Glycerides, tall-oil mono-

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:

06. Mar. 2018 to 15. Mar.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997 “Bacterial Reverse Mutation Test“

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”

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:

Glycerides, tall-oil mono-

EC Number:

263-034-7

EC Name:

Glycerides, tall-oil mono-

Cas Number:

61789-12-6

Molecular formula:

C19H38O4 to C21H42O4 * (mono-glycerides) # C37H66O5 to C39H76O4 * (di-glycerides) # C55H96O6 to C57H108O4 * (tri-glycerides) # * in H2 increments (i,e, decreasing # of double bonds)

IUPAC Name:

Glycerides, tall-oil mono-

Test material form:

liquid

Details on test material:

Name: LUMULSE GMT-K
Batch no.: 385932
Appearance: opaque amber liquid
Purity: 100%
Homogeneity: homogeneous
Expiry date: 26. Oct. 2019
Storage: Room Temperature: (20 ± 5°C)

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

other: TA97a, TA98, TA100, TA102 and TA1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

The following nominal concentrations were prepared for the first experiment:
5 μL/plate, 1.5 μL/plate, 0.5 μL/plate, 0.15 μL/plate and 0.05 μL/plate.
The following nominal concentrations were prepared for the second experiment:
5 μL/plate, 2.5 μL/plate, 1.25 μL/plate, 0.63 μL/plate, 0.31 μL/plate, 0.16 μL/plate, 0.08 μL/plate and 0.04 μL/plate.
Test concentrations for the second experiment based on the results from the first experiment.

Vehicle / solvent:

In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 mL/L in demineralized water, dimethyl sulfoxide (DMSO) and acetone.
The test item was sufficiently soluble in DMSO and acetone.
DMSO was chosen as vehicle, because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO), Demineralised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine; 2-Amino-Anthracene

Details on test system and experimental conditions:

Culture of Bacteria
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutrient broth. After incubation for eight hours at 37 ±1 °C, the cultures were used in the experiment.
During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Conduct of Experiment
Preparations
Different media and solutions were prepared preliminary (exact production dates are documented in the raw data).
On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table surface was disinfected.
The S9 mix was freshly prepared and stored at 0 °C.

Experimental Parameters
First Experiment
Concentrations tested: 5 / 1.5 / 0.5 / 0.15 / 0.05 μL/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tested strains: TA97a, TA98, TA100, TA102, TA1535
Method: plate incorporation method

Second Experiment
Concentrations tested: 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 / 0.08 / 0.04 μL/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tested strains: TA97a, TA98, TA100, TA102, TA1535
Method: pre-incubation method

Description of the Method
General preparation
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
-100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-500 μL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
-100 μL bacteria suspension
-2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
-100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
-500 μL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
-100 μL bacteria suspension
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate. The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.

Rationale for test conditions:

In accordance with test guidelines

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

Not specified

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the second experiment, at he two highest concentrations (5 and 2.5 μL/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

First Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 10e+9 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the test conditions.
To verify this result, a further experiment was performed.

Second Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter 15, page 41). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
The test item showed signs of toxicity towards the bacteria strain TA100 in both the absence and presence of metabolic activation in the two highest concentrations (5 and 2.5 μL/plate).
The bacterial background lawn was not reduced at any of the concentrations.
Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the test conditions.

Any other information on results incl. tables

Survey of the Findings

The mean revertant values of the three replicates are presented in the following tables.

Mean Revertants First Experiment

Strain Induction		TA97a		TA98		TA100		TA102		TA1535
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. Water	Mean	73	84	36	33	92	108	249	259	21	22
	sd	7.6	5.1	6.2	5.5	8.0	15.9	26.6	53.3	1.2	1.5
DMSO	Mean	74	110	38	36	89	91	239	249	26	18
	sd	5.2	21.1	4.4	11.9	9.2	1.2	28.9	46.0	1.7	0.6
Positive Controls*	Mean	479	463	268	176	379	1001	657	1323	220	175
	sd	67.2	132.3	28.0	24.3	62.3	0.0	9.2	60.0	10.6	31.1
	f(l)	6.47	4.21	7.05	4.89	4.12	11.00	2.75	5.31	10.48	9.72
5 μL/plate	Mean	89	78	26	34	74	80	245	211	39	28
	sd	7.8	14.4	3.5	4.0	6.2	5.1	22.7	52.1	5.7	4.5
	f(l)	1.20	0.71	0.68	0.94	0.83	0.88	1.03	0.85	1.50	1.56
1.5 μL/plate	Mean	66	95	38	40	90	104	203	233	23	21
	sd	14.4	15.3	1.0	0.0	10.8	10.6	38.4	39.5	4.7	3.1
	f(l)	0.89	0.86	1.00	1.11	1.01	1.14	0.85	0.94	0.88	1.17
0.5 μL/plate	Mean	74	85	33	37	79	95	200	307	24	29
	sd	1.2	13.0	4.0	4.0	7.0	13.2	28.8	26.6	4.7	6.6
	f(l)	1.00	0.77	0.87	1.03	0.89	1.04	0.84	1.23	0.92	1.61
0.15 μL/plate	Mean	86	71	35	41	84	110	252	272	27	21
	sd	7.2	11.7	4.4	5.1	4.0	4.0	42.1	31.2	6.1	3.1
	f(l)	1.16	0.65	0.92	1.14	0.94	1.21	1.05	1.09	1.04	1.17
0.05 μL/plate	Mean	68	105	32	30	103	82	276	307	37	29
	sd	6.4	15.3	5.1	3.1	11.4	3.0	38.6	46.0	2.0	2.6
	f(l)	0.92	0.95	0.84	0.83	1.16	0.90	1.15	1.23	1.42	1.61

f(l) = increase factor

* Different positive controls were used

1001 colonies per plate means the bacteria growth was too strong for counting

 

Mean Revertants Second Experiment

Strain Induction		TA97a		TA98		TA100		TA102		TA1535
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. Water	Mean	79	76	37	37	85	93	308	284	10	12
	sd	3.2	11.3	5.9	8.7	9.5	12.5	35.6	30.2	2.1	3.5
DMSO	Mean	79	95	37	42	75	84	308	261	11	12
	sd	15.9	5.8	3.6	3.1	18.9	7.6	55.6	64.0	2.5	4.0
Positive Controls*	Mean	577	433	621	264	699	717	1339	1181	249	113
	sd	182.5	32.3	56.8	18.3	25.7	37.0	72.6	257.2	72.0	29.5
	f(l)	7.30	4.56	16.78	6.29	8.22	8.54	4.35	4.52	24.90	9.42
5 μL/plate	Mean	74	98	39	33	3	5	315	221	10	11
	sd	13.1	10.8	3.5	4.0	1.5	6.9	52.2	32.6	1.5	1.0
	f(l)	0.94	1.03	1.05	0.79	0.04	0.06	1.02	0.85	0.91	0.92
2.5 μL/plate	Mean	72	93	36	34	20	28	235	191	11	11
	sd	12.4	14.2	2.6	6.1	11.4	11.0	12.2	22.0	1.2	1.7
	f(l)	0.91	0.98	0.97	0.81	0.27	0.33	0.76	0.73	1.00	0.92
1.25 μL/plate	Mean	75	89	43	38	82	76	261	216	7	10
	sd	9.5	15.5	2.9	6.4	12.2	2.1	42.8	22.3	0.6	1.0
	f(l)	0.95	0.94	1.16	0.90	1.09	0.90	0.85	0.83	0.64	0.83
0.63 μL/plate	Mean	86	88	41	34	82	84	256	261	10	11
	sd	5.2	21.1	7.0	2.3	10.4	10.6	21.2	103.2	2.5	1.0
	f(l)	1.09	0.93	1.11	0.81	1.09	1.00	0.83	1.00	0.91	0.92
0.31 μL/plate	Mean	73	98	31	39	87	75	229	227	10	11
	sd	10.1	17.9	3.1	6.1	7.4	10.0	32.6	16.2	0.6	2.9
	f(l)	0.92	1.03	0.84	0.93	1.16	0.89	0.74	0.87	0.91	0.92
0.16 μL/plate	Mean	72	82	33	40	77	77	253	275	10	11
	sd	8.0	0.6	3.0	8.1	13.0	2.1	12.9	9.2	1.7	1.0
	f(l)	0.91	0.86	0.89	0.95	1.03	0.92	0.82	1.05	0.91	0.92
0.08 μL/plate	Mean	79	80	34	39	81	78	224	279	12	12
	sd	13.8	1.0	4.6	6.0	4.6	9.3	43.3	25.7	0.6	2.5
	f(l)	1.00	0.84	0.92	0.93	1.08	0.93	0.73	1.07	1.09	1.00
0.04 μL/plate	Mean	73	78	38	37	84	83	248	269	13	11
	sd	22.0	7.6	3.1	8.1	10.0	8.1	8.0	14.0	2.5	1.0
	f(l)	0.92	0.82	1.03	0.88	1.12	0.99	0.81	1.03	1.18	0.92

f(l) = increase factor

* Different positive controls were used

 

Positive Controls

-Without metabolic activation:

4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 20 μg/plate

Sodium azide (Na-azide) in demineralized water, 1 μg/plate

-With metabolic activation:

2-Amino anthracene (2-AA) in DMSO, 1 μg/plate

Benzo-a-pyrene (BaP) in DMSO, 20 μg/plate

 

Diagnostic Mutagens (colonies per plate) – First Experiment

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Substance	NPD	2-AA	NPD	BaP	Na-azide	2-AA	NPD	2-AA	Na-azide	2-AA
Repl. 1	552	612	236	164	444	1001	652	1264	228	200
Repl. 2	464	416	288	160	320	1001	668	1320	208	140
Repl. 3	420	360	280	204	372	1001	652	1384	224	184
Mean	479	463	268	176	379	1001	657	1323	220	175
sd	67.2	132.3	28.0	24.3	62.3	0.0	9.2	60.0	10.6	31.1
f(l)	6.47	4.21	7.05	4.89	4.12	11.00	2.75	5.31	10.48	9.72
Rev. abs.	405	353	230	140	287	910	418	1074	199	157

f(l) = increase factor

Rev. abs. = absolute revertants

1001 colonies per plate means the bacteria growth was too strong for counting.

 

Diagnostic Mutagens (colonies per plate) – Second Experiment

Strain	TA97a		TA98		TA100		TA102		TA1535
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Substance	NPD	2-AA	NPD	BaP	Na-azide	2-AA	NPD	2-AA	Na-azide	2-AA
Repl. 1	788	468	632	268	688	696	1272	888	252	115
Repl. 2	476	404	560	280	728	760	1328	1368	320	142
Repl. 3	468	428	672	244	680	696	1416	1288	176	83
Mean	577	533	621	264	699	717	1339	1181	249	113
sd	182.5	32.3	56.8	18.3	25.7	37.0	72.6	257.2	72.0	29.5
f(l)	7.30	4.56	16.78	6.29	8.22	8.54	4.35	4.52	24.90	9.42
Rev. abs.	498	338	584	222	614	633	1031	920	239	101

f(l) = increase factor

Rev. abs. = absolute revertants

 

Historical Data of Spontaneous Revertants

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	89	94	20	22	92	96	279	297	18	18
	Min	60	63	6	8	51	64	85	67	6	7
	Max	144	138	52	51	141	141	425	511	31	33
	SD	18	16	11	10	15	14	58	69	6	6
	Exp 1	73	84	36	33	92	108	249	259	21	22
	Exp 2	79	76	37	37	85	93	308	284	10	12
DMSO	Mean	88	97	20	21	89	92	278	294	18	17
	Min	58	67	7	8	44	62	79	80	8	6
	Max	135	144	47	50	1369	199	393	459	33	32
	SD	17	17	11	10	16	17	56	61	6	6
	Exp 1	74	110	38	36	89	91	239	249	26	18
	Exp 2	79	95	37	42	75	84	308	261	11	12
Positive Controls*	Mean	537	520	407	113	490	764	1110	1213	264	132
	Min	264	237	100	39	220	273	491	408	55	45
	Max	1165	1181	1001	487	984	1912	2331	6083	515	7212
	SD	173	160	163	90	153	288	423	585	88	82
	Exp 1	479	463	268	176	379	1001	657	1323	220	175
	Exp 2	577	433	621	264	699	717	1339	1181	249	113

*Different positive controls were used

1001 colonies per plate mean the bacteria growth was too strong for counting.

Applicant's summary and conclusion

Conclusions:

The test item LUMULSE GMT-K showed no increase in the number of revertants in all bacteria strains in both experiments.
All negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that LUMULSE GMT-K is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Executive summary:

Title of Study: Determination of the mutagenic potential of LUMULSE GMT-K with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14

 

Findings and Results:

Two valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item LUMULSE GMT-K was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

Experiment 1:

In the first experiment, the test item (dissolved in DMSO) was tested up to concentrations of 5 μL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2:

Based on the first experiment, the test item was tested up to concentrations of 5 μL/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The test item showed signs of toxicity towards the bacteria strain TA100 in both the absence and presence of metabolic activation in the two highest concentrations (5 and 2.5 μL/plate).

The bacterial background lawn was not reduced at any of the concentrations.

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded that LUMULSE GMT-K is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.