[No public or meaningful name is available]

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:

05 May 2008 to 29 May 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Signature: 15/10/2007; Date of Inspection: 21/08/2007

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of disodium N-(1-oxooctadecyl)-L-glutamate and disodium N-(1-oxohexadecyl)-L-glutamate

EC Number:

700-014-3

Molecular formula:

See remarks

IUPAC Name:

Reaction mass of disodium N-(1-oxooctadecyl)-L-glutamate and disodium N-(1-oxohexadecyl)-L-glutamate

Method

Target gene:

Histidine operon for Salmonella triphimurium
Tryptophan operon for Escherichia coli

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test:
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 1 (Range finding test)
Salmonella strains, with and without S9: 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
E. coli strain WP2uvrA-, with and without S9: 50, 150, 500, 1500, 5000 µg/plate.

Experiment 2 (Main test)
Salmonella strains, with and without S9: 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
E. coli strain WP2uvrA-, with and without S9: 50, 150, 500, 1500, 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 12.5 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-Nitrosoguanidine 2 µg/plate for WP2uvrA-, 3 µg/plate for TA100 and 5µg/plate for TA1535.

Remarks:

Without S9

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 Migrated to IUCLID6: 80 µg/plate for TA1537

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide 0.2 µg/plate for TA98

Remarks:

Without S9

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2 Aminoanthracene (2AA) at 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 and 10 µg/plate for WP2uvrA-

Remarks:

With S9

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 Migrated to IUCLID6: 5 µg/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 to 72 hours

NUMBER OF REPLICATIONS: triplicate plating

DETERMINATION OF CYTOTOXICITY
- Method: lawn deficiency and colony reduction

Evaluation criteria:

Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method with historical control ranges from 2006 and 2007 were used.
The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10^9 bacteria per mL.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The positive control historical ranges were used from 2006 and 2007.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation Criteria
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (5) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none reported
- Effects of osmolality: none reported
- Evaporation from medium: none reported
- Water solubility: The test material was fully soluble in sterile distilled water at 12.5 mg/mL in solubility checks performed in-house.
- Precipitation: A pale flakey precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity test:
The test material caused a reduction in the frequency of TA100 revertant colonies and/or a weakening of the background lawn, initially from 500 µg/plate. Substantial reductions in the frequency of WP2uvrA- revertant colonies were also noted at 5000 µg/plate. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
None

CONTROLS
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused a visible reduction in the growth of the bacterial background lawn and/or a substantial decrease in the frequency of revertant colonies for all of the Salmonella strains in both the presence and absence of S9, initially from 500 µg/plate. Small decreases in revertant colony frequency were also noted for E.coli strain WP2uvrA- at 5000 µg/plate in both the presence and absence of S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposure with or without S9 and experiment number. 
A pale flakey precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a substantial decrease in the frequency of revertant colonies for all of the Salmonella strains in both the presence and absence of S9, initially from 500 µg/plate. Small decreases in revertant colony frequency were also noted for E .coli strain WP2uvrA^- at 5000 µg/plate in both the presence and absence of S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposure with or without S9 and experiment number. 

A pale flakey precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1: Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
84		14		29		19		7
95	(94)	18	(16)	22	(26)	20	(19)	5	(6)
102		16		26		19		7

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
137		21		34		15		12
140	(136)	18	(19)	26	(35)	8	(11)	11	(12)
131		19		45		11		13

Table2: Test Results: Range-Finding Test– Without Metabolic Activation

Test Period		From: 19 May 2008					To: 22 May 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
-	0	108 91 97	(99) 8.6#	12 14 13	(13) 1.0	37 20 15		(24) 11.5	12 11 16	(13) 2.6	3 5 8	(5) 2.5
-	5	86 106 92	(95) 10.3	13 15 13	(14) 1.2	N/T			11 11 12	(11) 0.6	4 5 4	(4) 0.6
-	15	97 90 111	(99) 10.7	11 9 18	(13) 4.7	N/T			15 18 15	(16) 1.7	3 7 8	(6) 2.6
-	50	118 112 88	(106) 15.9	19 10 10	(13) 5.2	14 32 19		(22) 9.3	16 15 14	(15) 1.0	5 2 3	(3) 1.5
-	150	87 106 89	(94) 10.4	7 7 11	(8) 2.3	26 24 15		(22) 5.9	13 11 10	(11) 1.5	1 5 4	(3) 2.1
-	500	38 41 41	(40) 1.7	2 10 2	(5) 4.6	20 22 15		(19) 3.6	8 2 2	(4) 3.5	1 1 5	(2) 2.3
-	1500	10 P* 9 P* 14 P*	(11) 2.6	1 P* 1 P* 1 P*	(1) 0.0	15 P 13 P 13 P		(14) 1.2	5 P 8 P 0 P	(4) 4.0	0 P 0 P 1 P	(0) 0.6
-	5000	0 P* 0 P* 0 P*	(0) 0.0	0 P* 0 P* 0 P*	(0) 0.0	9 P 5 P 10 P		(8) 2.6	0 P* 0 P* 0 P*	(0) 0.0	0 P* 0 P* 0 P*	(0) 0.0
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		682 696 685	(688) 7.4	354 375 305	(345) 35.9	881 841 654		(792) 121.2	588 129 155	(291) 257.8	1168 1144 1107	(1140) 30.7

ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

N/T = Not tested at this dose level

P = Precipitate

* = Partial or complete absence of bacterial background lawn

# = Standard deviation

Table3: Test Results: Range-Finding Test– With Metabolic Activation

Test Period		From: 19 May 2008					To: 22 May 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
+	0	92 97 77	(89) 10.4#	15 15 12	(14) 1.7	22 29 18		(23) 5.6	26 30 32	(29) 3.1	7 7 7		(7) 0.0
+	5	71 90 90	(84) 11.0	9 7 8	(8) 1.0	N/T			31 22 24	(26) 4.7	13 9 3		(8) 5.0
+	15	75 99 96	(90) 13.1	3 9 10	(7) 3.8	N/T			16 21 15	(17) 3.2	10 4 4		(6) 3.5
+	50	80 81 77	(79) 2.1	8 4 8	(7) 2.3	31 21 22		(25) 5.5	21 23 12	(19) 5.9	7 7 5		(6) 1.2
+	150	80 95 80	(85) 8.7	9 7 2	(6) 3.6	21 22 14		(19) 4.4	20 14 19	(18) 3.2	9 8 3		(7) 3.2
+	500	97 89 75	(87) 11.1	3 2 4	(3) 1.0	18 14 18		(17) 2.3	16 27 16	(20) 6.4	4 3 4		(4) 0.6
+	1500	26 P 30 P 43 P	(33) 8.9	4 P* 2 P* 4 P*	(3) 1.2	14 P 12 P 7 P		(11) 3.6	8 P 13 P 10 P	(10) 2.5	8 P 4 P 4 P		(5) 2.3
+	5000	0 P* 0 P* 0 P*	(0) 0.0	0 P* 0 P* 0 P*	(0) 0.0	4 P 16 P 14 P		(11) 6.4	0 P* 0 P* 0 P*	(0) 0.0	0 P* 0 P* 0 P*		(0) 0.0
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		2094 1811 1780	(1895) 173.0	310 198 288	(265) 59.3	508 475 435		(473) 36.6	216 222 224	(221) 4.2	275 365 197	(279) 84.1

BP = Benzo(a)pyrene

2AA = 2-Aminoanthracene

N/T = Not tested at this dose level

P = Precipitate

* = Partial absence of bacterial background lawn

# = Standard deviation

Table4: Test Results: Main Test– Without Metabolic Activation

Test Period		From: 26 May 2008					To: 29 May 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
-	0	135 172 134	(147) 21.7#	20 20 22	(21) 1.2	33 31 31		(32) 1.2	12 21 13	(15) 4.9	9 22 11		(14) 7.0
-	5	153 142 123	(139) 15.2	16 20 15	(17) 2.6	N/T			19 10 14	(14) 4.5	9 16 13		(13) 3.5
-	15	129 112 140	(127) 14.1	25 20 15	(20) 5.0	N/T			11 14 10	(12) 2.1	14 14 7		(12) 4.0
-	50	121 136 119	(125) 9.3	25 18 24	(22) 3.8	31 36 20		(29) 8.2	19 12 12	(14) 4.0	15 9 9		(11) 3.5
-	150	119 111 146	(125) 18.3	20 10 18	(16) 5.3	27 29 32		(29) 2.5	9 19 15	(14) 5.0	11 11 9		(10) 1.2
-	500	56 74 68	(66) 9.2	2 3 7	(4) 2.6	38 48 36		(41) 6.4	13 9 9	(10) 2.3	0 0 0		(0) 0.0
-	1500	27*P 18*P 25*P	(23) 4.7	0*P 0*P 0*P	(0) 0.0	23 P 25 P 30 P		(26) 3.6	7 P 10 P 2 P	(6) 4.0	0*P 0*P 0*P		(0) 0.0
-	5000	0*P 0*P 0*P	(0) 0.0	0*P 0*P 0*P	(0) 0.0	25P 25P 13P		(21) 6.9	0*P 0*P 0*P	(0) 0.0	0*P 0*P 0*P		(0) 0.0
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		472 498 578	(516) 55.2	255 183 196	(211) 38.4	715 713 730		(719) 9.3	112 111 121	(115) 5.5	2294 2350 1477	(2040) 488.7

ENNG = N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-Aminoacridine

N/T = Not tested at this dose level

P = Precipitate

* = Partial or complete absence of bacterial background lawn

# = Standard deviation

Table5: Test Results: Main Test– With Metabolic Activation

Test Period		From: 26 May 2008					To: 29 May 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
+	0	99 126 128	(118) 16.2#	8 16 16	(13) 4.6	40 42 41		(41) 1.0	34 29 23	(29) 5.5	13 21 18		(17) 4.0
+	5	128 117 117	(121) 6.4	9 10 14	(11) 2.6	N/T			27 26 23	(25) 2.1	16 18 21		(18) 2.5
+	15	125 114 118	(119) 5.6	10 11 18	(13) 4.4	N/T			23 21 23	(22) 1.2	14 10 15		(13) 2.6
+	50	154 115 120	(130) 21.2	13 8 11	(11) 2.5	34 37 31		(34) 3.0	22 22 16	(20) 3.5	12 12 14		(13) 1.2
+	150	109 109 113	(110) 2.3	19 10 9	(13) 5.5	38 40 32		(37) 4.2	27 21 24	(24) 3.0	26 13 16		(18) 6.8
+	500	122 123 141	(129) 10.7	8 9 11	(9) 1.5	29 43 38		(37) 7.1	C 26 33	(30) 4.9	9 9 10		(9) 0.6
+	1500	95 P 102 P 76 P	(91) 13.5	7*P 12*P 4*P	(8) 4.0	24 P 24 P 23 P		(24) 0.6	22 P 24 P 30 P	(25) 4.2	12*P 11*P 9*P		(11) 1.5
+	5000	0*P 0*P 0*P	(0) 0.0	0*P 0*P 0*P	(0) 0.0	17P 26P 28P		(24) 5.9	0*P 0*P 0*P	(0) 0.0	0*P 0*P 0*P		(0) 0.0
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		2319 4009 2002	(2777) 1078.9	397 379 340	(372) 29.1	451 494 472		(472) 21.5	148 179 177	(168) 17.3	330 419 653	(467) 166.8

BP = Benzo(a)pyrene

2AA = 2-Aminoanthracene

C = Contaminated

N/T = Not tested at this dose level

P = Precipitate

* = Partial absence of bacterial background lawn

# = Standard deviation

Applicant's summary and conclusion

Conclusions:

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

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA^- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels (5 and 15 µg/plate) were included (where applicable) to allow for test material induced toxicity, in Salmonella typhimurium strains only, ensuring that at least four non‑toxic doses were achieved.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn and/or a substantial decrease in the frequency of revertant colonies for all of the Salmonella strains in both the presence and absence of S9, initially from 500 µg/plate. Small decreases in revertant colony frequency were also noted for E.coli strain WP2uvrA^-at 5000 µg/plate in both the presence and absence of S9. The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposure with or without S9 and experiment number. 

A pale flakey precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.