to aquatic amphibian development stages (see section on “Toxicity to other aquatic organisms”), the protocol has been evaluated an unsuitable test system for the assessment on toxicity to reproduction (NICATM 2000). Accordingly it is not reliable in the context of this section and rated here “not reliable” (Klimisch 3).

The test item was tested together with a series of compounds in order to develop a Quantitative Structure-Activity Relationship (QSAR), which is presented in the publication. Mekenyan et al (1996) re-investigated this QSAR under inclusion of additional data from other substances, which was published together with the original first author. No new measurements were made and thus the latter publication is a secondary source of the original measurements, which is referred here to avoid confusion as the data origin is not easily identifiable and the values are presented there in logarithmic form. The test organisms in the stage of early to mid-blastula embryos were exposed via the aquatic medium during 96 hours to a series of not further specified graduated concentrations of the test chemicals or pure medium (control). The effects were compared to the reference substance (positive control) Semicarbazide hydrochloride (CAS 563-41-7), from which teratogenic effects were well known as evidenced by the authors, and which represents the common structural element of the substances investigated by Schultz et al (1988). The specific effect of the reference is Osteolathyrism, a collagen cross-linking deficiency. Accordingly histological and electron microscopical examinations were conducted on representative embryos with emphasis on the axial skeleton. Additionally developmental stage, length, macroscopic malformations and mortality were recorded.

The test item was able to induce the connective tissue defect osteolathyrism (Mekenyan et al 1996) and gave thus a positive teratogenic response with a 96 h EC50 of 12.72 g/L or 109.51 mmol/L. This EC50 indicates a 2190 times less effectiveness than the positive control on molar basis. The 96 h LC50 of the test item was determined to be 18.96 g/L (163.3 mmol/L), which is less toxic than the reference substance by a factor of 2.86 (molar basis). The authors found a Teratogenic Index (TI) calculated as molar LC50/EC50 of 1.5, which is by a factor of 762 lower than the one of the positive control substance for osteolathyrism.

Nonetheless the findings may indicate an affiliation to a respective category/mode of action this is considered not relevant as the effect level was close to the lethal concentration as expressed by the TI. In addition the effect levels were in the order of 10 to 20 grams per litre.

In conclusion, under the conditions of this assay, the submission item does not evidence a relevant teratogenic hazard.

• NICEATM (NTP Interagency Center for the Evaluation of Alternative Toxicological Methods) (2000). Background Review Document (BRD) Frog Embryo Teratogenesis Assay – Xenopus (FETAX). Prepared by The National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). Edited by National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC 27709, U.S.A. 273 p