Registration Dossier

Administrative data

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:
23 October 2009 - 21 January 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to official test guidelines, and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
other: JMHW Genotoxicity Testing Guideline, PAB Notification No. 1604 (1 November 1999)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The study report includes a statement of GLP compliance signed by the study director, a certificate of quality assurance, and a certificate of GLP Compliance issued by the UK GLP Monitoring Authority.
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,3-Bis(aminomethyl)cyclohexane
- Substance type: organic, clear liquid
- Physical state: liquid
- Storage condition of test material: room temperature in the dark

Test animals

Species:
mouse
Strain:
other: Crl:CD1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: UK
- Age at study initiation: Micronucleus test (main test) approximately 43 days old.
- Weight at study initiation: 29.1 g to 33.0 g
- Assigned to test groups randomly:Yes
- Diet (e.g. ad libitum): Free access to pelleted rat and mouse maintenance diet.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C.
- Humidity (%): 40 to 70% relative humidity.
- Photoperiod (hrs dark / hrs light): 12 hours light per day.

IN-LIFE DATES: From: 28 October 2009 To: Approx. 11 December 2009 (assumes final sacrifice of animals 48 hours after start of Micronucleus test).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water; pH was adjusted (from pH 12 to a pH which could be tolerated by the rodents, pH<9) using Hydrochloric Acid and / or Sodium Hydroxide
- Justification for choice of solvent/vehicle: The test material is highly soluble in water.
- Concentration of test material in vehicle: 0 (control), 43.75, 87.5, 175 mg/mL.
- Amount of vehicle (if gavage or dermal): 10 mL/kg/day
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations were sonicated or heated to 37°C where required to form a solution.
Duration of treatment / exposure:
Two treatments, approximately 24 hours apart. Sacrifice was performed approximately 24 hours after the second treatment.
Frequency of treatment:
Doses were administered approximately 24 hours apart.
Post exposure period:
24 Hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day (Vehicle control)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
437.5 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
875 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
1750 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
6 Males (test only conducted in males, as no difference in toxicity was found between sexes in a preliminary test).
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Justification for choice of positive control(s): Mitomycin C is a recommended positive control material suggested in the OECD test guideline.
- Route of administration: Oral gavage
- Doses / concentrations: 5 Males dosed at 12 mg/kg/day (0.6 mg/mL, 20 mL/kg dose). Positive control group was dosed once only, 24 hours prior to termination.

Examinations

Tissues and cell types examined:
Bone marrow from both femurs of each rat was collected. Polychromatic erythrocytes were examined.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.
The resulting cell suspensions were centrifuged at 1000 rpm (150 × g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976).


METHOD OF ANALYSIS:
Fixed for a minimum of 10 minutes in methanol and allowed to air-dry, rinsed in purified water, stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes. Cells were then washed in purified water for 5 minutes, and rinsed in cold tap water for 2 minutes. Slides were stored at room temperature protected from light until required. Immediately prior to scoring, slides are wet mounted with coverslips using purified water.
Evaluation criteria:
Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded.
Statistics:
For the proportion of polychromatic erythrocytes at 24 hours, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3, then on Groups 1 and 2. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.

Results and discussion

Test resultsopen allclose all
Sex:
male
Genotoxicity:
ambiguous
Remarks:
statistically significant decrease in the proportion of polychromatic erythrocytes was observed at 1750 mg/kg/day only
Toxicity:
yes
Remarks:
The decrease was considered indicative of toxicity.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Remarks:
No statistically signifcant increase in the induction of micronucleated polychromatic erythrocytes in male Crl:CD1TM mice compared to control.
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
A preliminary toxicity (range-finding) test was conducted to determine the maximum tolerated dose of the test material. 2 males and 2 females in each group were dosed at 500, 1000, 2000, 1500, and 1750 mg/kg/day, then observed for 48 hours after the first dose. Clinical signs and mortalities were recorded. At the end of the observation period, surviving animals were killed and discarded.
Excessive clinical signs were seen at 2000 mg/kg/day (the standard limit for this test), and so 1750 mg/kg/day was chosen as the highest concentration for the main (micronucleus) test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):The test substance did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes in male animals.
- Ratio of PCE/NCE (for Micronucleus assay):The test substance did cause a statistically significant decrease (p<0.05) in the proportion of polychromatic erythrocytes at 1750 mg/kg/day in male animals only. This decrease is considered as being indicative of toxicity. All individual and group mean values were within the laboratory historical control range.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance is not considered to be genotoxic as no clear statistically significant increases in micronuclei (MPCE) were observed in any treatment groups relative to the control. 
Executive summary:

An In-Vivo Mammalian Erythrocyte Micronucleus Test was conducted to assess the mutagenic potential of the test substance 1,3-Bis(aminomethyl)cyclohexane. The study was conducted according to a number of international test guidelines including OECD test guideline 474 and EC test guideline B12. The study was conducted in compliance with GLP.

A range finding study test was performed to determine the maximum tolerated dose, and then the main study was conducted at levels 437.5, 875, and 1750 mg/kg/day. No difference in response was seen between males and females in the range finding test, and so the dose groups consisted of six male mice only. A vehicle control group and positive control group were tested contemporaneously.

No statistically significant increase was seen in the induction of micronucleated polychromatic erythrocytes. A statistically significant decrease was seen in the proportion of polychromatic erythrocytes at 1750 mg/kg/day only, but this observation was believed to be indicative of toxicity.