3,7,11,15-tetramethylhexadecane-1,2,3-triol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 30 June 2003 and 04 July 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,7,11,15-tetramethylhexadecane-1,2,3-triol

EC Number:

277-923-2

EC Name:

3,7,11,15-tetramethylhexadecane-1,2,3-triol

Cas Number:

74563-64-7

Molecular formula:

C20H42O3

IUPAC Name:

3,7,11,15-tetramethylhexadecane-1,2,3-triol

Test material form:

liquid: viscous

Details on test material:

Substance name: Phytantriol
Appearance: Light yellow, viscous liquid
Batch No.: UQ20318007
Storage conditions: Room temp. protected from light (dry place)
Expiry date: Best use before 03. 2004

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535, TA97, TA98, TA100 and TA102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 50, 158, 500, 1580 and 5000 µg/plate

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene and ICR 191

Details on test system and experimental conditions:

Bacterial strains
Nutrient broth cultures of each strain, supplemented with 9 % DMSO were stored in liquid nitrogen. Subcultures with a normal spontaneous frequency were stored in NB + 9 % DMSO at -75°C± 20°C. The strain identities and characteristics were periodically checked by the recommended procedures.
For use in tests, cultures of the strains were grown overnight at 37°C in a shaking water bath in a NB liquid medium. The strain TA102 was incubated with 0.3 μg tetracycline per ml NB medium in order to ensure the presence of an adequate number of plasmids.
The growth of the overnight cultures was controlled by taking a check of the bacterial density with a photometer (Lumetron Colorimeter) at 650 nm. Each bacterial strain was diluted 106 fold in 0.85 % sodium chloride, and 100 μl of the last dilution step was plated on a NB complete medium. Two replicate plates were incubated at 37°C, upside down, for two days. The number of colonies was registered and the number of cells plated on VB medium was calculated.
The sensitivity of the Salmonella typhimurium strains was verified using the following positive controls: sodium azide with strains TA1535 and TA100, ICR 191 with strain TA97, 2-nitrofluorene with strain TA98 and MMC with strain TA102. Moreover, 2-aminoanthracene was used with all strains with and without metabolic activation to examine the activity of the S9 mix.


Toxicity Pre-screen and Dose Selection
Toxicity of the test item was assessed in a preliminary toxicity assay (not subjected to GLP conditions) by evaluating the growth on Vogel-Bonner minimal agar plates (determination of the growth of the background lawn and/or frequency of spontaneous revertants). Each test item dose, as well as the appropriate solvent control, was evaluated in duplicate in strain TA100 in the plate incorporation version of the assay. The highest test dose for the main experiments is chosen to either produce signs of toxicity (reduction in the revertant colony number and/or observation of thinning or absence of the background lawn) or to be evidently insoluble in the aqueous medium.
There is much debate about selection of highest dose for badly soluble compounds. Homogeneous suspensions are included and only particulate aggregation is considered for limiting the dose range to be evaluated. If the compound is soluble and non-toxic, then 5 mg/plate is chosen in general as the highest dose level. The solubility of the compound in the solvent, the occurrence of precipitation in the test tube after addition of the soft agar and on the agar plates after the incubation period are noted in the toxicity test with TA100. Since precipitation under the conditions of the pre-incubation version might differ from the plate incorporation version an examination of precipitation after addition of the phosphate buffer (as in the pre-incubation test) is included in the pre-screen. In this case cells are not added to the mixture.


Standard Plate Incorporation Test Procedure
Test tubes containing 2 ml of 0.7 % agar medium were autoclaved and kept in a pre-warmed water bath at 42°C to 45°C. The following solutions were added in order:
• 0.2 ml of the histidine/biotin mixture corresponding to 21 μg L-histidine and 24.4 μg biotin
• 0.1 ml of the test item solutions at different concentrations or of the solvent or 0.05 ml of the different reference items which were thawed shortly before use
• 0.1 ml of the overnight cultures of the bacterial strain
• 0.5 ml of the S9 mixture where metabolic activation was needed. The S9 mixture was replaced by 0.5 ml sodium phosphate buffered saline 0.2 M, pH 7.4 in the treatment without metabolic activation.
The contents of the tubes were mixed and poured immediately onto Vogel-Bonner minimal agar plates. Three replicate plates for the test item and negative control or two replicate plates for the positive controls were incubated at 37°C, upside down, for 2 days.


Liquid Preincubation Assay
In the preincubation procedure the following solutions are added in order:
0.0316 ml of the test item solutions or of the solvent or 0.05 ml of the different reference items which were thawed shortly before use
0.5 ml of sodium phosphate buffered saline 0.2 M, pH 7.4 or 0.5 ml of the S9 mixture
0.1 ml of the overnight cultures of the bacterial strain.
The test tubes were incubated and shaken for 30 minutes at 37°C. 2.2 ml soft agar supplemented with 21 μg L-histidine and 24.4 μg biotin was added afterwards and the content of the tubes were mixed and poured on Vogel-Bonner minimal agar plates. The volume of organic solvent was reduced to 31.6 μl/plate to avoid poisoning of the metabolic enzymes. Three replicate plates for the test item and negative control or two replicate plates for the positive controls were incubated at 37°C, upside down, for 2 days.

Evaluation criteria:

There is as yet no generally accepted statistical treatment of Ames test data. In most tests, the results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of his+ revertants. The increase should reach at least a doubling of the number of spontaneous revertants for Salmonella typhimurium strains TA1535 and TA98. For strains TA97, TA100 and TA102 a 1.5 - fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation and biological relevance should always be taken into account. A negative result is defined as the absence of a reproducible increase in the number of his+ revertant colonies. Since it is impossible to define criteria that would apply to every configuration of data generated by the mutation assay, the study director is responsible for the ultimate decision in the evaluation of the results. The factors considered in making the decision are discussed in results and discussion.

Results and discussion

Test results

Species / strain:

other: TA1535, TA97, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item was evaluated for mutagenic activity in the standard plate incorporation and in the pre-incubation versions of the Ames test. Five S. typhimurium trains were employed and the compound was tested in the presence and absence of a metabolic activation system derived from the livers of phenobarbital / 5,6 benzoflavone treated male Sprague Dawley rats.

Based on the range finder assay the dose ranges of 50 to 5000 µg/plate and 15.8 to 1508 µg/plate were selected for the standard and pre-incubation versions of the assay. Toxic effects were generally not visible with the exception of a reduced background growth in the pre-incubation test (-S9) for strain TA98 at 1580 µg/plate and for strain TA102 (-S9) at the four highest concentrations.
The mutant frequencies of the controls were in the range of historical control values and the data published in the literature. The positive controls induced significant increases in the mutant frequencies verifying the sensitivity of the strains used.
The test item did not induce any dose related increase of the number of revertant colonies / plate in any of the five tester strains. Thus it can be concluded that the test substance is devoid of mutagenic activity in the Ames test under the described experimental conditions

Any other information on results incl. tables

Dose selection

Upon addition to the aqueous medium formation of milky suspensions started at ≥ and ≥ 158 µg/plate in the standard and pre-incubation tests respectively, and precipitation was visible at 5000 and ≥ 500 µg/plate. A range finder assay with strain TA100 (standard plate incorporation version, ± S9) was performed. Toxic effects were nor apparent but precipitation hindered an unambigous evaluation of the background growth at 5000 µg/plate.

Concentration µg/plate	Precipitation in standard assay at plating time (on plate)	Precipitation in pre-incubation assay (in pre-inc. mix)	Revertants per plate (two plates average)		Background growth; precipitation at counting time
			-S9	+ S9	-S9	+ S9
0	Clear	Clear	102	121	- ; -	- ; -
50	Clear	Clear	106	121	- ; -	- ; -
158	Clear	Turbidity	101	135	- ; -	- ; -
500	Starting turbidity	Precipitation	121	120	- ; -	- ; -
1580	Turbidity	Precipitation	104	101	- ; -	- ; -
5000	Precipitation	Precipitation	119	103	NE; PAC	NE; PAC

- ; -: No reduction of background growth; no precipitate

NE: Background growth not evaluable

PAC: Precipitation of the test item without influence on automatic counting

Data reporting

Colonies are usually counted electronically using a DOMINO automatic image analysis system, (Perspective Instruments, Haverhill, England) after having inspected the background lawn for signs of toxicity. Plates exhibiting precipitate or contamination may be counted manually.

The microscopic examination of the bacterial lawns, resulting from the trace histidine added to the plates, is an aid to determine the toxicity of the test item and is essential to the interpretation of results. The reduction of the density of the background lawn of bacteria or its total absence (with possible appearance of isolated, non-mutated, enlarged micro-colonies) is noted.

Summary data: Ames plate incorporation assay

Summary of the results of the reverse mutation assay using bacteria of the indicated strains.

Strain	TA1535		TA 97		TA98		TA100		TA102
Activation	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
Concentration (µg/plate)
0	10± 3	8± 4	198± 10	205± 8	15± 5	20± 2	106± 14	119± 10	393± 27	359± 20
50	9± 2	8± 4	193± 17	208± 1	13± 4	19± 2	108± 4	110± 3	371± 12	402± 14
158	11± 4	9± 2	179± 20	226± 16	15± 6	17± 3	105± 14	122± 8	386± 16	434± 17
500	8± 3	4± 3	216±11	212± 16	16± 4	24± 7	111± 13	116± 7	375± 13	393± 21
1580	7± 3	7± 1	180±5	194± 4	16± 3	21± 5	97± 4	98± 5	385± 14	375± 28
5000	13± 6a	5± 4a	167± 15a	199± 12a	18± 2a	21± 2a	103± 9a	97± 5a	401± 36a	380± 30a

a = precipitation of compound, electronic counting, backgrounds growth no evaluable.

Note: Because of computer problems during data storage / retrieval, all plates were re-counted (re-evaluation 1) on the same day.

 

Summary data: Pre-incubation assay

Summary of the results of the reverse mutation assay using bacteria of the indicated strains.

Strain	TA1535		TA 97		TA98		TA100		TA102
Activation	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9	-S9	+ S9
Concentration (µg/plate)
0	15± 4	10± 2	290±8	345± 23	23± 4	26± 1	164± 12	169± 8	440± 32	520± 59
15.8	11± 3	10± 1	247± 3	357± 11	17± 8	21± 4	159± 17	178± 4	298± 19	561± 44
50	12± 3	12± 1	250± 19	289± 9	14± 5	26± 5	142± 11	144± 21	243± 22	471± 12
158	14± 3a	11± 3	245± 18a	322± 24	19± 8a	21± 6	146± 2a	156± 16	288± 26 t	567 ± 24
500	13± 1 b	13± 4a	269± 18a	287±41 a	13± 1 b	19± 7a	135± 12a	164 ± 6a	312±48 t	502± 8a
1580	17± 1 b	9± 2b	264± 11a	345± 34a	19± 3c	30± 2b	131± 6a	202± 11a	264± 11t	573± 15a

t = toxic effect

a = precipitation of the test compound without influence on automatic counting

b = precipitation of the test compound necessitating manual counting

c = precipitation of test compound, manual count necessary, background growth reduced

At the two highest concentrations precipitations occurred in a form which was not distinguished by the image analysis systems from colonies. At 500 µg/plate, a value of about 5 and at 1580 µg/plate a value of about 10 would have to be subtracted from the electronic plate count. Since this does not influence the assessment for plates with high colony counts (TA97, TA100 and TA102) plates were counted electronically, no correction was made. For the plates with low colony counts (strain TA1535 and TA98) counting was done by hand.

Applicant's summary and conclusion

Conclusions:

There was no increase in the number of revertant colonies for any of the five tester strains after treatment with the test substance. It can therefore be concluded that neither the test substance, nor any of the metabolites formed by the metabolic activation system are mutagenic in the Ames test under the conditions of the experiment.

Executive summary:

The genotoxic potential of the test substance was assessed according to OECD 471 using a bacterial reverse mutation method. There was no increase in the number of revertant colonies for any of the tester strains after treatment with the test substance. It can therefore be concluded that neither the test substance, nor any of the metabolites formed by the metabolic activation system are mutagenic in the Ames test under the conditions of the experiment and the test substance is not classified as a mutagen.