Decahydro-2-naphthyl acetate

Genetic toxicity: in vitro

Administrative data

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:

17 November 2000 - 15 January 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Column 1 of REACH Annex VII informs on the standard mutagenicity information requirements, for substances produced or imported in quantities >1 tpa. The Bacterial Reverse Mutation Test (OECD 471, EU B.13/14) detects gain of function point mutations and frameshifts in vitro, and is required to fulfil Annex VII information requirements.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Decahydro-2-naphthyl acetate

EC Number:

234-054-3

EC Name:

Decahydro-2-naphthyl acetate

Cas Number:

10519-11-6

Molecular formula:

C12H20O2

IUPAC Name:

decahydronaphthalen-2-yl acetate

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system is a microbial assay which measures mutation in the histidine operon. Gain of function his- to his+ mutation is induced by chemicals which cause base changes or frameshift mutations in the genome of this organism. S. typhimurium strains TA98 and TA1537 primarily respond to frameshift mutations at the histidine gene locus his D 3052 and his C 3076, respectively. Strains TA100, TA102 and TA1535 respond to base-pair substitutions in the his G 46, his G 428 and his G 46 locus.

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: Strain TA1537 contains the hisC3076 frameshift mutation, uvrB- mutation and rfa mutation. Strain TA98 contains the hisD3052 frameshift mutation, uvrB- mutation, rfa mutation and a resistance factor on the pKM101 plasmid. Strain TA1535 contains the hisG46 base pair mutation, uvrB- mutation and rfa mutation. Strain TA100 contains the hisG46 substitution mutation, uvrB- mutation, rfa mutation and a resistance factor on the pKM101 plasmid. Strain TA102 contains the his(delta)G8476 base pair mutation, hisG428 substitution mutation on plasmid pAQ1, rfa mutation and a resistance factor on the pKM101 and pAQ1 plasmids.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Oxoid nutrient broth No.2 (2.5%)
- Properly maintained: yes, tester strain cultures were grown on a shaker at 37°C for 11-12 hours to a density of 1 to 3x10^9 cells per mL and maintained at 4°C thereafter.
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from Sprague-Dawley male rats induced with Aroclor 1254 (500 mg/kg body weight)

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence of S9 and 1.5, 5, 15, 50 and 150 µg/plate in the absence of S9, chosen based on an initial toxicity test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO, µL/plate

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable):1 to 3 x 10^9 cells/mL

DURATION
- Preincubation period: None
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of revertant colonies and/or a diminution of the background lawn was taken as an indication of bacteriotoxicity.

- OTHER: The plates were also examined for precipitates and microcolony growth. When there is any question about the nature of colonies scored as revertants and when positive mutagenic results are obtained, the genotype of revertant colonies are spot-checked by picking and streaking on histidine free plates.

Evaluation criteria:

Significant increase in the mutation frequency in the absence and presence of a metabolic activation system.

Statistics:

Chi2-test (Mohn and Ellenberger, 1977)

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

500 µg/plate in the absence of S9, 1500 µg/plate in the presence of S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

150 µg/plate in the absence of S9, 5000 µg/plate in the presence of S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

150 µg/plate in the absence of S9, 500 µg/plate in the presence of S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

500 µg/plate in the absence of S9, 1500 µg/plate in the presence of S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

150 µg/plate in the absence of S9, 500 µg/plate in the presence of S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound an the plates was not observed.

RANGE-FINDING/SCREENING STUDIES: Test concentrations were determined after an initial toxicity test.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: For TA1537, revertant frequencies for 50 µg/plate 9-aminoacridine was 259±81 in the absence of S9 and for 1.5 and 1.7 µg/plate 2-aminoanthracene were 298±160 and 169±15 respectively, in the presence of S9. For TA1535, revertant frequencies for 0.7 µg/plate sodium azide was 513±137 in the absence of S9 and for 1.5 µg/plate 2-aminoanthracene was 487±150 in the presence of S9. For TA98, revertant frequencies for 2.5 µg/plate 2-nitrofluorene was 361±126 in the absence of S9 and for 0.7 and 0.8 µg/plate 2-aminoanthracene were 980±399 and 615±47 respectively, in the presence of S9. The historical revertant frequency for 5.0 µg/plate benzo[a]pyrene for TA98 was 523±30. For TA100, revertant frequencies for 0.7 µg/plate sodium azide was 434±83 in the absence of S9 and for 0.7 µg/plate 2-aminoanthracene and 5.0 µg/plate benzo[a]pyrene (2 different S9 lots)were 884±382 and 869±100 / 801±58 respectively, in the presence of S9. For TA102, revertant frequencies for 0.15 µg/plate mitomycin C was 81±156 in the absence of S9 and for 1.5 and 1.7 µg/plate 2-aminoanthracene were 1080±345 and 1046±44 respectively, in the presence of S9.
- Negative (solvent/vehicle) historical control data: Spontaneous revertant frequencies for TA1535, TA1537, TA98, TA100 and TA102 were 30±14, 10±4, 29±8, 134±31 and 279±41 respectively, in the absence of S9, and were 19±5, 16±5, 40±10, 118±26 and 307±45 respectively, in the presence of S9. Solvent control revertant frequencies for TA1535, TA1537, TA98, TA100 and TA102 were 31±13, 11±4, 28±8, 127±26 and 264±41 respectively, in the absence of S9, and were 19±6, 16±5, 38±9, 114±26 and 300±47 respectively, in the presence of S9.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Reduction in the number of revertant colonies and/or a diminution of the background lawn

Any other information on results incl. tables

Table 1. Induction of revertants in S. typhimurium by the test item in the absence of a metabolising system, experiment 1

		Mean number of revertants per plate (SD)
Substance	Concentration (µg/plate)	TA98	TA100	TA102	TA1535	TA1537
Control	0	32 (9)	141 (16)	298 (14)	31 (4)	10 (4)
Solvent control	0	24 (2)	129 (1)	332 (14)	31 (5)	9 (4)
Test item	5	16 (2)	126 (11)	268 (19)	31 (3)	-
Test item	15	20 (3)	119 (8)	300 (10)	33 (3)	8 (3)
Test item	50	16 (5)	118 (5)	291 (12)	26 (5)	13 (1)
Test item	150	12 (4) T	105 (10)	209 (37) T	18 (3) T	9 (2)
Test item	500	2 (3) T	38 (16) T	39 (29) T	2 (3) T	2 (3) T
Test item	1500	-	-	-	-	0 (0) T
Sodium azide	0.7	-	535 (178)	-	1065 (37)	-
2 -nitrofluorene	2.5	323 (23)	-	-	-	-
9 -aminoacridine	50	-	-	-	-	583 (77)
Mitomycin C	0.15	-	-	583 (17)	-	-

T: bacteriotoxic

Table 2. Induction of revertants in S. typhimurium by the test item in the absence of a metabolising system, experiment 2

		Mean number of revertants per plate (SD)
Substance	Concentration (µg/plate)	TA98	TA100	TA102	TA1535	TA1537
Control	0	28 (4)	107 (20)	310 (6)	27 (2)	5 (4)
Solvent control	0	17 (3)	103 (3)	296 (4)	19 (6)	8 (1)
Test item	1.5	20 (3)	104 (11)	286 (14)	23 (3)	8 (2)
Test item	5	20 (3)	101 (8)	316 (14)	22 (2)	5 (4)
Test item	15	27 (3)	94 (9)	319 (19)	26 (4)	7 (4)
Test item	50	19 (4)	91 (8)	313 (6)	22 (3)	7 (2)
Test item	150	10 (4) T	99 (2)	262 (15) T	22 (1)	7 (2)
Sodium azide	0.7	-	366 (20)	-	1084 (108)	-
2 -nitrofluorene	2.5	547 (40)	-	-	-	-
9 -aminoacridine	50	-	-	-	-	281 (32)
Mitomycin C	0.15	-	-	813 (50)	-	-

T: bacteriotoxic

Table 3. Induction of revertants in S. typhimurium by the test item in the presence of a metabolising system, experiment 1

		Mean number of revertants per plate (SD)
Substance	Concentration (µg/plate)	TA98	TA100	TA102	TA1535	TA1537
Control	0	36 (2)	129 (14)	398 (13)	17 (3)	11 (1)
Solvent control	0	28 (2)	117 (9)	376 (9)	6 (3)	15 (3)
Test item	5	32 (7)	115 (7)	359 (18)	-	-
Test item	15	24 (5)	112 (21)	344 (17)	8 (2)	19 (4)
Test item	50	28 (5)	124 (5)	343 (3)	7 (3)	16 (2)
Test item	150	25 (6)	110 (9)	340 (14)	8 (3)	13 (2)
Test item	500	21 (6) T	107 (6)	294 (19) T	8 (4)	11 (3)
Test item	1500	-	-	-	5 (0)	3 (3) T
2 -aminoanthracene	0.8	481 (27)	817 (20)	576 (29)	176 (16)	-
2 -aminoanthracene	1.7	-	-	-	-	231 (43)

T: bacteriotoxic

Table 4. Induction of revertants in S. typhimurium by the test item in the presence of a metabolising system, experiment 2

		Mean number of revertants per plate (SD)
Substance	Concentration (µg/plate)	TA98	TA100	TA102	TA1535	TA1537
Control	0	28 (4)	127 (3)	407 (30)	9 (2)	20 (3)
Solvent control	0	27 (3)	109 (6)	366 (20)	10 (2)	14 (3)
Test item	5	23 (7)	-	355 (3)	-	-
Test item	15	24 (4)	113 (13)	388 (22)	-	15 (7)
Test item	50	24 (6)	116 (7)	359 (4)	10 (3)	11 (3)
Test item	150	25 (3)	102 (8)	338 (23)	13 (3)	14 (4)
Test item	500	15 (2) T	94 (16)	311 (9) T	11 (2)	12 (2)
Test item	1500	-	83 (11) T	-	6 (2)	7 (4) T
Test item	5000	-	-	-	2 (2) T	-
2 -aminoanthracene	0.8	467 (12)	719 (73)	709 (56)	159 (14)
2 -aminoanthracene	1.7	-	-	-	-	154 (37)

T: bacteriotoxic

Applicant's summary and conclusion

Conclusions:

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. The Bacterial Reverse Mutation Test (OECD 471, EU B.13/14) detects gain of function point mutations and frameshifts in vitro, and is required to fulfil REACH Annex VII information requirements. Under the test conditions, the test item did not present any cytotoxic or mutagenic properties in the Bacterial Reverse Mutation Test. Conducted according to the aforementioned guidelines and GLP, the Ames Test passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Executive summary:

An Ames test was conducted on the test item using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102. The test was performed by the plate incorporation assay at test item concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence of S9 and 1.5, 5, 15, 50 and 150 µg/plate in the absence of S9, alongside negative, solvent and positive controls. The test item was incubated for 48 to 72 hours and mutagenicity was assessed based on the number of revertant colonies. The test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolising system. In the absence of S9-mix the test item was bacteriotoxic towards the strains TA1535, TA98, and TA102 at a concerntration of 150 µg/plate and towards the strains TA1537 and TA100 at 500 µg/plate. In the presence of S9-mix the test item was bacteriotoxic towards the strains TA98 and TA102 at 500 µg/plate, towards the strains TA1537 and TA100 at 1500 µg/plate, and towards the strain TA1535 at 5000 μg/plate. The study is reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD Guideline 471.