Reaction products of 1,4-cyclohexanedimethanol with propylene oxide and ammonia

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-12-19 - 2012-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH S3A (1995), ICH S2A (1996), ICH S2B (1997)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 1,4-cyclohexanedimethanol, propylene oxide and ammonia

Cas Number:

1220986-58-2

Molecular formula:

C11H24N2O

IUPAC Name:

Reaction products of 1,4-cyclohexanedimethanol, propylene oxide and ammonia

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Jeffamine RFD-270 Amine
- Substance type: Colourless liquid
- Physical state: Liquid
- Lot/batch No.: 8802-8-9
- Analytical purity: 92%
- Composition of test material, percentage of components: 0.02 wt% water
- Purity test date: 2010-07-15
- Storage condition of test material: Sample stored in cool, well-ventilated storage area prior to testing

Specific details on test material used for the study:

- Name of test material (as cited in study report): Jeffamine RFD-270 Amine
- Substance type: Colourless liquid
- Physical state: Liquid
- Lot/batch No.: 8802-8-9
- Analytical purity: 92%
- Composition of test material, percentage of components: 0.02 wt% water
- Purity test date: 2010-07-15
- Storage condition of test material: Sample stored in cool, well-ventilated storage area prior to testing

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan
- Age at study initiation: 6 weeks old
- Weight at study initiation: dose range finding study: male mice: 30.9 - 36.5 grams, female mice: 30.0 - 25.0 grams; definitive micronucleus study: male mice: 29.5 - 34.0 grams, female mice: 24.4 - 28.6 grams
- Assigned to test groups randomly: yes, based on equalization of group mean body weights. At the time of randomization, the weight variation of animals did not exceed ± 20% from their group mean.
- Fasting period before study: No
- Housing: Animals were housed by sex, with up to five animals per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-Vent full ventilation, HEPA filtered system.
- Diet (e.g. ad libitum): Certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet); ad libitum
- Water (e.g. ad libitum): Tap water; Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ± 22.2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: All test substance dose formulations were freshly prepared prior to administration. An appropriate amount of the test substance was weighed separately for each formulation, and ~ 80% of the required vehicle was added to the respective containers. Each formulation was vortexed briefly, and the remaining volume of the vehicle was added to achieve the required final volume. All formulations appeared clear formulations.

Duration of treatment / exposure:

Animals were treated once.

Frequency of treatment:

Animals were treated once, by oral gavage, using a dose volume of 20 mL/kg

Post exposure period:

Bone marrow was harvested at 24 or 48 hours after treatment and evaluated microscopically for the presence of micronucleated polychromatic erythrocytes (MPCEs), as well as for the proportion of PCEs (relative to total erythrocytes) as an indication of bone marrow cytotoxicity.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5, with exception of the high dose group (500 mg/kg) where 15 mice/sex were tested (including 5 replacement mice/sex)

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:

bone marrow ; polychromatic erythrocytes were examined for the presence of micronuclei

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: 24 hours post dose: 5 animals per group, 48 hours post dose: 5 animals in the vehicle control group and 5 animals in the high dose group

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): sampling at 24 and 48 hours post dose

DETAILS OF SLIDE PREPARATION: The bone marrow was transferred to a labeled centrifuge tube containing approximately 1 mL fetal bovine serum, the cells were pelleted by centrifugation at approximately 100 x g for five minutes, and the supernatant was removed, leaving a small amount of serum with the remaining pellet.

The cells were re-suspended and a small drop of the suspension was spread onto a clean glass slide. Two slides were prepared from each mouse, labeled with the experiment and animal numbers, air dried, and fixed in methanol. One set of slides was stained with acridine orange and anallyzed microscopically.

METHOD OF ANALYSIS: Slides were initially scanned using a fluorescent microscope and medium magnification (400X; blue 440-490 nm excitation filter and 520 nm barrier filter) and an area acceptable quality was selected such that the cells were well spread and stained. Once selected, the following cell populations and cellular components were evaluated under high power magnification (1000X oil immersion).

- Polychromatic erythrocytes (PCEs):
PCEs are young erythrocytes in the early stage of erythropoiesis that stain orange-red; 2000 PCEs/animal (10000 PCEs/group) were scored for the presence of micronuclei.
- Normochromatic erychtrocytes (NCEs):
NCEs are mature erythrocytes, the final cell population formed during erythropoiesis, that stain light green; the number of NCEs/1000 total erythrocytes also was determined for each animal, to calculate the proportion of PCEs to total erythrocytes, as an index of bone marrow cytotoxicity. PCE proportions less than 20% of control values are considered excessively toxic.
- Micronuclei (M)
Micronuclei are round nuclear (chromosome) fragments, generally with a sharp contour and diameters of approximately 1/20 to 1/5 of an erythrocyte, that stain green. Micronuclei may occur in PCEs (MPCEs) or NCEs (MNCEs). The frequency of MNCEs observed while scoring 2000 PCEs was determined, but the results are not presented or used in analysis of genotoxic response since the primary target cells are the PCEs.

Evaluation criteria:

- A test substance is considered to be positive for the induction of micronuclei if it induces a significant increase in MPCE frequency (p<=0.05) at any dose level of sampling time
- A test substance is considered to be negative for the induction of micronuclei if no significant increase in MNPCE frequency is observed (p>0.05).
- If criteria for either a positive or negative clastogenic response were not met, the resuls would have been judged as equivocal.
Other criteria also may be used in reaching a conclusion about the study results(e.g. magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.).

Statistics:

Statisical significance, as compared to the concurrent vehicle control, was determined for each group using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately by sex and sampling time.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Complete mortality was observed on day 0 in all mice treated with Jeffamine RFD-270 at doses of 750, 1000 and 2000 mg/kg. All mice treated at a dose of 500 mg/kg exhibited piloerection after treatment and on day 1; males in this group also exhibited lethargy on day 0. All mice in the 100 and 250 mg/kg treatment groups appeared normal throughout the study, except for piloerection noted in the 100 mg/kg dose group on day 3.

RESULTS OF DEFINITIVE STUDY
Five males treated with Jeffamine RFD-270 at a dose of 500 mg/kg were found dead on day 0. Males from the replacement group were reallocated to ensure sufficient animals were available for each sampling time. Piloerection also was observed in some or all males at all dose levels on day 0 and 1, and in some or all females at a dose of 500 mg/kg on day 0 and 1. Lethargy also was observed in all males at a dose of 500 mg/kg on day 0.

Applicant's summary and conclusion

Conclusions:

The results of this test indicate the test substance was negative in the mouse bone marrow erythrocyte micronucleus.