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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AH salt was not mutagenic in the Ames test in five Salmonella strains (TA 1535, TA 1537, TA 1538, TA 98, TA 100) both in the presence and in the absence of metabolic activation (liver S-9 mix; up to 2500 µg/plate tested).

AH salt did not induce unscheduled DNA synthesis in primary rat hepatocytes in vitro in a test performed according to current standards.

The components adipic acid and 1,6 -hexanediamine were negative in the HPRT test with and without metabolic activation.

AH salt is therefore considered to be non genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980-01-15 to 1980-01-17 (experiment 1); 1980-02-02 to 1980-02-04 (experiment 2)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): AH Salz (Hexamethylene diaminadipate)
- Analytical purity: ca. 100%
- Storage condition of test material: 4°C

Target gene:

his

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S-9

Test concentrations with justification for top dose:

4, 20, 100, 500, 2500 µg/plate

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrososguanidine, 4-nitro-ophenylenediamine, 9-aminoacridinium chloride monohydratrate

Remarks:

positive control substance depending on the strain and activation condition

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h (37°C)

NUMBER OF REPLICATIONS: 4 plates per dose and control

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: decreased his- background growth

OTHER:
For better evaluation, the test was repeated with tester strain TA 100 with metabolic activation, only.

Evaluation criteria:

In general, a test substance has to be judged as positive, if the following criteria are fulfilled:
- doubling of the spontaneous mutation rate (control),
- dose-response relationship,
- reproducibility of results.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

No mutagenic activity (increase in the number of his+ revertants) and no bacteriotoxicity (decreased his- background growth) was observed at any concentration with any tester strain both with and without metabolic activation. For details, see attached files.

Conclusions:

Interpretation of results:
negative

The substance, hexamethylenediamine adipate was not mutagenic in the Ames test, when assayed at concentrations of up to 2500 µg/plate in the presence and absence of metabolic activation and has not to be classified according to EU GHS classification and according to annex VI-Directive
67/548/EEC.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Principles of method if other than guideline:

This assay is based on the procedures described by Williams (1977, 1980).
- Williams GM (1977). Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell culture. Cancer Res 37: 1845-1851.
- Williams GM (1980). The detection of chemical mutagens-carcinogens by DNA repair and mutagenesis in liver cultures. In: Chemical Mutagens, Vol. 6. Edited by DeSerres F and Hollaender A. Plenum Press, NY, pp. 61-79.

GLP compliance:

not specified

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Specific details on test material used for the study:

- Name of test material (as cited in study report): Hexamethylenediamine adipate
- Physical state: white powder
- Date Received: 1981-10-27

Species / strain / cell type:

hepatocytes: rat

Details on mammalian cell type (if applicable):

- Type and identity of media:
The cell cultures are established in Williams' Medium E supplemented with 5% fetal bovine Serum, 2mM L-glutamine, 2.49 µM dexamethasone, 100 U/ml penicillin, 100 µg/ml Streptomycin sulfate, and 150 µg/ml gentamicin. After the establishment period, the dexamethasone and serum components are removed. This latter culture medium is referred to simply as WME.

Metabolic activation:

without

Test concentrations with justification for top dose:

5, 10, 25, 50, 100, 250, 500, 1000 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: medium (Williams' Medium E)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 h
- Exposure duration: 18-19 h
- Fixation time (start of exposure up to fixation or harvest of cells): 20-24 h after the initiation of the treatment

SELECTION AGENT (mutation assays): ³H-thymidine (1 µCi/ml)
STAIN (cytotoxicity): trypan blue
STAIN (DNA damage): hematoxylin, eosin

NUMBER OF REPLICATIONS: 5

NUMBER OF CELLS EVALUATED: 150 total cells

DETERMINATION OF CYTOTOXICITY
- Method: number of viable cells relative to vehicle control (2/5 replicates were used for determination of cytotoxicity)

Evaluation criteria:

The test material is considered active in the UDS assay at applied concentrations that cause:
1) An increase in the mean nuclear grain count to at least 6 grains/ nucleus in excess of the concurrent negative control value, and/or
2) The percent of nuclei with 6 or more grains to increase above 10% of the examined population, in excess of the concurrent negative control, and/or
3) The percent of nuclei with 20 or more grains to reach or exceed 2% of the examined population.

Species / strain:

hepatocytes: rat

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

The test substance (Hexamethylenediamine adipate), did not induce significant changes in the nuclear labeling of primary rat hepatocytes for an applied concentration range of 5 - 1000 µg/ml. The test substance was not toxic at any of the applied concentrations. None of the criteria used to indicate UDS were even approached and a dose-related response was not observed. Therefore, the test substance was evaluated as inactive in the Primary Rat Hepatocyte UDS Assay.

For details, see attached file.

Conclusions:

Interpretation of results:
negative without metabolic activation

The test substance, Hexamethylenediamine adipate was evaluated as inactive in the Primary Rat Hepatocyte UDS Assay and has not to be classified as mutagenic according to EU GHS classification and according to annex VI-Directive 67/548/EEC.

Executive summary:

The test substance (Hexamethylenediamine adipate) was evaluated as negative in the Primary Rat Hepatocyte UDS Assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from male Wistar rats

Test concentrations with justification for top dose:

0.1, 0.25, 0.5, 1, 2.5, 5, 7.5 and 10 mM without metabolic activation
0.5, 0.75, 1, 2,5, 4, 7, 8.5 and 10 mM with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Executive summary:

The test substance was investigated in an OECD TG 476 study in Chinese hamster V79 cells in the absence and in the presence of metabolic activator (S9) up to concentrations of 10 mM. No precipitation of the test item was noted in any experiment; no biological relevant growth inhibition was observed with and without metabolic activation. In both experiments no biologically relevant increase of mutants was found after treatment with the test item. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequencies.

In conclusion, the test substance is considered to be non-mutagenic in the HPRT locus using V79 cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

AH salt was neither clastogenic nor aneugenic in a mouse micronucleus test (OECD 474) after intraperitoneal administration.

AH salt is therefore considered to be non genotoxic.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(July 21, 1997)

Deviations:

no

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

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

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

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

Dose / conc.:

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

Remarks:

total dose: 800 mg/kg bw

Dose / conc.:

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

Remarks:

total dose: 1600 mg/kg bw

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

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.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

IN VITRO

AH salt was not mutagenic in the Ames test in five Salmonella strains (TA 1535, TA 1537, TA 1538, TA 98, TA 100) both in the presence and in the absence of metabolic activation (liver S-9 mix; up to 2500 µg/plate tested). The positive controls induced appropriate responses in the corresponding strains. The test did not include cytotoxic exposure levels (BASF AG 1980). AH salt did not induce unscheduled DNA synthesis in primary rat hepatocytes in vitro in a test performed according to current standards (BASF AG, 1982). The test substance was not toxic at any of the applied concentrations (5-1000 µg/ml). The positive control gave the expected results.

The components adipic acid and 1,6-hexanediamine were negative in the HPRT test with and without metabolic activation. Adipic acid was investigated in an OECD TG 476 study in Chinese hamster V79 cells in the absence and in the presence of metabolic activator (S9) up to concentrations of 10 mM. No precipitation of the test item was noted in any experiment; no biological relevant growth inhibition was observed with and without metabolic activation. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (Lanxess, 2009).

1,6-hexanediamine was evaluated in the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay at dose levels of 25, 50, 100, 175 and 250 µg/mL of treatment volume without metabolic activation preparation and at dose levels of 50, 100, 300, 450 and 600 µg/mL of treatment volume with a 5% concentration of metabolic activation preparation. No statistically significant increases of mutants in test substance treated cultures were observed when compared to the negative controls (Solutia, 1985).

IN VIVO

In a GLP micronucleus test performed via the intraperitoneal route (2 applications with a 24 h interval) in male mice (5 males/group) according to OECD TG 474, the administration of AH-salt (purity 99%) led to clinical signs of toxicity at 800 and 1600 mg/kg bw. No clinical signs were observed at 400 mg/kg bw. Because no differences in toxicity between the two sexes were found in a pre-test, only males were used in the main study. There was no increase in the number of polychromatic erythrocytes containing either small or large micronuclei. Therefore the test substance had no 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 (BASF AG, 2001).

Justification for classification or non-classification

There is no need to classify AH-Salt for mutagenicity according to the Directive 67/548/EC or GHS criteria (Regulation (EC) N° 1907/2006).