Adipic acid, compound with hexane-1,6-diamine (1:1)

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:

2001-05-07 to 2001-11-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(July 21, 1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(May 19, 2000)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Adipic acid, compound with hexane-1,6-diamine (1:1)

EC Number:

222-037-3

EC Name:

Adipic acid, compound with hexane-1,6-diamine (1:1)

Cas Number:

3323-53-3

Molecular formula:

C6H16N2.C6H10O4

IUPAC Name:

hexanedioic acid - hexane-1,6-diamine (1:1)

Specific details on test material used for the study:

- Name of test material (as cited in study report): AH Salz kristallisiert (hexamethylenediamine adipate, crystalline)
- Physical state: white crystals
- Analytical purity: 99%
- Purity test date: 2001-01-16
- Lot/batch No.: 30.10.2000, date of manufacture: 2000-10-30
- Stability under test conditions: confirmed by analysis
- Storage condition of test material: Room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
male Crl:NMRI mice
- Source: Charles River Deutschland GmbH
- Age at study initiation: ca. 5 - 8 weeks
- Weight at study initiation: ca. 27 g mean weight
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: individually in Makrolon cages, type Ml
- Diet (ad libitum): Standardized pelleted feed (Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (ad libitum): bottled water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, purified water was selected as the vehicle.
- Concentration of test material in vehicle: 40, 80, 160 mg/ml for the low, mid, and high dose group, respectively.
- Dosing volume: 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS (i.p.):
All test substance formulations were prepared immediately before administration. The amount of substance or volume to be administered was related to the specific weight of the individual animals an the day of the first administration.

Duration of treatment / exposure:

twice at a 24-hour interval (vehicle controls and groups dosed with the test substance);
single dose (positive control groups)

Frequency of treatment:

twice at a 24-hour interval (vehicle controls and groups dosed with the test substance);
single dose (positive control groups)

Post exposure period:

24 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

total dose: 800 mg/kg bw

Doses / concentrations 2

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Remarks:

total dose: 1600 mg/kg bw

Doses / concentrations 3

Dose / conc.:

1 600 mg/kg bw/day (actual dose received)

Remarks:

total dose: 3200 mg/kg bw

No. of animals per sex per dose:

5 males per group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): The stability of CPP is weIl-defined under the selected conditions, since the positive control article is a well-defined clastogen.
- Route of administration: i.p.
- Doses / concentrations: 20 mg/kg bw

vincristine
- Justification for choice of positive control(s): The stability of VCR is weIl-defined under the selected conditions, since the positive control article is a well-defined aneugen.
- Route of administration: i.p.
- Doses / concentrations:0.15 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow of the two femora; polychromatic erythrocytes and normocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest for determination of acute intraperitoneal toxicity, deaths were observed following two treatments down to a dose of 1700 mg/kg bw. 1600 mg/kg were survived by all animal, but led to evident signs of toxicity. There were no distinct symptomatic differences between the male and female animals. Thus, only male animals were used in the main study, 1600 mg/kg bw was selected as the highest dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals of the vehicle control and the dose groups were treated twice at a 24-hour interval and samples of bone marrow were taken 24 hours after the last treatment. Animals of the positive control groups were treated only once and samples of bone marrow were taken after 24 hours.

DETAILS OF SLIDE PREPARATION:
1) Preparation of the bone marrow
- The two femora were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 31°C (about 2 mI/femur).
- The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µI fresh FCS.
- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

2) Staining of the slides
The slides were stained in eosin and methylene blue solution for 5 minutes (Mayer Grünwald solution modified = Wrights solution), rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes.
After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS:
Microscopic evaluation
In general, 2000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
- Ratio of polychromatic to normochromatic erythrocytes
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter)

OTHER:
Clinical examinations
After the administration of the vehicle, the test substance and the positive controls, the animals were examined for any evident clinical signs of toxicity.

Evaluation criteria:

1) Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i.e. >= 2000 polychromatic erythrocytes.
- The proportion of cells with micronuclei in negative control animais was within the normal range of the historical control data.
- The two positive control chemicals induced a significant increase in the number of cells containing small and large micronuclei within the range of the historical control data.

2) Evaluation criteria
The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and significant increase in the number of micronucleated polychromatic erythrocytes was observed.
- The proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range.
A test substance is generally considered negative in this test system if:
- There was no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies.
- The frequencies of cells containing micronuclei were within the historical control range.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN.
The number of micronuclei in polychromatic erythrocytes was analyzed.
A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (* for p <= 0.05, ** for p <= 0.01) were printed with the group means in the tables. This test was performed one-sided.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Systemic toxicity:

The administration of the test substance led to signs of toxicity in the mid- and high-dose groups (800 mg/kg bw: squatting posture; 1600 mg/kg bw: squatting posture and poor general state).

Chromosome aberrations:

There was no statistically significant increase in the number of  polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was close to the range of the concurrent negative control in all dose groups and within the range of the historical control data. (The number of PCE's (%o) were: control 1.5; 400 mg/kg 1.6; 800 mg/kg  1.7; 1600 mg/kg 2.1; Cyclophosphamid 14.1; Vincristine 60.6; range of  historical control: 1.0-2.7, mean 1.7).
No inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected.
The test substance had no chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Control data:
Both of the positive control chemicals, i.e. cyclophosphamide for clastogenic effects and vincristine for induction of spindle poison effects, induced the expected significant  increases in the rate of polychromatic erythrocytes containing small or  large micronuclei.
The result for the negative control was within the historical control range.

See also attached document: table 1.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
AH salt does not have any impairment of chromosome damaging or chromosome distributing effect. The substance was not clastogen.
The substance has not to be classified as genotoxic according to EU GHS classification and according to annex VI-Directive 67/548/EEC.

Executive summary:

Report summary

The substance AH-Salz kristallisiert was tested for chromosomal damage (clastogenicity) and for the ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in purified water, was administered twice intraperitoneally, with a 24-hour interval between administrations, to male animals at dose levels of 400 mg/kg, 800 mg/kg and 1,600 mg/kg body weight in a volume of 10 ml/kg body weight in each case.

As a negative control, purified water was administered to male mice, by the same route, and gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.

Both of the positive control chemicals, i.e. cyclophosphamide for clastogenic effects and vincristine for induction of spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity.

The administration of the test substance led to signs of toxicity.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours after the second administration. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also registered.

According to the results of the present study, the two intraperitoneal administrations of AH-Salz kristallisiert did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was close to the range of the concurrent negative control in all dose groups and within the range of the historical control data.

No inhibition of erythropoiesis, determined from the ratio of polychromatic to normochromatic erythrocytes, was detected.

Thus, under the experimental conditions chosen here, the test substance AH-Salz kristallisiert does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.