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Genetic toxicity: in vitro

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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:
13 to 27 June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear, pale yellow liquid
Details on test material:
Identification: Hatcol 2352
Chemical name: Isononanoic acid, mixed esters with heptanoic acid and pentaerythritol
CAS Number: 118685-29-3
Description: Clear pale yellow liquid
Batch: K16156
Test substance storage: At room temperature in the dark
Stability under storage conditions: Not indicated
Expiry date: 24 January 2004 (allocated by NOTOX, 1 year after receipt of the test substance)
Specific Gravity: 0.97
Stability in vehicle: Ethanol: not indicated

Method

Target gene:
histidine and tryptophan
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Test System Salmonella typhimurium bacteria and Escherichia coli bacteria Rationale Recommended test system in international guidelines (e.g. EPA, OECD, EEC).
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD30521R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cell coat)
gg : mutation in the galactose metabolism
chi : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicilin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (-196.C).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
In the dose range finding test, Hatcol 2352 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Hatcol 2352 emulsified on the plates at dose levels of 1000 µg/plate and upwards.
In the first and in the second mutation assay, Hatcol 2352 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix.
Vehicle / solvent:
Ethanol
Controls
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
9-aminoacridine
sodium azide
methylmethanesulfonate
other: daunomycin (DM), 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
Test substance preparation: The test substance was dissolved in ethanol (Lichrosolv, Merck). Test substance concentrations were prepared directly prior to use and used within 4 hours after preparation.

Cell culture
Preparation of bacterial cultures: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.
Permeabilization of the Escherichia coli strain: WP2uvrA bacteria were washed twice in 0.25 the original volume of ice-cold 0.12 M Tris-HCL buffer pH 8.0, then gently resuspended in 0.2 vol. 0.12 M Tris-HCL, 0.5 mM EDTA pH 8.0, and shaken for 2.5 min at 37°C. MgCl2 was then added to a final concentration of 10 mM. The cells were centrifuged and resuspended in the original volume of nutrient broth.
Agar plates: Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purifed agar (Oxoid, code L28) in Vogel-Bonner Medium E, 20 g glucose.
N.B. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin and 15 µg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan.
Top agar: Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid, code L 11) and 0.5% (w/v)
Sodium Chloride was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 1°C.
Environmental conditions: All incubations were carried out in the dark at 37 ± 1°C. The temperature was monitored during the experiment.

Metabolic activation system: Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany.

Study design
Dose range finding test: Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations, 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of Hatcol 2352 used in the subsequent mutation assay was the level at which the test substance exhibited limited solubility.
Mutation assay: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1°C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Colony counting: The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
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) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) 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:
No formal hypothesis testing was done.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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 applicable
Positive controls validity:
valid
Additional information on results:
Dose range finding test
Hatcol 2352 was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.
This dose range finding test is reported as a part of the first experiment of the mutation test.
Precipitate: The test substance precipitated in the top agar at concentrations of 333 µg/plate and upwards.
Precipitation of Hatcol 2352 on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 ¡.g/plate and upwards. This precipitate was judged as an emulsion of Hatcol 2352 at the end of the incubation period.
Toxicity: To determine the toxicity of Hatcol 2352, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutation assay
Based on the results of the dose range finding test, Hatcol 2352 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Precipitate: Hatcol 2352 precipitated in the top agar at the concentrations of 333 and 1000 µg/plate.
Precipitation of Hatcol 2352 on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 µg/plate. This precipitate was judged as an emulsion of Hatcol 2352 at the end of the incubation period.
Toxicity: The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
Number of revertants: All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1: Mutagenic response of Hatcol 2352 in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

324± 26

340± 81

460± 98

938± 42

596± 16

Solvent control

7± 2

8± 4

20± 3

118± 5

10± 5

3

 

 

 

141± 13

9± 3

10

9± 1

15± 5

16± 5

127± 11

9± 4

33

7± 4

8± 2

15± 5

123± 6

12± 2

100

7± 2

11± 2

18± 3

130± 2

8± 3

333

7± 1

10± 2

14± 1

149± 7

11± 2

1000E

8± 8

11± 4

13± 3

142± 7

11± 4

3330E

 

 

 

132± 13

9± 2

5000E

 

 

 

130± 15

14± 4

With S9-mix1

Positive control

195± 26

202± 29

679± 36

942± 42

94± 13

Solvent control

8± 2

9± 4

18± 4

138± 2

12± 2

3

 

 

 

159 V 9

11± 4

10

7± 2

8± 3

21± 2

147± 13

12± 12

33

6± 1

11± 5

22± 1

142± 3

11± 3

100

8± 3

7± 2

22± 7

153± 10

13± 4

333

8± 2

11± 2

22± 2

131± 7

13± 3

1000E

11± 1

6± 3

20± 1

149± 13

15± 1

3330E

 

 

 

146± 7

11± 1

5000E

 

 

 

141± 9

11± 3

Solvent control: 0.1ml ethanol

1 The S9-mix contained 5% (v/v) S9 fraction

E Hatcol 2352 emuslified on the plates

 

Experiment 2: Mutagenic response of Hatcol 2352 in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

TA1535

TA1537

TA98

TA100

WP2uvrA

Without S9-mix

Positive control

231± 16

244± 31

588± 29

546± 56

763± 75

Solvent control

10± 3

8± 1

19± 1

127± 6

10± 3

10

14± 1

5± 1

19± 2

123± 6

11± 2

33

12± 2

5± 0

21± 4

132± 13

8± 3

100

11± 2

5± 1

14± 2

124± 9

10± 2

333

12± 5

5± 1

20± 3

119± 15

9± 2

1000E

7± 1

6± 1

13± 3

128± 9

9± 2

With S9-mix1

Positive control

114± 13

148± 18

340± 55

492± 15

65± 8

Solvent control

11± 2

6± 2

19± 2

122± 5

8± 2

10

12± 1

5± 2

20± 2

102± 4

10± 3

33

12± 3

6± 3

24± 1

108± 6

11± 2

100

10± 2

7± 2

21± 2

109± 4

14± 5

333

10± 2

6± 2

22± 3

113± 13

9± 3

1000E

12± 3

7± 2

22± 3

108± 5

9± 1

Solvent control: 0.1ml ethanol

1 The S9-mix contained 5% (v/v) S9 fraction

E Hatcol 2352 emuslified on the plates

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Based on the results of this study it is concluded that Hatcol 2352 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of Hatcol 2352 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

Hatcol 2352 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

The study procedures described in this report were based on the following guidelines:

- OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (Adopted July 21, 1997).

- European Economic Community (EEC). Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity; B.13/14: "Mutagenicity: "Reverse Mutation Test using bacteria". EEC Publication Commission Directive (Published June 8, 2000).

Batch K16156 of Hatcol 2352 was a clear pale yellow liquid. The test substance was soluble in ethanol.

In the dose range finding test, Hatcol 2352 was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Hatcol 2352 emulsified on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first and in the second mutation assay, Hatcol 2352 was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix. Hatcol 2352 emulsified on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings. Hatcol 2352 did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that Hatcol 2352 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.