Fatty acids, C10-12, esters with polylactic acid, sodium salts

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:

11 - 27 March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The lack of some identifiers of the test substance given in the study report is caused by the status of the test substance at the time of the test period. At that time the test substance was under development with regard to its detailed composition. At a later stage the test substance was identified as UVCB according to REACH regulation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Str. 7, 55116 Mainz, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Fatty acids, C10-12, esters with polylactic acid, sodium salts

EC Number:

700-937-1

Cas Number:

1312021-45-6

Molecular formula:

not available

IUPAC Name:

Fatty acids, C10-12, esters with polylactic acid, sodium salts

Test material form:

liquid: viscous

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: hisG46, uvrB, rfa

Species / strain 2

Species / strain / cell type:

S. typhimurium, other: TA 97a

Additional strain / cell type characteristics:

other: his D6610, uvrB, pKM 101, rfa

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: hisD3052, uvrB, pKM 101, rfa

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: hisG46, uvrB, pKM101, rfa

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: hisG428, pKM101, rfa

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

First experiment (plate incorporation method): 502, 151, 50, 15, 15, 5, 1.5 µg/plate
Second experiment (pre-incubation method): 498, 149, 50, 15, 5 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent:
DMSO was chosen as solvent, because the test item was completely soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants. For solution of the test item the stock solution was used. Each solution was membrane filtrated to accomplish sterility.

Controls

Untreated negative controls:

yes

Remarks:

Water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-o-phenylenediamine; 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
First experiment: in agar (plate incorporation)
Second experiment: preincubation method

DURATION
- Preincubation period: 10 h at 37 °C
- Exposure duration: 48 h at 37 °C
- Expression time (cells in growth medium): Befor estarting the experiment cell titre should give a density of 10 9 cells/mL at the least.

SELECTION AGENT (mutation assays): Depending on deficinecy of the individual strain used: ampicillin or ampicillin in combination with tetracyclin were added. TA 1535 was incubated without addition of antibiotica

NUMBER OF REPLICATIONS: Per strain and dose four strains with and four plates without S9 mix were used.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity control was performed by adding 500, 1500 and 500 µg/plate of the test item to the overnight culture diluted in sodium chloride solution with and without S9 on maximal soft agar. Normal growth was observed at a concentration not exceeding 500 µg/plat. Therefor this concentration was chosen as highest concentration in the test.

CONTROL TESTS TO CONFIRM COMPLIANCE OF THE RESULTS OBTAINED IN THE STUDY
- Genotype Confirmation. Genotype confirmation is performed once a term.
- Histidine requiremen: Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized wire loop.
- Ampicillin-Resistance (pKM 101) resp. ampicillin-tetracycline-resistance (pAQ1): The strains were streaked on ampicillin agar, TA102 on ampicillin-tetracycline agar. TA1535 was taking the function of control strain, since it is not ampicillin resistant.
- UV-sensitivity (uvrB): Two plates were streaked with the five strains, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates were irradiated for 8 seconds with a germicidal lamp (254 nm, 30W), keeping a distance of 33 cm. Incubation over night at 37 °C followed.
- Crystal violet sensitivity (deep rough): For each strain, two plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs (Æ9 mm), each soaked with 10 µl of crystal violet solution (0.1%) were placed into the middle of each plate, followed by incubation over night.
- Spontaneous revertants: Four replicates, with/without S9, for each solvent which was used in the test.
- Determination of Titre: The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. It should give a density of 109cells/mL (at the least).
- Toxicity Control: Performed analogously to the titre control with the maximum dose of test item with and without S9 on maximal-soft agar.
- Sterility Control: Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar.
- Positive Controls: Using diagnostic mutagens, four replicates were prepared. The stock solutions of the substances were diluted to effect an application volume of 0.1 mL/plate.
Without metabolic activation system
4-Nitro-1,2-phenylene diamine Concentration per plate: 80 µg; strains 97a, 98 and 102.
Sodium azide Concentration per plate: 6 µg; strains 100 and 1535.
With metabolic activation system
Benzo(a)pyrene Concentration per plate: 40 µg; strain 98.
2-Aminoanthracene (2-AA) Concentration per plate: 3 µg; strains 97a, 100, 102 and 1535.

Evaluation criteria:

A test item can be considered to have mutagneic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor > = 2) in at least one strain can be observed. A concentration - related increase over the range tested can also be taken as a sign of mutagenetic activity.

Statistics:

The colonies were counted visually, the numbers were recorded. A spreadsheet software (microsoft excel) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Exp. 2 highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Exp. 2 highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Exp. 2 highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Exp. 2 highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Exp. 2 highest concentration

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: see result on cytotoxicity test
COMPARISON WITH HISTORICAL CONTROL DATA:
In the following table, the mean of the spontaneous revertants and positive controls of all performed experiments in the year 2009 is stated (Number of experiments: 3) in compari-son with the experiments performed within this study.

Strain TA97a TA98 TA100 TA102 TA1535
Induction - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9
H2O Mean 105 105 7 8 119 112 215 225 11 13
Min 92 99 6 7 107 100 165 205 8 10
Max 124 109 9 10 130 122 261 237 14 16
Exp 1 92 108 6 10 120 113 220 237 8 10
Exp 2 124 109 9 7 107 100 165 205 14 14
DMSO Mean 123 95 7 8 121 123 247 223 10 11
Min 106 86 6 5 111 115 219 162 8 9
Max 133 108 8 10 138 131 269 253 13 13
Exp 1 133 86 6 8 111 115 219 253 8 12
Exp 2 129 90 7 10 115 131 269 162 9 9
Pos. Contr. Mean 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
Min 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
Max 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
Exp 1 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
Exp 2 1001 1001 1001 1001 1001 1001 1001 1001 1001 1001
In this table, “> 1000” is represented by “1001”.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
The toxicity of the following concentrations were tested: 5005, 1502 and 501 µg/plate.
Per strain, four plates were incubated with the corresponding dose of the test item on maximal soft agar.
16.1 Experimental Parameters
Date of treatment 11. March 2009
Concentrations tested 5005 / 1502 / 501µg/plate
Incubation time 48 hours
Incubation temperature 37 °C
Tester strains TA97a, TA98, TA100, TA102, TA1535
Method Plate incorporation method
In the two higher concentrations (5003 and 1502 µg/plate), the test item showed cytotoxic-ity towards all the strains. In the lower concentration (501 µg/plate), normal growth oc-curred.
On the base of these results, the first experiment was performed with 500 µg/plate as maximum dose for all strains.

Any other information on results incl. tables

Table 1: Mean revertants Experiment 1

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H20	Mean	92	108	6	10	120	113	220	237	8	10
	sd	32.0	23.5	1.7	3.8	7.2	4.4	46.5	43.4	3.7	2.4
DMSO	Mean	133	86	6	8	111	115	219	253	8	12
	sd	31.7	27.0	1.0	3.2	14.5	26.3	22.2	19.1	2.4	2.4
Pos.Contr.	Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
	sd	0	0	0	0	0	0	0	0	0	0
	f(I)	7.53	11.64	166.8	125.1	8.34	8.70	4.57	3.96	125.1	83.42
501 µg/pl.	Mean	107	100	6	7	139	133	206	220	6	9
	sd	7	25	3	4	9	23	4	9	4	2
	f(I)	0.80	1.16	1.00	0.88	1.25	1.16	0.94	0.87	0.75	0.75
151 µg/pl.	Mean	124	99	8	7	92	105	292	252	11	10
	sd	17	19	3	2	23	41	21	71	1	2
	f(I)	0.93	1.15	1.33	0.88	0.83	0.91	1.33	1.00	1.38	0.83
50 µg/pl.	Mean	80	112	7	5	111	102	250	257	9	5
	sd	28	36	1	1	13	11	99	53	2	2
	f(I)	0.60	1.30	1.17	0.63	1.00	0.89	1.14	1.02	1.13	0.42
15 µg/pl.	Mean	112	109	9	9	101	94	213	200	13	10
	sd	32	34	1	2	14	22	16	35	3	4
	f(I)	0.84	1.27	1.50	1.13	0.91	0.82	0.97	0.79	1.63	0.83
5 µg/pl.	Mean	114	112	8	9	89	98	201	189	7	11
	sd	19	37	2	2	24	13	27	26	4	2
	f(I)	0.86	1.30	1.33	1.13	0.80	0.85	0.92	0.75	0.88	0.92
1.5 µg/pl.	Mean	97	103	8	6	119	92	244	243	9	12
	sd	29	41	2	1	12	5	62	81	5	4
	f(I)	0.73	1.20	1.33	0.75	1.07	0.80	1.11	0.96	1.13	1.00

In this table, "> 1OOO" is represented by "1001". Induction factors for the respective treatments are stated as lower limit.

Table 2: Mean revertants Experiment 2

Strain				TA97a			TA98			TA100		TA102		TA1535
Induction				-S9		+S9	-S9		+S9	-S9	+S9	-S9	+S9	-S9	+S9
H20		Mean		99		108	10		9	122	130	220	232	11	13
		sd		9.8		10.7	3.1		3.5	13.4	15.2	8.3	8.2	1.7	2.9
DMSO		Mean		103		100	8		10	117	117	237	265	13	14
		sd		11.5		10.5	1.5		1.3	12.6	12.5	25.7	14.7	3.2	2.6
Pos.Contr.		Mean		1001		1001	1001		1001	1001	1001	1001	1001	1001	1001
		sd		0		0		0	0	0	0	0	0	0	0
		f(I)		9.72		10.01	125.1		100.1	8.20	8.56	4.22	3.78	91.00	71.50
498 µg/pl.		Mean			15	12	7		5	20	51	16	24	3	10
		sd			15	7	3		4	10	5	4	5	1	2
		f(I)		0.15		0.12	0.88		0.50	0.17	0.44	0.07	0.09	0.23	0.71
149 µg/pl.		Mean		129		112	9		5	119	121	256	228	10	11
		sd		26		9	1		1	24	49	40	27	4	1
		f(I)		1.25		1.12	1.13		0.50	1.02	1.03	1.08	0.86	0.77	0.79
50 µg/pl.			Mean	90		109	7		7	103	69	236	150	11	10
			sd	15		6	4		5	12	6	27	17	2	2
		f(I)		0.87		1.09	0.88		0.70	0.88	0.59	1.00	0.57	0.85	0.71
15 µg/pl.		Mean		137		113	9		5	93	98	151	186	8	8
		sd		7		23	1		2	21	15	13	28	2	2
		f(I)		1.33		1.13	1.13		0.50	0.79	0.84	0.64	0.70	0.62	0.57
5 µg/pl.		Mean		126		135	10		9	89	94	147	187	7	9
		sd		23		14	3		3	9	14	14	52	2	5
		f(I)		1.22		1.35	1.25		0.90	0.76	0.80	0.62	0.71	0.54	0.64

In this table, "> 1OOO" is represented by "1001". Induction factors for the respective treatments are stated as lower limit.

Table 3: Historical control data

Strain			TA97a		TA98		TA100		TA102			TA1535
Induction			-S9	+S9	-S9	+S9	-S9	+S9	-S9		+S9	-S9	+S9
H20		Mean	105	105	7	8	119	112	215		225	11	13
		Min	92	99	6	7	107	100		165	205	8	10
		Max	124	109	9	10	130	122	261		237	14	16
		Exo 1	92	108	6	10	120	113	220		237	8	10
		Exp 2	124	109	9	7	107	100	165		205	14	14
DMSO		Mean	123	95	7	8	121	123	247		223	10	11
		Min	106	86	6	5	111	115	219		162	8	9
		Max	133	108	8	10	138	131	269		253	13	13
		Exo 1	133	86	6	8	111	115	219		253	8	12
		Exo 2	129	90	7	10	115	131	269		162	9	9
		Mean	1001	1001	1001	1001	1001	1001	1001		1001	1001	1001
Pos. Contr.		Min	1001	1001	1001	1001	1001	1001	1001		1001	1001	1001
		Max	1001	1001	1001	1001	1001	1001	1001		1001	1001	1001
		Exp 1	1001	1001	1001	1001	1001	1001	1001		1001	1001	1001
		Exo 2	1001	1001	1001	1001	1001	1001	1001		1001	1001	1001

In this table, "> 1OOO" is represented by "1001".

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Executive summary:

The test item did not show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only). Cytotoxicity of the test item was detected in the second experiment in the highest concentration 498 µg/plate. The background lawn was visible but the number of revertants was decreased. In the lower concentrations, normal growth occurred.

The test item is considered not mutagenic for the reasons given above.

In the cytotoxicity experiment, the test item showed cytotoxicity towards the bacteria in the follow concentrations: 5000 µg/plate and 1500 µg/plate; on the base of these results the highest concentration in the first experiment was chosen with 500 µg/plate. The test item showed any cytotoxicity towards the bacteria in the second experiment (using the pre-incubation method) in the highest concentration (500 µg/plate). The lower concentrations showed normal growth.

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value. The number of revertant colonies of the positive controls was in the range of the historical data of the laboratory and the revertants were increased in comparison with the negative controls, as well as showing mutagenous potential of the diagnostic mutagenes. Some of the spontaneous revertants are lower than the values given by Prof. Ames; in comparison with the historical data of the LAUS GmbH, they were within the normal range. For these reasons, the result of the test is considered valid.

Therefore it can be stated, that under the test conditions, the test item is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.