Cyclododeca-1,5,9-triene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames

1996: Comparable to OECD 471; GLP; 10, 50, 100, 500, 1000, 2500, and 5000 µg/plate; TA 1535, TA 97a, TA 98, TA 100, and E. coli WP2 uvrA; no evidence for mutagenicity (K2)

Chromosome aberration

1997: Comparable to OECD 471; GLP; 0.25-5.0 mg/ml; human lymphocytes; no evidence for clastogenicity (K1)

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

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)

Principles of method if other than guideline:

Method: see reference

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium TA97a, TA98, TA100, TA1535; E. coli strain WP2 uvrA (pKM101)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

0; 10; 50; 100; 500; 1000; 2500; 5000 ug/plate

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Remarks:

solvent (vehicle) DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (see "Negative control")

True negative controls:

no

Positive controls:

yes

Remarks:

2-aminoanthracene, 2-nitrofluorene, sodium azide, ICR 191  acridine, methyl methanesulfonate; solvent DMSO except last three  positive controls: deionized water

Positive control substance:

other: 2-aminoanthracene, 2-nitrofluorene, sodium azide, ICR 191  acridine, methyl methanesulfonate; solvent DMSO except last three  positive controls: deionized water

Details on test system and experimental conditions:

Ames test

SYSTEM OF TESTING
- Metabolic activation system:   arochlor induced rat liver S9 mix ADMINISTRATION: 
- Number of replicates: 3
- Application: solvent dimethyl sulfoxide
- Positive and negative control groups and treatment:    positive: 2-aminoanthracene, 2-nitrofluorene, sodium azide, ICR 191  acridine, methyl methanesulfonate; solvent DMSO except last three  positive controls: deionized water; negative: DMSO (solvent)

-further details: see reference

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS: Positive if the average number of  revertants in any strain at any test substance concentration studied was  at least 2 times greater than the average of the negative control, and  there was a positive dose-response relationship in that same strain.

Statistics:

no data

Species / strain:

other: Salmonella typhimurium TA97a, TA98, TA100, TA1535; E. coli strain WP2 uvrA (pKM101)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not reported

Additional information on results:

see reference

Conclusions:

The test substance proved to be non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Principles of method if other than guideline:

details: see reference

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

other: Human Lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

exogenous metabolic S9 activation

Test concentrations with justification for top dose:

0.25 - 5.0 mg/ml

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Remarks:

acetone

Negative solvent / vehicle controls:

yes

Remarks:

=see negative control (acetone)

True negative controls:

no

Positive controls:

yes

Remarks:

positive control without activation: mitomycin C (in water);   positive control with activation: cyclophosphamide (in water)

Positive control substance:

other: positive control without activation: mitomycin C (in water);   positive control with activation: cyclophosphamide (in water)

Details on test system and experimental conditions:

SYSTEM OF TESTING:
- Metabolic activation system: arochlor-induced rat liver S9 fraction
- No. of metaphases analyzed: 50 per dose and replicate where possible

ADMINISTRATION: 
- Dosing:    
Trial 1: 0;  0.25; 0.5;  0.75; 1.0; 2.5 mg/ml   
Trial 2: 0;  0.5;  0.75; 1.0,  2.5; 3.5; 5.0 mg/ml  
Supplemental harvest 24 hours later from trial 2: 1.0; 2.5; 3.5 mg/ml
- Number of replicates: 2
- Positive and negative control groups and treatment: positive, without activation: mitomycin C (in water)   positive, with activation: cyclophosphamide (in water)  
- negative control: solvent (acetone)
-further details: see reference

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS: The test substance was classified as  clastogenic (positive) if the test substance produced a statistically  significant increase in percent abnormal cells as compared to the  negative control at one or more test concentrations and there was a  statistically significant dose-related increase in percent of abnormal  cells.

Species / strain:

other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Moderate cytotoxicity observed as measured by the decrease of MI in concentrations of >= 2.5 mg/ml

Remarks on result:

other: other: Human Lymphocytes

Remarks:

Migrated from field 'Test system'.

MITOTIC INDEX:  - Trial 1, 2.5 mg/l: reduced to 43% (non-activated) and 41% (activated)  of the negative control - Trial 2, 5.0 mg/l: reduced to 30% (non-activated) and 48% (activated)  of the negative control

CYTOTOXIC CONCENTRATION:  - With metabolic activation: At 1 mg/l, average generation time (AGT) 

was  increased 18-19 hours with activation. 

Significant increases in AGT were  not observed in the non-activated system. - Without metabolic activation: At 1 mg/l, the mitotic index was reduced  to 38% of the negative control without 

metabolic activation, and 52% of  the negative control with activation

Conclusions:

Under the conditions of the study the substance 1,5,9-cyclododecatriene was not clastogenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

1996: Comparable to OECD 474; GLP; 500 ppm; at least 5 male Sprague Dawley; no induction of chromosomal aberration; Non-clastogenic, non-aneugenic (K1)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Method: see references

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

other: Crl:CD (SD) BR

Sex:

male

Details on test animals or test system and environmental conditions:

- Age: 51 days
- Weight at study initiation: mean 231.4 g

Route of administration:

inhalation

Vehicle:

air

Details on exposure:

ADMINISTRATION: nose-only
- vapour atmosphere generated by metering the liquid test substance into a nebulizer
Sampling times and number of samples:   
- Test atmosphere: one sample during each exposure to determine  concentration and particle size distribution; result: 3200 +/- 280 mg/m3;  mass median aerodynamic diameter 3.6 µm   

-atmospheric concentration measured by gas chromatography and gravimetric analysis

-further details: see references

Duration of treatment / exposure:

6 hours each on 2 consecutive days

Frequency of treatment:

once per day on 2 consecutive days

Dose / conc.:

500 ppm (nominal)

Remarks:

3300 mg/m3 (aerosol/vapor); 3200 +/- 280 mg/m3 (analytical concentration)

No. of animals per sex per dose:

No. of animals per dose: 10 exposed, 5 negative control

Control animals:

yes

Positive control(s):

Control groups and treatment:
- positive: 40 mg cyclophosphamide/kg by oral intubation
-negative: concurrent houseline air   

Tissues and cell types examined:

Bone marrow: At least 3 slides per rat (fixed, and stained with acridine orange), 2000 PCEs per rat scored for  micronuclei, 1000 erythrocytes scored for PCE/NCE ratio ; Representative slides from each rat were examined blindly.

further EXAMINATIONS: 
- Clinical observations: during exposure (including alerting stimulus)
- Organs examined at necropsy: marrow  from 1 femur
- Body weights: yes

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

no mortalities

Additional information on results:

MORTALITY: no test substance-induced mortality

CLINICAL SIGNS:   
- During exposure: no clinical signs, response to alerting stimulus  diminished or absent   
- After exposure: lethargy and/or irregular respiration. 

BODY WEIGHT CHANGES: losses in treated and positive control rats

EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO:    No statistically significant depressions in the proportion of PCEs  among 1000 erythrocytes were observed.

GENOTOXIC EFFECTS:    There were no statistically significant increases in the MNPCE  frequency in rats exposed to the test substance. A statistically  significant increase in the MNPCE frequency was found in the positive  control rats.

Conclusions:

Under the conditions tested, the test substance did not induce chromosomal aberration in experimental animals and thus, was suggested to be non-clastogenic and non-aneugenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

IN VITRO

Bacterial reverse mutation assay

Two Ames test investigating the genotoxic potential of the test substance are available.

An Ames test using the method by Ames BN et al. (1975) was performed using strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 1538. Strains were treated both with and without metabolic activation (Aroclor induced rat S9 liver homogenate) to 10 – 5000 µg/plate of the test substance. The vehicle was DMSO. Appropriate negative and positive controls were included. Mutagenic effects were evaluated if ratio of revertant rates treated/control was >= 2 at <= 5000 µg/plate with generally positive dose-response relationship in any strain. No evidence for a mutagenic potential was found in either strain of S. typhimurium neither with nor without metabolic activation. Cytotoxicity was not reported. Thus, the test substance was suggested to be non-mutagenic under the conditions chosen (1988, K2).

An Ames test was performed comparable to OECD 471 and in compliance with GLP using strains of Salmonella typhimurium TA 1535, TA 97a, TA 98, TA 100, and E. coli WP2 uvrA (pKM101). Strains were treated both with and without metabolic activation (Aroclor induced rat S9 liver) to 10, 50, 100, 500, 1000, 2500, and 5000 µg/plate of the test substance. The vehicle was DMSO. Appropriate negative and positive controls were included. Mutagenic effects were evaluated if ratio of revertant rates treated/control was >= 2 at <= 5000 µg/plate with generally positive dose-response relationship in any strain. No evidence for a mutagenic potential was found in either strain of S. typhimurium or E. coli neither with nor without metabolic activation. Cytotoxicity was not reported. Thus, the test substance was suggested to be non-mutagenic under the conditions chosen (1996, K2).

In addition, a study which was only available in the original language (Japanese) was available. Strains used were S. typhimurium TA 100, TA 1535, TA 98, and TA 1537 as well as E. coli WP2 uvrA. Strains were treated with concentrations from 0.15 to 5000 µg/plate. In all strains, no evidence of a genotoxic potential was found (2008, K4).

Chromosome aberration

A chromosome aberration test was performed comparable to OECD 473 and in compliance with GLP using human lymphocytes. Cells were treated both with and without metabolic activation to 0.25-5.0 mg/ml of the test substance. The vehicle was acetone. Appropriate negative and positive controls were included. The test substance was classified as clastogenic (positive) if the test substance produced a statistically significant increase in percent abnormal cells as compared to the negative control at one or more test concentrations and there was a statistically significant dose-related increase in percent of abnormal cells. No evidence for a mutagenic potential was found in human lymphocytes neither with nor without metabolic activation. Moderate cytotoxicity observed as measured by the decrease of mitotic index in concentrations of >= 2.5 mg/ml. In trial 1, mitotic index was reduced to 43% (without S9) and 41% (with S9) in comparison to negative control at 2.5 mg/l. In trial 2, mitotic index was reduced to 30% (without S9) and 48% (with S9) in comparison to negative control at 5.0 mg/l. Thus, the test substance was suggested to be non-clastogenic under the conditions chosen (1997, K1).

In addition, a study only available in the original language (Japanese) was available. Chinese hamster lung cells (CHO/IU) were treated with concentrations ranging from 13.3 to 1700 µg/ml. Chromosome aberration was observed in short-term treatment with S9 mix at 27.1, 37.9, and 53.1 µg/ml. However, positive response was associated with high cytotoxicity and thus, was regarded as not substance related (2007, K4).

IN VIVO

Chromosome aberration

To investigate the genotoxic potential of the test substance, an in vivo micronucleus assay comparable to OECD 474 and in compliance with GLP was performed. Five and ten male Sprague Dawley rats in the control and treatment groups, respectively, were exposed via inhalation to 500 ppm of the test substance once per day for six hours on two consecutive days. Cyclophosphamide was included as positive control. At least three slides per rat for bone marrow were fixed and stained with acridine orange and 2000 PCEs per rat were scored for micronuclei and 1000 erythrocytes were scored for PCE/NCE ratio. Representative slides from each rat were examined blindly. Clinical observations were performed during exposure (including altering stimulus) and organs that were examined at necropsy were marrow from one femur. In addition, body weights were also examined. No animal died during the study. No clinical signs were observed during exposure and response to alerting stimulus were diminished or absent. After exposure, lethargy and/or irregular respiration were reported. Decreases in body weight were observed in treated and positive control animals. No statistically significant depressions in the proportion of PCEs among 1000 erythrocytes were observed. There were no statistically significant increases in the MNPCE frequency in rats exposed to the test substance. A statistically significant increase in the MNPCE frequency was found in the positive control rats. Under the conditions tested, the test substance did not induce chromosomal aberrations in experimental animals and thus, was suggested to be non-clastogenic and non-aneugenic (1996, K1).  

Justification for classification or non-classification

The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended for the second time in Directive EC 286/2011, classification as a mutagen is not warranted.