Reaction mass of 2-octyldodecyl 2-hydroxypropanoate, 2-Octyldodecan-1-ol and 2-octyldodecyl 2-[(2-hydroxypropanoyl)oxy]propanoate

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:

11 September - 14 October 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study performed according GLP and protocol cmpliant with OECD Guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-octyldodecan-1-ol; 2-octyldodecyl 2-[(2-hydroxypropanoyl)oxy]propanoate; 2-octyldodecyl 2-hydroxypropanoate

EC Number:

700-839-9

IUPAC Name:

2-octyldodecan-1-ol; 2-octyldodecyl 2-[(2-hydroxypropanoyl)oxy]propanoate; 2-octyldodecyl 2-hydroxypropanoate

Details on test material:

The test article below was received from the sponsor and assigned the test article number M08-4389.03.
It was stored as indicated by the client-supplied storage conditions until testing commenced. Test article derivation,
characterization and stability was the responsibility of the sponsor.
Name: Cosmol 13
Lot Number: 7-10-A
Storage Conditions: Room Temperature
CPTC ID No.: M08-4389.03

Specific details on test material used for the study:

Lot-Nr. 7-10-A
Storage at room temperature

Method

Target gene:

his-

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver microsomes (S9)

Test concentrations with justification for top dose:

5.0, 1.0, 0.5, 0.1, 0.05 mg/plate

Vehicle / solvent:

Isopropyl Alcohol

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: Strain TA100 & 1535

Controls 2

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Migrated to IUCLID6: Strain TA98

Controls 3

Positive controls:

yes

Positive control substance:

other: ICR 191 Acridine: Strain TA1537

Controls 4

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: Strain E. coli WP2 uvrA

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene w/S9 all strains

Details on test system and experimental conditions:

Test Procedure
A Pre-test
Assemble the supplies and equipment needed to perform the test on the day before.
Remove the QUAD plates form the refrigerator, cut off the plastic sleeve and allow to dry upright at room temperature overnight.
Label Minimal Glucose Agar plates and Top Agar tubes with strain number, test identification adn dose, S9 (+/-) and date of test. Be sure to include the diagnostic positive and negative controls.
Adjust the temperature of your water bath or dry block heater to approximately 45 °C.

One Day Prior to the Assay
Label sterile flasks with the strain number (e.g., TA98, TA100, TA1535, TA1537, E.coli WP2 uvrA).
Using aseptic technique, carefully decant approximately 20-25 ml of Oxoid #2 nutrrient broth into Erlenmeyer flasks. To all flasks add ampicillin to final concentration of 25 pg/ml (to 25 ml nutrient broth, 78 µl of an 8 mg/ml solution ).
Remove the STDisc vials from the refrigerator and warm to room temperature before opening.
Remove the vial closure.
Using a sterile loop/needle, pick up one or more discs and drop intro the appropriately labeled flask containing nutient broth.
After the flasks are inoculated, place them on the shaker and incubate at 37°C with the shaker operating at approximately 150 rpm. Incubation must not exceed 16 hours.
After incubation (e.g. on the morning of the assay) remove the flask cultrues and place them in the refrigerator until needed.

The Day of the Assay
Melt the top atar. After melting, place the top agar bottles into a 45°C water bath allow at least 45 minutes for temperature equilibration.
Cultures and the S9 must be placed on ice prior to use and kept on ice throughout the assay.
Prepare the positive controls:
Add one ml of the appropriate solvent to each of the CONTROLCHEM tubes.
Mutagen Strain
Sodium Azide TA100 & 1535
2-Nitrofluorene TA98
ICR 191 Acridine TA1537
Methyl Methanesulfonate E. coli WP2uvrA
2-Aminoanthracene All strains
Perform the dilutions of your test article.
Load the 45°C heating block with sterile 13 *100 mm tubes with closures equal to the number of labeled minimal glucose agar plates. Pipette 2 ml of molten,
45°C, top agar into each tube (top agar containing histidine for all Salmonella strains; top agar with tryptophan for the E. coli strain).
Arrange your previously labeld Minimal Glucose Agar plates by strain and condition.
Decide which strain you are going to begin with. In the scheme shown below it is assumed that TA98 will be used first in a triplicate plate, + and - S9 assay. The S9 Concentration used in the assay will be 10 %.
Assay the test article:
Without S9
Begin with the solvent control; add 100 µl of water or DMSO (or other solvent used to solubilize the test article) to the first three tubes preheated to 37+-2°C. Then, in ascending sequence, add 100 µl of each test article dilution to each additional trio of preheated tubes. Add 500 µl of sterile buffer to each tube.
Add 100 µl of the TA98 culture to the first three tubes (solvent control tubes).
Without delay, gently mix the tube contents using a vortex mixer. Decant the mixture onto the surface of the appropriately labeled Minimal Glucose Agar plate. Do one tube at a time. Immediately upon decantation, gentrly tilt the plate and rotate so as to obtain an even distribution of the plating mixture over the surface of the bottom atar. Place onto a level surface and allow to harden.
Repeat the procedure for each dose of the test article.

With S9
Begin with the solvent control; add 100 µl of water or DMSO (or other solvent used to solubilize the test article) to the first three tubes preheated to 37+-2°C. Then, in ascending sequence, add 100 µl of each test article dilution to each additional trio of preheated tubes. Add 500 µl of sterile buffer to each tube.
Add 500 µl of the previously prepared S9 mix to the first three tubes (solvent control tubes).
Without delay, gently mix the tube contents using a vortex mixer. Decant the mixture onto the surface of the appropriately labeled Minimal Glucose Agar plate. Do one tube at a time. Immediately upon decantation, gentrly tilt the plate and rotate so as to obtain an even distribution of the plating mixture over the surface of the bottom atar. Place onto a level surface and allow to harden.
Repeat the procedure for each dose of the test article.

Repeat the above procedure for each strain

Inoculate the Quad Plates with the Salmonella strains and the Tri Plates with E. coli WP2 uvrA.
Using a sterile loop or swab, wet with the appropriate culture and inoculate each of the four sectors of a Quad PCTM plate using a "Z" inoculation pattern and Tri plate using a "Z" inoculation pattern.
Repeat for each strain. After all plates are inoculated, open the vial containing the crystal violet discs and using forceps or an inoculating loop, place a single disc on the atar surface in Sector II of each of the Quad plates.

Determine the Titer of the Strain Cultures
Arrange sets of 3 sterile tubes with closures for each strain. Pipette 4.95 ml sterile water into each strain.
Usinge your positive displacement pipette, inoculate the first tube with 50 µl of the appropriate strain culture. Mix thoroughly. This tube contains 1: 100 dilution of the sampled culture. Add 50 µl of the 1:100 dilution to the second tube containign 4.95 ml sterile water and mix. The second dilution is 1:10000. Complete the diltions by adding 50 µl of the 1:10000 dilution to the third 4.95 ml tube and mix. The final dilutio is 1:1000000.
Arrange sets of 2 sterile tubes with closures for each strain and place in 45°C water bath. Add 2 ml of molten top agar to each tube.
Using the positive displacement pipette, inoculate the top agar containing tubes with 50 µl of the 1:10000 and 1:000000 dilutions in water, respectively.
Incubate the Assay
Invert the plates and arrange in stacks correspongin to each experimental condition.
Place in a 37°C incubator and continue incubation for approximately 48 h.

Read the Assay
After the incubation period, remove the inverted plates and allow them to come to room temperature.
Colony counting can be performed manually with the aid of a magnifying counter or with an automatic colony counter. depending on the activity of your test, large numbers of colonies may develop in certain dose groups. In some cases, it may be desirable to utilize sector-counting techniques rather than full plate counts.
After counting and recording the results for the test treatments, the diagnostic positive control plates should be counted. Plates should also be examined for bacterial growth inhibition or cytotoxicity in the noted dose level producing the inhibition should be recorded. If any precipitation of the test article is noted in the plates, this information should also be recorded for each dose level.
Examine the cell titer plates. The 5*10^-6 plates should be too numerous to count. In contrast, the 5*10^-8 plates should contaoin approximately 25-100 colonies;

Evaluation criteria:

Negative (solvent) control Counts
The colonies that grew o the Minimal Glucose Agar plates developed from single cells that had regained their ability to grow in the absence of added histidine. The genetic reversion, form histidine auxotrophy to prototrophy, that enabled those cells to grow in the absence of exogenous histidine might have arisen spontaneously or as the result of a mutation induced by the treatments.
Diagnosic Positive Control Counts
In general, the positive control frequencies should be at least 2.5 times the negative contol counts. Large deviations usually indicate problems with cell management; e.g., high spontaneous frequencies often are paralleled by low induced frequencies. Such eventualities reduce the resolving power of the assay and raise questions regarding the interpretation ot the results of the test treatments.
Phenotypic Confirmation
The Quad plates are prepared with fou different media that provide basic information concerning the genotypes of the strain provided in the kit. Tri plate are used for E. coli WP2 uvrA. By sector, the results should be:
Quad Plate
Sector Observation Genotype
I No growth (all strains) his-
II Zonal inhibition around CV disc (all) rfa
III Profuse growth (all) pKM101
IV No growth pAQ1
Triplate
Sector Observation Genotype
I No growth (all strains) trp-
II Luxuriant Growth trp-
III Inhibition R-factor
Test Article Results
In general, the 2 or 2.5 times over the background "rule-of-thumb" serves as a useful way of distinguishing active mutagens from non-mutagenic test articles.

Statistics:

The mean and standard deviations will be calculated at each dose level of test article for each test organism.

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table #1: Number of revertants without S-9 activation

Test Strain #	Solvent Control	Positive Control	5.0 mg sample	1.0 mg sample	0.5 mg sample	0.1 mg sample	0.05 mg sample
TA 98	24	770	23	25	24	21	23
	24	784	24	23	23	25	22
	23	798	23	23	22	24	25
Average =	23	784	23	24	23	23	23
Std. Deviation =	1.15	14.00	0.58	1.15	1.00	2.08	1.53

Test Strain #	Solvent Control	Positive Control	5.0 mg sample	1.0 mg sample	0.5 mg sample	0.1 mg sample	0.05 mg sample
TA 100	88	1026	87	83	89	80	86
	85	1040	81	84	85	82	82
	84	1069	84	83	81	85	84
Average =	86	1045	84	83	85	82	84
Std. Deviation =	2.08	21.93	3.00	0.58	4.00	2.52	2.00

Test Strain #	Solvent Control	Positive Control	5.0 mg sample	1.0 mg sample	0.5 mg sample	0.1 mg sample	0.05 mg sample
TA 1535	9	456	9	10	12	9	12
	10	499	9	9	10	12	10
	11	485	12	12	8	11	12
Average =	10	480	10	10	10	11	11
Std. Deviation =	1.00	21.93	1.73	1.53	2.00	1.53	1.15

Test Strain #	Solvent Control	Positive Control	5.0 mg sample	1.0 mg sample	0.5 mg sample	0.1 mg sample	0.05 mg sample
TA 1537	9	399	7	8	9	9	7
	9	385	9	8	8	9	10
	8	413	9	10	9	8	9
Average =	9	399	8	9	9	9	9
Std. Deviation =	0.58	14.00	1.15	1.15	0.58	0.58	1.53

Test Strain #	Solvent Control	Positive Control	5.0 mg sample	1.0 mg sample	0.5 mg sample	0.1 mg sample	0.05 mg sample
WP2 uvrA	27	698	28	25	26	27	25
	25	670	26	29	28	24	26
	28	684	24	27	28	29	29
Average =	27	684	26	27	27	27	27
Std. Deviation =	1.53	14.00	2.00	2.00	1.15	2.52	2.08

Table#2: Number of revertants with S-9 activation

Test Strain #	Solvent Control	Positive Control	5.0 mg sample	1.0 mg sample	0.5 mg sample	0.1 mg sample	0.05 mg sample
TA 98	26	877	28	28	26	27	29
	29	869	26	27	26	28	30
	26	884	28	26	30	28	28
Average =	27	877	27	27	27	28	29
Std. Deviation =	1.73	7.51	1.15	1.00	2.31	0.58	1.00

Test Strain #	Solvent Control	Positive Control	5.0 mg sample	1.0 mg sample	0.5 mg sample	0.1 mg sample	0.05 mg sample
TA 100	97	1083	97	96	98	94	96
	94	1112	93	94	93	98	97
	93	1097	93	98	96	98	95
Average =	95	1097	94	96	96	97	96
Std. Deviation =	2.08	14.50	2.31	2.00	2.52	2.31	1.00

Test Strain #	Solvent Control	Positive Control	5.0 mg sample	1.0 mg sample	0.5 mg sample	0.1 mg sample	0.05 mg sample
TA 1535	13	570	10	12	13	13	15
	11	556	10	11	13	9	11
	11	542	13	10	10	14	14
Average =	12	556	11	11	12	12	13
Std. Deviation =	1.15	14.00	1.73	1.00	1.73	2.65	2.08

Test Strain #	Solvent Control	Positive Control	5.0 mg sample	1.0 mg sample	0.5 mg sample	0.1 mg sample	0.05 mg sample
TA 1537	10	456	12	11	11	10	12
	10	426	10	10	10	9	11
	11	485	9	10	11	13	10
Average =	10	456	10	10	11	11	11
Std. Deviation =	0.58	29.50	1.53	0.58	0.58	2.08	1.00

Test Strain #	Solvent Control	Positive Control	5.0 mg sample	1.0 mg sample	0.5 mg sample	0.1 mg sample	0.05 mg sample
WP2 uvrA	29	755	27	30	32	29	30
	30	741	26	28	30	31	31
	28	770	31	27	28	27	29
Average =	29	755	28	28	30	29	30
Std. Deviation =	1.00	14.50	2.65	1.53	2.00	2.00	1.00

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The results in Tables 1 and 2 show that the test strains are sensitive to the positive control mutagens and showed the appropriate mutagenic response (i.e. positive control counts were greater than 2.5 times the negative solvent control). The spontaneous reversion rate was well within the accepted values of each strain, indicating that under the test conditions, the strains were sensitive to the detection of potentially genotoxic agents. the data in Tables 1 and 2 shows that the test article was not cytotoxic to the test system at 5.0, 1.0, 0.5, 0.1 and 0.05 mg. There was no precipitation of the test article noted at any test concentration either with or without S-9 for the test system.
The metabolic activation using the S9 activation mixture shows an active microsomal preparation.
Using the same test conditions, there was no detectable genotoxic activity at the concentrations shown above (i.e. the test article did not show a 2.5 fold increase in counts over the negative solvent control) associated with Cosmol 13 neither in the absence (Table 1) or presence (Table 2) of the S9 enzyme activation.