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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
A mouse micronucleus test was negative with RDX, a chemically similar substance to HMX suitable for reading across to the test substance. Several supporting studies also gave negative results. These included five Ames tests, a mouse lymphoma forward mutation assay and a mammalian cell mutagenicity assay. The in vivo a dominant lethal test in rats was also negative. HMX therefore shows no signs of genetic toxicity.
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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
It is a GLP study following international guidelines and has been published in a peer reviewed journal. The restriction is also due to the use of the read across approach: the test was performed not with HMX but with RDX, a substance which has been demonstrated to be very similar in structure, physical/chemical properties and toxicological profile .
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: well known breeder
- Age at study initiation:
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):18-26°C
- Humidity (%):30% to 70%
- Photoperiod (hrs dark / hrs light):12hrs dark /12 hrs light

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle:
- Amount of vehicle (if gavage or dermal): 10ml/kg
- Lot/batch no. (if required):lot no. 12-394
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test article wasdissolved in corn oil
Duration of treatment / exposure:
24h and 48h
Frequency of treatment:
single dose
Remarks:
Doses / Concentrations:
62.5 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
125 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5 males/dose for the negative and positive control and the 62.5 and 125 mg/kg and 10/sex/dose for the 250 mg/kg
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide in sterile deionized water
- Route of administration: gavage
- Doses / concentrations: 80mg/kg
Tissues and cell types examined:
Bone marrow. micronuclei in polychromatic erythrocytes (PCEs) and norchromatic erythrocytes (NCEs)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:Based upon the result of the dose range-finding study, the estimated maximum tolerated doses was 250 mglkg after one oral gavage administration

SAMPLING TIMES ( in addition to information in specific fields): 24h and 48h

DETAILS OF SLIDE PREPARATION:Bone marrow smears were prepared and allowed to air dry. The slides were then fixed in methanol, stained in May-Grunwald-Giemsa stain and protected by permanently mounted cover slips.

METHOD OF ANALYSIS:The slides were blind scored for micronuclei in polychromatic erythrocytes (PCEs) and norchromatic erythrocytes (NCEs) and determination of PCE/NCE ratios to access possible bone marrow cytotoxicity.
The micronucleus frequency (expressed as percent micronucleated cell) wasdetennlned by analyzing number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least the first 500 erythrocytes per animal.

Evaluation criteria:
The criteria for the identification of micronuclei were those of Schmid (1976).
Statistics:
Yes but no details
Sex:
male
Genotoxicity:
negative
Remarks:
no significant decreases in the PCE:NCE ratios observed at any RDX dose or bone marrow sampling time point
Toxicity:
no effects
Remarks:
RDX at doses 62.5, 125, and 250 mg/kg were not cytotoxic to bone marrow (i.e.no statistically significant decrease in PCE:NCE ratios)
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: up to 500 mg/kg
- Clinical signs of toxicity in test animals: Mice dosed at all dose levels showed neurotoxic signs (hyperactive) and deaths at high dose of 500 mg/kg.


Summary data from micronuleus assay for RDX in bone marrow of CD-l mouse

 Treatment  Dose  Harvest time  % Micronucleated PCE (mean of 2000 per animal ±SE)  Ratio PCE:NCE (mean ± S.E.)
     
  Positive control   CP 80 mg/kg  24h   2.39 ± 0.31*  0.57 ± 0.05
  Test article   62.5 mg/kg  24h 0.04 ± 0.02 0.47 ± 0.07 
    125 mg/kg  24h  0.03 ± 0.01  0.36 ± 0.04
    250 mg/kg 24  0.02 ± 0.01  0.60 ± 0.05
     48h  0.10 ± 0.02  0.69 ± 0.11
   Vehicle control   Corn oil 24h    0.05 ± 0.02    0.50 ± 0.07
     48h    0.08 ± 0.03   0.71 ± 0.06

*Significantly greater than the corresponding vehicle control, P =0.01

CP = cyclophosphamide.

PCE = polychromatic erythrocyte.

NCE = normochromatic erythrocyte.

Conclusions:
Interpretation of results (migrated information): negative
RDX was found to be negative in the in vivo mouse bone marrow micronucleus assay.
Executive summary:

HMX and RDX, which is tested for its genotoxicity in a mouse micronucleus assay, are both explosive compounds used in military munitions formulations.

These substances have been demonstrated to be very similar in structure, physical/chemical properties and toxicological profile in the Analogue Approach - Read Across High Melting Explosive (HMX) (2013) document (see Section 13). Due to the fact that HMX and RDX have nearly the same chemical structure, the same mode of interaction with bio-macromolecules, living cells and tissue and metabolic pathway is expected. Therefore, a read-across from HMX to data obtained with RDX is scientifically justified.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro studies

Gene mutations

A key study, a mouse lymphoma forward mutation assay, was performed using RDX, a chemically similar substance to HMX for which read-across can be scientifically justified. The study was performed according to GLP guidelines and OECD guideline 476 (Reddy et al, 2005). Pure RDX (99.99%) af concentrations ranging from 3.93 to 500 μg/mL showed no cytotoxicity and no mutagenicity in forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, with and without metabolic activation. The finding was confirmed by repeat assays under identical conditions. A supporting mammalian cell mutagenicity assay was done (Lachance et al, 1999). V79 chinese hamster lung cells were used. There was no evidence of mutagenic activity in the mammalian cells tested, either in the presence or absence of metabolic activation at concentrations up to 33 μM.

Five bacterial reverse cell mutation (Ames) test results were available. Results were negative in every case, therefore there is enough weight of evidence in these supporting studies to suggest that the negative result arising from the read-across from RDX is justified and scientifically valid.

In vivo studies

Two in vivo studies were available, both using RDX, from which a read-across approach is justified. One study was a mouse micronucleus assay (Reddy et al. 2005). The study was conducted according to OECD guideline 474. Groups of CD-1 male mice were dosed orally with 62.5, 125 and 250 mg/kg RDX. Bone marrow samples were taken and slides were scored for micronulei in polychromatic erythrocytes (PCE's) and norchromatic erythrocytes (NCE's). PCE/NCE ratios were determined. There were no significant decreases in the PCE:NCE ratios observed at any RDX dose or bone marrow sampling time point. RDX gave a negative result in the in vivo mouse bone marrow micronucleus assay, therefore HMX can be said to also be negative using the read-across approach.

The other in vivo study was a dominant lethal assay in rats using RDX. Four groups of 22 male and 22 female rats were fed diets containing RDX at daily doses of 0, 5, 16 or 50 mg/kg. The F0 generation was treated for 13 weeks. After 13 weeks F0 males were mated with nontreated females. There were no statistically significant effects on any parameters examined, therefore RDX was not mutagenic and by using read-across, HMX can be said to be non-mutagenic also.


Justification for selection of genetic toxicity endpoint
Study conducted to international guidelines and to GLP. Read-across from chemically similar substance RDX.

Justification for classification or non-classification

HMX is not classified for mutagenic effects as all the tests undertaken (including Ames test, mammalian cell mutagenicity, mouse lymphoma forward mutation, mouse micronucleus and dominant lethal assay in rats) were negative.