Decahydronaphthalene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two published and one unpublished microbial mutagenicity assay demonstrated the lack of mutagenic activity of decahydronaphthalene. In addition, published in vitro studies on mammalian mutagenicity assay (mouse lymphoma cells) and chromosomal aberration (Chinese Hamster lung cells) revealed no genotoxic activity of the test substance.

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:

1989-09-25 to 1989-10-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions: TA 102 or E.coli WP2 were not tested (not required by applied version of guideline).

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Cited as Directive 84/449/EEC, B.14

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci responsible for histidine auxotrophy

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

  Aroclor 1254 induced rat S9 liver, male Bor: WISW (SPF/Cpb) rats

Test concentrations with justification for top dose:

8; 40; 200; 1000; 5000 µg/plate

Vehicle / solvent:

solvent acetone (CAS No. 67-64-1)

Untreated negative controls:

yes

Remarks:

untreated 

Negative solvent / vehicle controls:

yes

Remarks:

solvent controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: Aminoacridine TA 1537

Details on test system and experimental conditions:

Ames test
SYSTEM OF TESTING
- Metabolic activation system:   Aroclor 1254 induced rat S9 liver, male Bor: WISW (SPF/Cpb) rats

ADMINISTRATION: 
- Dosing:    
main test: 8/40/200/1000/5000 µg/plate (+/- metabolic activation)   
pre-incubation test: 125/250/500/1000/2000 µg/plate (+/- metabolic  activation)
- Number of replicates: 3
- Application: 
solvent acetone (CAS No. 67-64-1)   main test 100 g/l, pre-incubation test 80 g/l
- Positive and negative control groups and treatment:    
positive, TA 98 and TA 1538: nitrofluorene   
positive, TA 100 and TA 1535: sodium azide   
positive, TA 1537: aminoacridine   
negative: untreated + solvent controls   
activity of metabolic system: aminoanthracene / TA 100
- Pre-incubation: 30 minutes at 30 °C   
incubation 96 hours at 37 °C

Evaluation criteria:

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

Statistics:

no data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: > 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100

GENOTOXIC EFFECTS: 
- With metabolic activation: None
- Without metabolic activation: Slight effects in TA 98 and TA 1537 in  the main test and slight effects in  TA 1537 in the pre-incubation test  were associated with very low revertant rates in solvent control and not  dose-related.

Thus they were not considered to be signs of mutagenicity:
  -------------------------------------------------------
  Revertant rates in pre-incubation test without metabolic activation:
  TA 1537, untreated: 8; solvent: 7; 125 µg: 7; 250 µg: 9; 500 µg: 7;  1000 µg: 15; 2000 µg: 15
  -------------------------------------------------------
  Revertant rates in main test without metabolic activation:
  TA 98, untreated: 11; solvent: 4; 8 µg: 4; 40 µg: 10; 200 µg: 4; 1000  µg: 5; 5000 µg: 15, precipitation
  TA 1537, untreated: 7; solvent: 4; 8 µg: 7; 40 µg: 4; 200 µg: 12; 1000  µg: 3; 5000 µg: 0, toxic and precipitation
  -------------------------------------------------------
  The positive controls were functional.
PRECIPITATION CONCENTRATION: 5000 µg/plate
CYTOTOXIC CONCENTRATION: 
  TA 98: None (+/- S9)
  TA 100: None (+/- S9)
  TA 1535: 5000 µg/plate (+/- S9)
  TA 1537: 5000 µg/plate (- S9)
  TA 1538: None (+/- S9)

Conclusions:

Decahydronaphthalene was not genotoxic in both the presence or absence of metabolic activation in a microbial mutagenicity assay.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 and TA1538 of Salmonella typhimurium were exposed to decahydronaphthalene using acetone as a vehicle at concentrations of 8 - 5000 µg/plate, with and without S-9 activation in a preincubation assay according to EEC guidance Dir. 84/449/EEC, B.14.

The test article was tested at six dose levels along with appropriate vehicle and positive controls on the tester strains mentioned above in the presence and absence of S9-mix. All dose levels, vehicle and positive controls were plated in triplicate.

Cytotoxicity was noted at 1000 -5000 µg/plate.

No positive mutagenic responses were observed with any of the strains used, in the presence as well as in the absence of microsomal enzymes.These results were confirmed in an independent assay.

Reference 2

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:

1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: published, peer reviewed study, not guideline mentioned

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung cell (CHL/IU)

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix

Test concentrations with justification for top dose:

continious treatment (24/48 h) without S9: 0.15, 0.30, 0.60 , 1.20 mg/mL
Pulse treatment (6h) with S9: 0.63, 1.25, 2.50, 5.00 mg/mL

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Mitomycin (without S9), Cyclophosphamide (with S9)

Details on test system and experimental conditions:

no data

Statistics:

no data

Species / strain:

other: Chinese hamster lung cells (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5.0 mg/L without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Chromosomal aberration test without metabolic activation (continuous treatment 24/48h)

	Concentration (mg/mL)	Treatment time
		24 h		48h
		Percentage of cells showing structural aberrations
		-gaps	+gaps	-gaps	+gaps
Solvent (Ethanol)	10 µl/mL	4.0	4.5	2.5	2.5
Decahydronaphthalene	0.15	0.5	0.5	0	0
	0.30	2.0	2.0	2.0	2.5
	0.60	2.4	2.4	2.0	2.0
	1.20	1.0	1.5	1.0	1.0
Positive control Mitomycin	0.02 (µg/mL)	28.5	29.5	37.5	38.0

 

Chromosomal aberration test without and with metabolic activation (6h treatment)

	Concentration (mg/mL)	Metabolic activation
		Without S9mix		With S9mix
		Percentage of cells showing structural aberrations
		-gaps	+gaps	-gaps	+gaps
Solvent (1% CMC)	0.1 µl/mL	2.0	2.0	1.5	1.5
Decahydronaphthalene	0.63	2.5	2.5	2.5	2.5
	1.25	3.0	3.5	1.5	1.0
	2.5	1.0	1.0	1.0	1.0
	5.0	Toxic		1.0	1.0
Positive control Cyclophosphamide	10.0 (µg/mL)	3.5	5.0	25.0	25.5

 

Conclusions:

negative;

No increase in chromosomal aberrations was observed both with and without metabolic activation.

Executive summary:

A chromosome aberration test was conducted with Chinese hamster CHL/IU cells to investigate whether decahydronaphthalene induces structural or numerical aberrations. The test was conducted with or without metabolic activation system (rat S9-mix). Results indicated that decalin induces no chromosomal aberrations, both structurally and numerically under the present experimental conditions

Reference 3

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:

1991-04-22 to 1991-08-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus in mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 mix

Test concentrations with justification for top dose:

50-450 mg/l (+ S9); 5-45 mg/l + 9-100 mg/l (- S9)

Vehicle / solvent:

Application: solvent ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

3-methylcholanthrene with S9

Migrated to IUCLID6: without S9

Details on test system and experimental conditions:

Mouse lymphoma assay
- Metabolic activation system: Aroclor 1254-induced rat liver S9 mix
ADMINISTRATION: 
- Dosing:    Initial toxicity test (1 replicate):   0.5; 1; 5; 10; 50; 100; 500; 1000; 5000 mg/l (+/- S9)   Main studies, selected for cloning:   250; 300; 350;  400; 450 mg/l (+ S9)   25; 30; 35; 40; 45 mg/l (- S9)   9; 22; 35; 48; 61 mg/l (- S9)
- Number of replicates: 2
- Application: solvent ethanol
- Positive and negative control groups and treatment:    
positive (+ S9): 3-methylcholanthrene, solvent DMSO   
positive (- S9): ethylmethanesulfonate,  solvent DMSO   
negative (+/- S9): 2 untreated + 4 solvent replicates
- Pre-incubation time: two-day expression period
DESCRIPTION OF FOLLOW UP REPEAT STUDY: see second concentration series -  S9

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS: dose-dependent increase in mutant  frequency

Statistics:

no data

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: >= 300 mg/l (+ S9); >= 48 mg/l (- S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS: 
- With metabolic activation: none
- Without metabolic activation: none  
 The positive controls were all as expected.
CYTOTOXIC CONCENTRATION: 
- With metabolic activation:    Initial toxicity test: >= 500 mg/l   Main test: >= 300 mg/l
- Without metabolic activation:   Initial toxicity test: >= 50 mg/l   
Main test: No significant cytotoxicity   
Follow-up: >= 48 mg/l

Remarks on result:

other: L5178Y TK+/-

Conclusions:

negative; Decahydronaphthalene was not genotoxic.

Executive summary:

No induction of micronuclei in peripheral blood NCEs was observed in female mice exposed to 25 to 400 ppm decalin for 3 months by inhalation. In contrast to the negative results in females, a small but statistically significant increase in the frequency of micronucleated NCEs was noted in peripheral blood of male mice. A slight increase over the exposure range in the percentage of PCEs was seen in males and females; all values, however, were within the normal range.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A mouse micronucleus assay following 3 months inhalation showed no clastogenic activity in female but a slight clastogenic activity in male mice was noted. No clastogenic effect was noted in dogs following 3 months inhalation.

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:

1996-09-16 to 1996-12-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to Guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

subchronic exposure

Principles of method if other than guideline:

NTP mouse peripheral blood micronucleus test protocol
MacGregor, J. T., Wehr, C. M., Henika, P. R., Shelby, M. D.(1990) Fund. Appl. Toxicol. 14, 513.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Source: Taconic Laboratory Animals and Services, Germantown (NY, USA)
- Age: 6 weeks at study initiation
- Weight at study initiation: males mean 21.9 g, females mean 19.6 g
- Number of animals: 10 males + 10 females per exposure concentration

Route of administration:

inhalation: vapour

Vehicle:

Vehicle: clean air

Details on exposure:

ADMINISTRATION:   
- Concentration in vehicle: established within 12 minutes, monitored  every 24 minutes   At 72 +- 3 °F (22.2 °C) the ppm concentrations 
correspond to: 143, 285,  570, 1141, 2282 mg/m3
- Frequency of treatment: 6 hours (+ 12 minutes concentration  buildup)/day, 5 days/week
- Control groups and treatment: concurrent vehicle
EXAMINATIONS: 
CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs: observed twice daily
- Mortality: observed twice daily
- Clinical findings: weekly
- Body weight: initially + weekly + end of study
- Hematology: Blood sampling at end of study. Determination of  hematocrit; packed red cell volume; hemoglobin; erthrocyte, reticulocyte,  nucleated  erythrocyte, and platelet counts; mean cell volume; mean cell  hemoglobin; mean cell hemoglobin concentration; and leukocyte count and  
differentials.
- Biochemistry: None
- Urinalysis: Sampling during week 12. Determination of creatinine,  glucose, glucose/creatinine ratio, protein, protein/creatinine ratio,  alkaline
phosphatase, alkaline phosphatase/creatinine ratio, alkaline  aminotransferase, aspartate aminotransferase, aspartate  aminotransferase/creatinine  ratio, lactate dehydrogenase, lactate  dehydrogenase/creatinine ratio, gamma-glutamyltransferase,  gamma-glutamyltransferase/creatinine ratio,   N-acetyl-beta-D-glucosaminidase, N-acetyl-beta-  D-glucosaminidase/creatinine ratio, volume, and specific gravity.
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Weights: heart, right kidney, liver, lung, right testis, thymus
- Macroscopic: yes, no details reported
- Microscopic: 0 and 400 ppm animals: gross lesions, adrenal gland, bone  with marrow, brain, clitoral gland, esophagus, gall bladder, heart, large  
intestine (cecum, colon, rectum), small intestine (duodenum, jejunum,  ileum), kidney, larynx, liver, lung, lymph nodes (mandibular, mesenteric,  
bronchial, and mediastinal), mammary gland (females only), nose, ovary,  pancreas, parathyroid gland, pituitary gland, preputial gland, prostate  
gland, salivary gland, skin, spleen, stomach (forestomach and glandular),  testis with epididymis and seminal vesicle, thymus, thyroid gland,  
trachea, urinary bladder, and uterus.
- Sampling times and number of samples: end of 3-month toxicity study,  peripheral blood samples   2000 NCEs (normochromatic erythrocytes) 
per animal were analysed for  micronuclei in up to 10 animals per exposure group.   1000 erythrocytes were counted to determine the percentage 
of  polychromatic (immature) erythrocytes (PCEs) among the total erythrocyte  population.
- Criteria for evaluating results:    
(1) one-tailed Cochran-Armitage trend test, followed by pairwise  comparisons between each exposed group and the control, for determination  
of concentration-response relationship   
(2) adjustment in case of excess binomial variation (binomial  dispersion test)   
(3a) P <= 0.025 in trend test OR   
(3b) single group with P <= 0.025/number of exposed groups   
(4) expert judgment by scientific staff
- Criteria for selection of M.T.D.: Low mortality and low impairment of  body weight development in preceding two-week study

Duration of treatment / exposure:

14 weeks; 1 additional day before end of study

Frequency of treatment:

6 hours (+ 12 minutes concentration  buildup)/day, 5 days/week

Post exposure period:

Post-exposure period: none

Remarks:

Doses / Concentrations:
25, 50, 100, 200, 400 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Evaluation criteria:

In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or
if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups.
A final call of positive for micronucleus induction is preferably based on reproducibly positive trials.

Statistics:

The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tailed Cochran- Armitage trend test, followed by pairwise comparisons between each exposed group and the chamber control group
(ILS, 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

MORTALITY: All mice survived to the end of the study.
CLINICAL SIGNS: There were no clinical findings related to exposure. 
NECROPSY FINDINGS: see subchronic toxicity study
BODY WEIGHT CHANGES: see subchronic toxicity study
GENOTOXIC EFFECTS: 
- males: small but statistically significant (P=0.001) increase in  frequency of micronucleated NCEs with exposure concentration
- female: no induction of micronuclei (P=0.917)
- both: slight increase over exposure range in percentage of PCEs,  however, all values were within the normal range
NOAEL (NOEL) (C) / LOAEL (LOEL) (C): see subchronic toxicity study

EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO: 
--------------------------------------------------------
Treatment  Mice scored  Sex  % Micron. in PCE   PCEs (%)
--------------------------------------------------------
    0 ppm          10       m       5.5 +/- 1.6       1.8
   25 ppm        10       m       4.5 +/- 1.6       1.8
   50 ppm         10       m     10.0 +/- 1.5       2.0
  100 ppm       10       m     10.0 +/- 2.2       1.9
  200 ppm       10       m     12.5 +/- 2.0       1.9
  400 ppm         9       m     13.3 +/- 2.0       2.6
--------------------------------------------------------
    0 ppm          9       f      6.7 +/- 2.0       1.6
   25 ppm         10       f      5.0 +/- 1.1       1.8
   50 ppm        10       f      7.0 +/- 1.9       1.6
  100 ppm       10       f      3.5 +/- 1.3       1.5
  200 ppm       10       f      3.0 +/- 0.8       2.0
  400 ppm       10       f      4.0 +/- 1.2       2.0
--------------------------------------------------------
GENOTOXIC EFFECTS: 
- males: small but statistically significant (P=0.001) increase in  frequency of micronucleated NCEs with exposure concentration
- female: no induction of micronuclei (P=0.917)
- both: slight increase over exposure range in percentage of PCEs,  however, all values were within the normal range
NOAEL (NOEL) (C) / LOAEL (LOEL) (C): see subchronic toxicity study

Conclusions:

No effects were noted in females, a slight statistically significant increase in micronucleated normochromatic erythrocytes was observed in males but the values were within the normal range.

Executive summary:

As part of the repeated dose study, following inhalation exposure to decahydronaphthalene for 5 days per week, 4 hours per day for a period of 3 months blood smears from 10 male and ten female mice were obtained. Dose levels were 0, 25, 50, 100, 200, 400. A slight but statistically significant increase in micronucleated normochromatic erythrocytes was observed in males ppm. However the values were within the normal range. No effects were noted in female mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

No genotoxic activity was noted in an array of in vitro tests. In vivo following three months inhalation exposure no clastogenicity was noted in male and female dogs or female mice while a statistically significant effect was noted in male mice. However, since the observed effect was very small, and no increased incidence of neoplastic effects were noted in male mice in the NTP 2-year study this is considered to be toxicologically irrelevant.

 

Justification for classification or non-classification

The in vitro genotoxicity studies with decahydronaphthalene revealed clearly negative results. The data base on investigations of clastogenic effects indicates that the test substance has only a weak genotoxic potential in vivo in male mice and therefore must not be classified according to CLP Regulation 1272/2008.