Decamethylenediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce gene mutations in bacteria (OECD TG 471; RCC, 2008) or in mammalian cells (OECD TG 476; Harlan, 2009).

However, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro (OECD TG 473; RCC, 2008) either with or without metabolic activation.

Together with the negative in vivo micronucleus test (OECD TG 473; Charles River, 2009), the overall conclusion is that the test is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-07-29 to 2008-08-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study; GLP study without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci responsible for histidine auxotrophy

Species / strain / cell type:

E. coli WP2

Additional strain / cell type characteristics:

other: Tryptophan-independent

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9; male Wistar rats

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I and II:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Remarks:

for details see below

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate; 2-aminoanthracene

Details on test system and experimental conditions:

Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment / Experiment I: plate incorporation test (+/- metabolic activation)
Experiment II: pre-incubation assay (+/- metabolic activation)
- Metabolic activation assay: Phenobarbital/ß-Naphthoflavone induced Wistar rat liver S9 (protein concentration in S9: 30.7 mg/ml)
ADMINISTRATION
- Dosing: Pre-Experiment and Experiment I/II : 3 - 5000 µg/plate
- Number of replicates: 3
- Positive and negative control groups and treatment:
- without metabolic activation:
sodium azide for TA 1535 and TA 100
4-nitro-o-phenylene-diamine for TA 1537 and TA 98
methyl methane sulfonate for WP2 uvrA
- with metabolic activation:
2-aminoanthracene for all strains
- negative control: untreated; solvent control: Ethanol
- Pre-incubation time: 60 min at 37 °C; incubation time: 48 h at 37 °C in the dark

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:
mutagenic effects (i.e ratio of revertant rates treated/control >=2) at <= 5000µg/plate with generally positive dose-response relationship in any
strain

Statistics:

According to the OECD Guideline 471, a statistical analysis of the data is not mandatory

Species / strain:

other: S. typhimurium TA 98; TA 100; TA1535 and TA 1537; E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENTOXIC EFFECTS:
- With metabolic activation: None (even at cyctotoxic concentration)
- Without metabolic activation: None (even at cytotoxic concentration)
PRECIPITATION CONCENTRATION: > 5000 µg/plate
CYTOTOXIC CONCENTRATION: see remarks on results

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Strain Experiment I Experiment II

without S9 mix       with S9 mix without S9 mix       with S9 mix

TA 1535 2500 -5000       2500 -5000 2500 -5000              5000

TA 1537        2500 -5000 2500 -5000 2500 -5000 5000

TA 98 1000 -5000 2500 -5000                   2500 -5000 2500 -5000

TA 100               1000 -5000        2500 -5000               2500 -5000               5000

WP2 uvrA        2500 -5000 2500 -5000                            /                            /

/ = no reduced background growth observed

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups at the following concentrations (µg/plate):

Strain Experiment I                                           Experiment II

without S9 mix with S9 mix              without S9 mix              with S9 mix

TA 1535                     5000              2500 -5000                     /                                       /

TA 1537                     5000                      /                             5000                                   /

TA 98                     1000 -5000       2500 -5000              2500 -5000                         5000

TA 100                   1000 -5000       2500 -5000               2500 -5000                      5000

WP2 uvrA               2500 -5000       2500 -5000                      /                                         /

/ = No toxic effects observed

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not
induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, decamthylenediamine is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of decamethylenediamine to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. No data were evaluated for strains TA 1535 without S9 mix and TA 1537 with and without metabolic activation in experiment I, since the data of the controls did not met the acceptability criteria of the assay. These parts of experiment I were repeated under identical conditions and the results are reported as part of experiment I. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced background growth at higher concentrations in all strains.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 1, 10-Decanediamine at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, decamethylenediamine is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-09-01 to 2008-11-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Assay for the detection of forward gene mutations at the autosomal thymidine kinase (TK) locus of heterozygous L5178Y/TK +/- cells to
TK mutants -/-

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

tymidine kinase locus in mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/Naphthoflavone induced rat liver S9 mix, prepared from 8 - 12 weeks old male Wistar rats

Test concentrations with justification for top dose:

Experiment I +/-S9: 14.1, 28.1, 56.3, 112.5, 168.8, 225.0 µg/ml;
Experiment II + S9: 14.1, 28.1, 56.3, 112.5, 168.8, 225.0 µg/ml;
- S9: 0.88, 1.8, 3.5, 7.0, 14.1, 28.1 µg/ml

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

cyclophosphamide with S9-mix  

Migrated to IUCLID6: without S9-mix

Details on test system and experimental conditions:

Mouse lymphoma assay
Metabolic activation system: phenobarbital/naphthoflavone induced rat liver S9 mix, prepared from 8 - 12 weeks old male Wistar rats
ADMINISTRATION: 
- Dosing:   
Experiment I +/-S9: 14.1, 28.1, 56.3, 112.5, 168.8, 225.0 µg/ml;
Experiment II + S9: 14.1, 28.1, 56.3, 112.5, 168.8, 225.0 µg/ml;
- S9: 0.88, 1.8, 3.5, 7.0, 14.1, 28.1 µg/ml
solvent: Etanol
- Number of replicates: 2
- Positive and negative control groups and treatment:    
positive: ethylmethanesulfonate (-S9); cyclophosphamide (+S9)   
negative: solvent 
- Pre-incubation time: 
Experiment I: treatment periode 4 h with and without S9
Experiment II: treatment periode 24 h without S9, 4 h with S9

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS: A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of
126 colonies per 106 cells above the corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11
(SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability
value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

see Results

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Relevant toxic effects indicated by a relative total growth of less than 50 % were observed in the first experiment at
168.8 μg/mL with and without metabolic activation. In the second experiment cytotoxic effects as described above
were noted at 14.1 μg/mL without metabolic activation and at 112.5 and 168.5 μg/mL with metabolic activation. The
recommended toxic range of approximately 10-20% RTG was covered with and without metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the first
experiment except at the maximum concentration of 225 μg/ml with metabolic activation. At this test point the mutation
frequency exceeded the threshold of 126 above the corresponding solvent control in both parallel cultures. However,
only the increase in culture 2 was dose dependent as indicated by the statistical significance.

Therefore, the data generated in the presence of metabolic activation were judged as inconclusive and verified in the
second experiment with metabolic activation. No relevant increase of the mutation frequency occurred in the second
experiment with metabolic activation at comparable levels of cytotoxicity. Therefore, the increase observed in the first
experiment with metabolic activation was judged as biologically irrelevant toxicity artefact. Another increase of the
mutation frequency exceeding the threshold described above was noted in both cultures of the first experiment without
metabolic activation at an intermediate concentration of 56.3 μg/mL and in the second culture of the second experiment
with metabolic activation at 28.1 μg/mL. However, none of these isolated increases was dose dependent as indicated by
the lacking statistical significance and consequently, judged as irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant
frequencies using SYSTAT® statistics software. A significant dose dependent trend of the mutation frequency indicated
by a probability value of <0.05 was solely determined in the second culture of experiment I with metabolic activation.
The increase was not considered relevant as discussed above.

Remarks on result:

other: other: L5178Y TK+/-

Remarks:

Migrated from field 'Test system'.

Conclusions:

In conclusion, it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not
induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic
activation. Therefore, decamethylenediamine is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study was performed to investigate the potential of decamethylenediamine to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. The main experiments were evaluated at the following concentrations:

Experiment I: without S9 mix: 14.1; 28.1; 56.3; 112.5; and 168.5 μg/mL

with S9 mix: 28.1; 56.3; 112.5; 168.5; and 225.0 μg/mL

Experiment II: without S9 mix: 0.9; 1.8; 3.5; 7.0; and 14.1 μg/mL

with S9 mix: 14.1; 28.1; 56.3; 112.5; and 168.5 μg/mL

The highest concentration applied in the pre-experiment was 1800 μg/mL equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by the cytotoxic potential of the test item. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item.

MMS (19.5 μg/mL in experiment I and 13.0 μg/mL in experiment II) and CPA (3.0 μg/mL and 4.5 μg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies.

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, decamethylene is considered to be non-mutagenic in this mouse lymphoma assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-07-30 to 2008-08-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

cells have a stable karyotype with a modal chromosome number of 22
supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, 64287 Darmstadt, Germany

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and betanaphthoflavone induced Wistar rat liver (S9 homogenate)

Test concentrations with justification for top dose:

+/- S9 mix: 6.7, 13.5, 26.9, 53.8, 107.7, 215.4, 430.8, 861.5, 1723.0 µg/ml

Vehicle / solvent:

Ethanol, final concentration of ethanol in the culture medium was 0.5 % (v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

positive control with metabolic activation: Cyclophoshamide

Migrated to IUCLID6: without metabolic activation

Details on test system and experimental conditions:

SYSTEM OF TESTING
- Species/cell type: Chinese hamster lung fibroblasts (V79) cells,  obtained from Laboratory for Mutagenicity Testing, LMP, Technical University
Darmstadt, 64287 Darmstadt, Germany
- Metabolic activation system:   S9 homogenate prepared from young male Wistar rat livers, co-induced  with phenobarbital and  betanaphthoflavone. 
- No. of metaphases analyzed:    100 / culture except 50 for positive controls with chromosomal  aberration rates > 50 % (excl. gaps)
ADMINISTRATION: 
- Dosing:    +/- S9 mix: 6.7, 13.5, 26.9, 53.8, 107.7, 215.4, 430.8, 861.5, 1723.0 µg/ml
- Pre-experiment :conditions were identical to those for the mutagenicity assay. Since the cultures could be used for cytogenetic evaluation, this
preliminary test was designated Experiment I.
- Number of replicates: 2
- Application:    Approx. 5x10E5 cells each flask   
Exposure periode: 4 hours, 
Recovery: 14 hours
Preparation interval: 18 hours
- Positive and negative control groups and treatment:    
negative/solvent: Ethanol, final concentration of ethanol in the culture medium was 0.5 % (v/v)
positive -S9: 900 µg/mL ethylmethanesulphonate
positive +S9: 1.4 µg/mL cyclophosphamide/l
- Preparation of cultures
Colcmid was added 15.5 hours after start of treatment, cells on the slides were treated 2.5 hours later with hypotonic solution for 20 min at 37°C . After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per
experiment two slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, 64293 Darmstadt, Germany).

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:
(i) statistically significant increases in the incidence of cells bearing  aberrations at any dose-level over the concurrent control, AND
(ii) the increases must exceed the historical control values, AND
(iii) the increases are reproduced in both replicate cultures

Statistics:

Fisher's Exact Test

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the following doses used for scoring of aberrations revealed cytotoxicity: 40 mg/l (cell viability reduced to 26% of the control); 20 mg/l (viability reduced to 55% of the control); other doeses: no relevant cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS: 
- the test item 1,10-Decanediamine induced structural chromosome aberrations in V79 cells in the absence and presence of S9 mix.
- No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to controls.
OTHER OBSERVATIONS: 
- No precipitation of the test item in culture medium was observed.
- No relevant influence of the test item of the osmolarity was observed , the pH was adjusted to physiological values using small amounts of 2 M HCl at the concentration of 215.4, 430.8, 861.5, and 1723 μg/mL in the absence and presence of S9 mix (pH 7.5 to 9.1).
- Toxic effects indicated by reduced cell numbers were observed after 4 hours treatment with 430.8 μg/mL (43.9 % of control) in the absence of
S9 mix and with 861.5 μg/mL (32.0 % of control) in the presence of S9 mix.

Remarks on result:

other: other: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

see attached document

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as
determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, decamethylenediamine is considered to be clastogenic in this chromosome aberration test in the absence and presence of S9 mix.

Executive summary:

This in vitro test was performed to assess the potential of decamethylenediamine to induce structural chromosome aberrations. Evaluation of cytogenetic damage induced in V79 cells (cell line from the lung of the Chinese Hamster) in the absence and the presence of metabolic activation was performed in one experiment at 18 hours preparation interval.

The test item, dissolved in ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in one experiment. The following study design was performed:

Preparation Exposure                                                         Concentration

interval period                                                                  in μg/m

18 hrs 4 hrs              Without S9 mix 6.7 13.5 26.9 53.8 107.7 215.4 430.8 861.5 1723.0

18 hrs 4 hrs               With S9 mix 6.7 13.5 26.9 53.8 107.7 215.4 430.8 861.5 1723.0

In each experimental group two parallel cultures were set up. 100 metaphases per culture were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (1723 μg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data.

In the absence and presence of S9 mix clear cytotoxicity was observed at the highest evaluated concentrations. Clastogenicity was observed in the absence and presence of S9 mix. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, decamethylenediamine is considered to be clastogenic in this chromosome aberration test in the absence and presence of

S9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test item did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 800 mg/kg/day in male and female CD-1 mice (CR, 2009).

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

Study period:

2009-01-26 to 2009-03-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

equivalent or similar to guideline

Guideline:

other: US Environmental Protection Agency Gene-Tox Program and the Japanese Collaborative Study Group for Micronucleus Testing (Mavournin et al 1990; CSGMT/JEMS.MMS, 1990).

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Source: Charles River UK
- Age: approx. 6 - 7 weeks
- Weight at study initiation: female: 22.3 - 27.2 g, male: 27.6 - 33.7 g
- No. of animals: 33 males + 18 females
- Housing: in groups
- Diet: ad libitum, exept for ca. 2-4 h prior to dosing and 1 - 2 h after dosing
- Water: ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 23.3 °C
- Humidity (%): 34 - 62 %
- Air changes (per hr): at least 15 cycles per hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

3 % ethanol in water

Details on exposure:

ADMINISTRATION: 
- Vehicle: 3 % ethanol in water
- Control groups and treatment:    
negative: vehicle (2 oral gavage administration separated by 24 hours)   
positive: 50 mg cyclophosphamide (CPA)/kg bw (2x oral gavage administration separated by 24 hours in  distilled water)
- Total volume applied: 20 ml/kg bw/administration
- Duration of test: 48 hours

Duration of treatment / exposure:

2 doses separated by 24 hours

Frequency of treatment:

2 times for vehicle and test item
2 times for positive control

Post exposure period:

24 hours

Remarks:

Doses / Concentrations:
200 / 400 / 800 mg/kg bw/day
Basis:
other: 20 ml 3% ethanol in water/kg bw

No. of animals per sex per dose:

vehicle: 5 m + 5 f
200 + 400 mg, positive control: 5 m
800 mg: 10 m + 10 f

Control animals:

yes, concurrent vehicle

Positive control(s):

50 mg cyclophosphamide (CPA)/kg bw  (2x oral gavage administration separated by 24 hours in  distilled water)

Tissues and cell types examined:

femora, bone marrow

Details of tissue and slide preparation:

EXAMINATIONS: 
- Criteria for selection of M.T.D.: In the toxicity study, 5 groups of male and female CD-1 mice received oral doses of 1, 10-decanediamine ranging
from 2000 to 800 mg/kg/day. The highest dose was defined as the maximum routine in vivo exposure level normally administered.
Based on these toxicity investigations, the maximum tolerated dose of was judged to be in the region of 800 mg/kg/day.
- Organs examined at necropsy: femur bone marrow   2000 PCE (polychromatic erythrocytes) per animal were analysed for  micronuclei   
1000 erythrocytes were scored for PCE/NCE ratio

Evaluation criteria:

- Criteria for evaluating results: Statistically significant (p<=0.05)  and biologically relevant increase in frequency of micronucleated  polychromatic 
erythrocytes of at least one test group as compared to the  negative control group

Statistics:

Mann-Whitney test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Main study
- Clinical observations: 2 animal deaths occurred in the 800 mg/kg group. Clinical signs of subdued behaviour, hunched and piloerection were
also seen at this dose level. No deaths or clinical signs occurred in any of the other groups
- Sampling times and number of samples: 48 hours after last treatment, one femur of each mouse
-Vehicle Control and Untreated Groups
The numbers of micronucleated bone marrow polychromatic erythrocytes (MN-PCE) in mice dosed with the vehicle, 20 mL 3% ethanol in water/kg,
averaged 0.05%. The MN-PCE frequency in untreated mice averaged 0.04%. These MN-PCE frequency conformed to the established in-house control range for vehicle treated mice of the CD-1 strain (=0.00-0.23% per 5 mice or 0.00-0.18% per 10 mice).
- Positive Control Group
Exposure of mice to the positive control agent, 50 mg cyclophosphamide/kg, induced large increases in bone marrow micronuclei. The mean
MN-PCE frequency for the mice was 1.29%. An evident increase in the number of MN-NCE was also observed. Bone marrow toxicity accompanied
these findings as shown by a suppression of the PCE/NCE ratios.
- Test Item Group
There was no indication that 1, 10-decanediamine induced bone marrow micronuclei in the treated mice. The highest MN-PCE frequency recorded
for the test item was in the low dose males, where an incidence of 0.05% was observed. There was no indication of bone marrow toxicity in any of
the test item dose groups.

see attached table

Conclusions:

The test item did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 800 mg/kg/day in male and
female CD-1 mice using a 0 h + 24 h oral dosing and 48 h sampling regimen.

Executive summary:

The in vivo genotoxic potential of decamethylenediamine was evaluated in a micronucleus test in bone marrow erythrocytes of young, male and female CD-1 mice following a 0 h + 24 h oral dosing and 48 h sampling regimen.

A toxicity study was undertaken to establish a suitable dose range for the micronucleus experiment. Based on the findings of the toxicity study, the maximum recommended dose of decamethylenediamine /kg/day was judged to be 800 mg/kg.

In the micronucleus test, a group of CD-1 mice were therefore dosed at 0 h and 24 h via the oral route with the test item at a concentration of 800 mg/kg/day, 400 mg/kg/day, and 200 mg/kg/day. Bone marrow samples were taken 48 h after the initial 0 h dose. Two control groups of CD-1 mice were dosed orally with either the vehicle; 20 mL 3% ethanol in water/kg/day, or the positive control agent, 50 mg cyclophosphamide/kg/day. The experimental schedule for the control groups followed that of the test item treated mice.

No micronucleus induction was detected in bone marrow erythrocytes of mice dosed with decamethylenediamine.

Animals treated with the vehicle alone showed normal background levels of micronuclei, while animals dosed with cyclophosphamide responded with substantial increases in the numbers of bone marrow micronuclei.

It was concluded that decamethylenediamine did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 800 mg/kg/day in male and female CD-1 mice using a 0 h + 24 h oral dosing and 48 h sampling regimen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Decamethylenediamine did not induce gene mutations in bacteria (OECD TG 471; RCC, 2008) or in mammalian cells (OECD TG 476; Harlan, 2009).

However, the test item induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro (OECD TG 473; RCC, 2008) either with or without metabolic activation.

Together with the negative in vivo micronucleus test (OECD TG 473; Charles River, 2009), the overall conclusion is that decamethylenediamine is not genotoxic.

Justification for selection of genetic toxicity endpoint
Although in one in vitro chromosome aberration test a positive result was observed (RCC, 2008), two other in vitro genotoxicity studies revealed clearly negative results. Together with the negative in vivo micronucleus test (Charles River, 2008), the overall conclusion is that decamethylendiamine is not genotoxic.

Justification for classification or non-classification

Two of three in vitro genotoxicity studies revealed clearly negative results. Together with the negative in vivo micronucleus test, the overall conclusion is that the test item is not genotoxic and therefore must not be classified according to the criteria of EC Regulation 1272/2008.