Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion
Endpoint conclusion:
no study available

Genetic toxicity in vivo

Description of key information

In vivo, when administered by intraperitoneal route, the substance is a clastogen due to induction of micronucleus in spermatids (germ cells), in spite of an absence of clastogenicity in bone marrow cells (somatic cells).

The following additional data were collected from the literature [IARC. Monographs on the Evaluation of Carcinogenic Risks to Humans - Volume 71: Re-evaluation of Some Organic Chemicals, Hydrazine and Hydrogen Peroxide - chapter “1,1-dimethylhydrazine"]:

In mammalian cells treated in vitro, 1,1-dimethylhydrazine induced gene mutations in Chinese hamster lung V79 cells and in mouse lymphoma L5178Y cells, chromosomal aberrations in Chinese hamster ovary cells and unscheduled DNA synthesis in mouse hepatocytes but not in rat hepatocytes. In a single study, it induced somatic mutations in Drosophila melanogaster. There is conflicting evidence as to its mutagenicity to bacteria.

In a single study, 1,1-dimethylhydrazine formed N7-methylguanine with DNA in the liver of rats treated in vivo. Given to mice in vivo, it did not induce sperm abnormalities, nuclear aberrations in the colon or micronucleus formation in the bone marrow, but, in single studies, it did induce micronucleus formation in spermatids, splenocytes and hepatocytes. In one study, 1,1-dimethylhydrazine induced DNA fragmentation in lung and in liver of mice in vivo. It failed to induce unscheduled DNA synthesis in kidney cells of rats in a single study conducted in vivo. It produced negative results in a host-mediated assay using mice.

Additionally, in a National Toxicology Program study (N° C05732; no report available), the substance was not clastogen by oral route at up to the Maximal Tolerated Dose of 100 mg/kg/day, in an vivo bone marrow micronucleus assay conducted in male B6C3F1 Mice. However, the fact that this corrosive and highly reactive substance was given by gavage, leads to some doubt about the possibility to evidence effects on a distant organ as the bone marrow.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not indicated in publication
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Some missing data (GLP, methods, test item characterization, clinical signs of toxicity). However the negative result (in bone marrow) is reliable and the positive result (in spermatids) must be taken into account for classification.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(not mentioned in the publication)
Deviations:
yes
Remarks:
Only one sex tested, without justification
Principles of method if other than guideline:
Micronucleus investigated both in somatic cells (erythrocytes as in guideline) and germ cells (spermatids: no applicable guideline)
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Saint-Aubin les Elbeuf, France
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 30g
- Fasting period before study: no
- Diet (e.g. ad libitum): UAR A 04, ad lib.
- Water (e.g. ad libitum): tap water, ad lib.
- Assignment to groups, Housing, Acclimation period: not indicated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Photoperiod (hrs dark / hrs light): 12/12
- Humidity, Air changes: not indicated

IN-LIFE DATES: not indicated
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: water for injection
- Justification for choice of solvent/vehicle: soluble in vehicle
- Concentration of test material in vehicle: not indicated
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
dissolved (soluble at >10% in water)
Duration of treatment / exposure:
Bone marrow micronucleus test: 2 days
Spermatid micronucleus test: 15 days
Frequency of treatment:
Bone marrow micronucleus test: 2 injections at 24-h interval

Spermatid micronucleus test: 6 injections (days 0+1 during DNA synthesis phase of preleptotene spermatocytes I; days 7+8 during residual DNA synthesis of pachytene spermatocytes I = crossing-over and chromosome pairing; days 13+14 during meiotic phase of spermatocytes I/II )
Post exposure period:
Time of sacrifice, after last injection:
Bone marrow micronucleus test: 24h
Spermatid micronucleus test: 2 days
Remarks:
Doses / Concentrations:
28 mg/kg/injection
Basis:
other: nominal injected dose
Remarks:
Doses / Concentrations:
56 mg/kg/injection
Basis:
other: nominal injected dose
Remarks:
Doses / Concentrations:
83 mg/kg/injection
Basis:
other: nominal injected dose
No. of animals per sex per dose:
5, males only
Control animals:
yes, concurrent vehicle
other: additional groups indicated as dosed at 0 mg/kg: will be considered as negative controls for this RSS
Positive control(s):
In the bone marrow micronucleus test only:
- mitomycin C
- Justification for choice of positive control(s): clastogen
- Route of administration: intraperitoneal
- Doses / concentrations: 1 mg/kg
Tissues and cell types examined:
- Bone marrow: polychromatic erythrocytes (PCE)
- Testes: Golgi-phase spermatids
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Doses were 25, 50 and 75% of the 14-day LD50 (one treatment)

DETAILS OF SLIDE PREPARATION / METHOD OF ANALYSIS / EVALUATION OF TOXICITY:

Bone marrow:
- smears fixed with methanol, stained with MGG
- 2 slides per mouse, at least 1000 PCEs examined for micronucleus per slide.
- Myelotoxicity assessed as the ratio of normochromatic erythrocytes (NE) and PCE , over 200 NE/mouse.

Spermatids:
- On day 16, spermatids isolated by enzymatic dissociation of the testes (trypsin/deoxyribonuclease I). Smears fixed with methanol, stained with 20% Giemsa in Sörensen buffer (pH 7.4)
- 2 slides per mouse, at least 1000 Golgi-phase spermatids examined for micronucleus per slide.
- Testes weight as an indicator of testicular toxicity.
Sex:
male
Genotoxicity:
negative
Remarks:
Bone marrow PCEs
Toxicity:
no effects
Remarks:
except for positive control (myelotoxic)
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
positive
Remarks:
Golgi-phase spermatids
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
RESULTS OF RANGE-FINDING STUDY:
- Clinical signs of toxicity in test animals: the dose-levels were based on the LD50
- Other: no other details indicated

RESULTS OF DEFINITIVE STUDY - Bone marrow assay
- Induction of micronuclei: no statistically significant increase
- Ratio of PCE/NCE: no statistically significant increase
- Appropriateness of dose-levels: justified by absence of excessive cytotoxicity and closeness to LD50

RESULTS OF DEFINITIVE STUDY - Spermatid assay
- Induction of micronuclei: dose-related and biologically significant (up to 5-fold) increase: 0.4, 0.9, 1.2 and 1.9 micronuclei per 1000 spermatids at 0, 28, 56 and 83 mg/kg; p<0.01 at the top-dose.
- Cytotoxicity: no statistically significant effect on testicular weight
- Appropriateness of dose-levels: justified by absence of excessive cytotoxicity and closeness to LD50
Conclusions:
Interpretation of results (migrated information): positive for spermatid assay
The test item was negative in the bone marrow micronucleus assay (2 treatments) but clearly positive in the spermatid micronucleus assay (6 treatments) carried out at the same daily dose-levels. The difference could be related to the number of administrations, leading to an excessive sensitivity of the spermatid assay, for which no guideline exists. However, this spermatid assay shows that the substance is able to induce genetic damage that can not be attributed to overt cytotoxicity (as far as can be judged from the endpoint used, testicular weight).

NB: The authors also obtained positive micronucleus results in the liver, in previous experiments with the test item.
Executive summary:

In order to analyze in vivo micronucleus induction in cells with metabolic enzyme activity, the author compared the sensitivity of somatic and germ cells to four carcinogens in the bone marrow and spermatid micronucleus test in the mouse. Three procarcinogens with a complex metabolic pattern (dimethylnitrosamine, diethylnitrosamine and 1,1-dimethylhydrazine) and one direct unstable mutagen (beta-propiolactone) were tested. All four carcinogens were not detected by the bone marrow micronucleus test but were detected in the mouse spermatid micronucleus test in which they induced clear clastogenic effects, as was the case in a previous study in liver micronucleus test. In conclusion, this study demonstrates that the bone marrow micronucleus test is not sufficient for the prediction of a clastogenic hazard in germ cells. In addition to a second in vivo test in an organ with metabolic enzymes, i.e., the liver, the spermatid micronucleus test can be performed when a specific risk to the testis is likely.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

The genotoxicity data mentioned by IARC lead to inconstant conclusions, with about half of the large variety of assays being positive and the other half negative. In a worst-case approach and since carrying out a new test would not help reach a conclusion in view of the large number of existing tests, it is considered that the substance is genotoxic.

Endpoint Conclusion: Adverse effect observed (positive)

Justification for classification or non-classification

Due to dose-related, biologically and statistically significant (5-fold increase in micronucleus frequency) genotoxicity to germ cells in an in vivo assay in mice, it is proposed to classify the substance as inducing heritable genetic damage: Muta 2; H341.