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Genetic toxicity in vivo

Description of key information
Numerous in vitro and in vivo genotoxicity tests indicate that this substance is not mutagenic.
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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was performed according to accepted but older guidelines and under GLP.
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Cited as Directive 84/449/EEC, B.12
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
- Age: 7 to 8 weeks
- Weight at study initiation: 33-40 g (males) & 30-35 g (females)
- Housing: Makrolon cages
- Food and water: free access to feed and water
Route of administration:
intraperitoneal
Vehicle:
DMSO
Details on exposure:
- The animals received a single intraperitoneal injection of the test substance dissolved in DMSO
- The dose level used as the test dose was selcted to produce mortality of about 10% (based on preliminary LD50 study)
- Volume injected: 0.1 mL/10 g bw
Duration of treatment / exposure:
single application
Frequency of treatment:
once
Post exposure period:
24, 48 and 72 h after administration, 6 animals of each group were sacrificed
Remarks:
Doses / Concentrations:
50 mg/kg
Basis:

No. of animals per sex per dose:
No. of animals per dose: at least 20/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (20 mg/kg)
Tissues and cell types examined:
femoral bone marrow smears
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION
- The bone marrow preparations were prepared, stained and evaluated based on the method of Schmid
- The two femora were excised from the test animals, the attached tissue was removed and the femora were dissected at the condyles
- The bone marrow was rinsed with fetal calf serum by means of a syringe and cannula and transferred to a centrifuge tube with 4 mL calf serum
- The cell suspension was centrifuged at 100 g for 5 minutes, and the serum was sucked off except for a small amount
- The cells were resuspended by careful pipetting, dropped onto clean microscopic slides and smeared

METHOD OF ANALYSIS
- After one day, the microscopic slide preparations were stained with May-Gruenwald and Giemsa solution, rinsed in distilled water, dried and mounted in DePeX via xylene
- The coded preparations were evaluated under the microscope at a 500 fold enlargement
- 1000 erythrocytes were counted and differentiated according to polychromatic (young) and normochromatic (old) erythrocytes
- The fraction of polychromatic cells indicates the function of erythropoiesis and allows detecting possible cytostatic or cytotoxic effects of the test substance
- The proportion of cells carrying miconuclei was determined in 2000 polychromatic erythrocytes
- The mean and standard deviation were calculated from the individual values of the 6 animals of each group
Evaluation criteria:
CRITERIA FOR EVALUATING RESULTS:
- Significant increase in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of male and female mice
- Statistical method: U test
Statistics:
- The test group and control group were investigated for differences by means of the U-test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
- Mortality : Male: 2 out of 22, Female: 3 out of 22
- Clinical signs: slight to moderate convulsion and piloerection

STATISTICAL RESULTS:
% PCE and %PCE with micronucleus were not increased in males or females at 24, 48, and 72 hours after treatment.

EFFECT ON PCE/NCE RATIO (%PCE):
Males
- Control group: 52.9, 53.3, 51.8 at 24, 48 and 72 hours, respectively
- Treatment group: 50.4, 48.4, 53.6 at 24, 48 and 72 hours, respectively
- Positive control: 52.2 at 24 hours

Females
- Control group: 53.4, 51.9, 52.2 at 24, 48 and 72 hours, respectively
- Treatment group: 53.0, 53.2, 54.1 at 24, 48 and 72 hours, respectively
- Positive control: 51.7 at 24 hours

GENOTOXIC EFFECTS:
Mean number of micronucleated PCE:
Males
- Control group: 0.16, 0.27, 0.21 at 24, 48 and 72 hours, respectively
- Treatment group: 0.26, 0.24, 0.20 at 24, 48 and 72 hours, respectively
- Positive control: 1.40 at 24 hours

Females
- Control group: 0.13, 0.25, 0.19 at 24, 48 and 72 hours, respectively
- Treatment group: 0.17, 0.22, 0.21 at 24, 48 and 72 hours, respectively
- Positive control: 1.39 at 24 hours
Conclusions:
Interpretation of results (migrated information): negative
According to the test results available, there are thus no significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the dose group (50 mg Dicykan/kg body weight) or between the different sacrifice intervals (24, 48 and 72 hours). Thus, under the experimental conditions chosen here, the test substance Dicykan has no chromosome damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

in vitro studies

PACM (chemical name: 4,4'-Methylenebis(cyclohexylamine), trade name Dicykan) was tested for mutagenicity in the Ames test (GLP study) according to OECD 471 (Huels AG, 1997). The tested strains were TA 1535, TA 100, TA 1537, TA 98. The test conditions were without and with metabolic activation. The test substance was dissolved in DMSO. An increase in the number of his+ revertants could not be observed either without S-9 mix or after the addition of a metabolizing system. According to the results of the present study, the test substance 4,4'-Methylenebis(cyclohexylamine) is not mutagenic in the Ames test under the experimental conditions chosen.

There are four additional genotoxicity tests available according or similar to OECD 471. The results gave no hints for any genotoxic effects.

PACM was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (Huels AG, 1995). The experiments were carried out both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). The test concentrations were 0, 20, 60, 200, 600 and 1000 µg/ml medium. Under the experimental conditions of this study, the substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

PACM was investigated for its potential to induce chromosome-damaging effects in an in vitro cytogenetic study according to OECD 473 (Huels AG, 1994). The test substance did not lead to a biologically relevant increase in the number of structural chromosomal aberrations (including and excluding gaps) either without S9 mix or after the addition of a metabolizing system. The types and frequencies of structural chromosome aberrations were close to the range of the concurrent vehicle control values and within the range of the historical negative control data. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances Mitomycin and Cyclophosphamide clearly demonstrated the sensitivity of the test system and the metabolic activity of the used S9 mix. Thus, PACM is not a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation.

in vivo studies

The substance PACM was tested for its mutagenic potential in NMRI mice using the micronucleus test method (key study; GLP; BG Chemie, 1984). The acute LD50 of PACM was determined in a pretest in male mice after a single intraperitoneal injection. A dose of 50 mg/kg bw was selected to be used in the micronucleus test (mortality was expected to be 10%). During the main study, clinical signs observed were slight to moderate convulsions and piloerection; 2 of 22 males and 3 of 22 females died in test substance treated groups. The proportion of polychromatic erythrocytes in the bone marrow was not affected after administration of Dicykan. The administration of the positive control Cyclophosphamide (20 mg/kg bw) has also not affected the proportion of polychromatic erythrocytes, but led to the expected increase in the number of polychromatic erythrocytes containing micronuclei. After single intraperitoneal administration of 50 mg/kg bw Dicykan, the rate of micronuclei was always in the same range as that of the concurrent control at all 3 sacrifice intervals (24, 48 and 72 h). Thus, under the experimental conditions chosen, the test substance does not have any chromosome damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis.

 

In a supporting study, PACM was tested for its mutagenic potential in NMRI mice using the micronucleus test method (non GLP; BASF, 1983). For this purpose, PACM, dissolved in DMSO, was administered once intraperitoneally to male and female animals at a dose level of 50 mg/kg bw. As a control, male and female mice were administered merely the solvent by the same route. As a positive control, male and female animals were administered 40 mg Cyclophosphamide/kg bw dissolved in distilled water once by intraperitoneal administration. Animals which were administered the solvent DMSO or the positive control substance Cyclophosphamide did not show any clinical signs of toxicity. However, 50 mg PACM/kg bw led to piloerection, apathy, irregular respiration and spastic gait in some cases. After 30 - 60 minutes, a narcotic-like state was observed. On the day after test substance administration, the animals still showed clinical signs such as apathy and piloerection; in some cases, these symptoms were observed throughout the observation period. A total of 5 male and 4 female mice died during the study period. The positive control substance Cyclophosphamide, as expected, led to the clear increase in the number of polychromatic erythrocytes containing micronuclei. The single intraperitoneal administration of PACM at a dose of 50 mg/kg bw did not Iead to any increase in the number of polychromatic erythrocytes containing micronuclei. The rate of micronuclei was always in the same range as that of the concurrent control at all 3 sacrifice intervals (24, 48 and 72 h). A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected in individual animals of the 48 h sacrifice interval and in most animals of the 72 h sacrifice interval. Thus, under the experimental conditions chosen, the test substance Dicykan is not considered mutagenic.

 

There are further in vivo micronucleus tests available. In a non-GLP study (BASF, 1981), 5 male and 5 female NMRI-mice were treated with a single intraperitoneal dose of 4,4'-methylenebis(cyclohexylamine) at10 mg /kg bw. No significant increase in the number of polychromatic erythrocytes containing micronuclei was observed. Only 1000 PCEs were evaluated (the current OECD guideline requires an evaluation of 2000 PCEs) and no positive control group was investigated.

In another non-GLP study (BG Chemie, 1982), NMRI mice were treated with a single intraperitoneal dose of 50 mg/kg bw. The treated animals showed slight to moderate paresis a few hours after the administration; 6 of 25 animals died in the group of the males within 3 days, whereas none of the 25 females died. The number of micronuclei showed a slight increase up to twice the control values after administration of PACM. This increase was however statistically significant only in the females on days 1 and 2 after treatment. Only 1000 PCEs were evaluated in this study, the current OECD guideline requires an evaluation of 2000 PCEs. Due to the lack of historical control data, the relevance of the effect cannot be assessed.

The combination of a negative in vitro chromosomal aberrations test, with 3 of 4 in vivo mutagenicity tests (including a modern guideline assay according to GLP), offer a weight of evidence that the substance is not clastogenic or otherwise genotoxic.


Justification for selection of genetic toxicity endpoint
In vitro studies of genotoxicity were all negative. The in vivo micronucleus study in mice was performed according to a guideline and under GLP conditions.

Justification for classification or non-classification

The classification criteria of EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 are not met.