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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Jun - 09 Aug 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl linoleate
EC Number:
208-868-4
EC Name:
Ethyl linoleate
Cas Number:
544-35-4
Molecular formula:
C20H36O2
IUPAC Name:
ethyl octadeca-9,12-dienoate
Test material form:
liquid

Method

Target gene:
his operon for S. typhimurium and trp operon for E. coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9-mix 5 µg/plate for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9-mix 60 µg/plate for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9-mix 10 µg/plate for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9-mix 650 µg/plate for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9-mix 10 µg/plate for WP2 uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene: 1 µg/plate for TA98 and TA100; 2.5 µg/plate for TA1535 and TA1537; 10 µg/plate for WPA2uvrA
Remarks:
with 5% S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene: 1 µg/plate for TA98; 2.5 µg/plate for TA100 and TA1535; 5µg/plate for TA1537; 10 µg/plate for WPA2uvrA
Remarks:
with 10% S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Two independent experiments were performed in triplicates each.

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically.

In the first experiment S9-mix at 5% (v/v) was used. In the second experiment S9-mix at 10% (v/v) was used.

The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present.
If more than 40 colonies were present, these were counted automatically with a Biocount 4000 Pro-S-colony counter.
Plates with abundant test article precipitate which interfered with automated colony counting were counted manually and the evidence of test substance precipitate on the plates was recorded.
Evaluation criteria:
No formal hypothesis testing was done. A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment. A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final
evaluation decision.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test substance slightly precipitated at concentrations ≥1000 µg/plate

RANGE-FINDING/SCREENING STUDIES: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed based on preliminary tests in TA 100 and WP2uvrA up to 5000 µg/plate in triplicate. At the highest concentration the test substance exhibited limited solubility.

COMPARISON WITH HISTORICAL CONTROL DATA: positive and solvent controls were in the range of historical control data

Any other information on results incl. tables

Table 1: Maximum number of revertants at the first experiment with 5% S9 -mix

 

Maximum number of revertants

solvent Control

positive control

treatment

(at dose level [µg/mL])

Strain

With S9

Without S9

With S9

Without S9

With S9

Without S9

TA 1535

8

12

132

893

12 (100)

9 (33)

TA 1537

6

7

417

325

10 (33)

9 (1000)

TA 98

28

18

963

1025

34 (33)

21 (333)

TA 100

77

69

890

1248

81 (33)

86 (100)

WPA­2uvrA

23

19

465

1159

35 (5000)

32 (3330)

Table 2: Maximum number of revertants at the second experiment with 10% S9 -mix

 

Maximum number of revertants

solvent Control

positive control

treatment

(at dose level [µg/mL])

Strain

With S9

Without S9

With S9

Without S9

With S9

Without S9

TA 1535

7

11

134

838

12 (10)

13 (100)

TA 1537

6

6

413

154

6 (333)

8 (100)

TA 98

25

16

517

981

28 (33)

21 (100)

TA 100

88

89

1418

925

83 (10)

101 (10)

WPA­2uvrA

23

18

200

1195

31 (33)

27 (10)

Applicant's summary and conclusion

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.