Alcohols, C18-unsatd and ethoxylated C18-unsatd., phosphates (5 moles ethoxylation)

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:

From June 14, 2017 to July 20, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

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:

Alcohols, C18-unsatd and ethoxylated C18-unsatd., phosphates (5 moles ethoxylation)

Molecular formula:

C18H37O4P (representative of phosphate monoesters; i.e., mono- C18-unsatd. PSE) C28H57O9P1 (representative of ethoxylated phosphate monoesters; i.e., mono- C18-unsatd. AE5 PSE) C36H71O4P1 (representative of phosphate diesters; i.e., di- C18-unsatd. PSE) C46H91O8P1 (representative ethoxylated phosphate diesters; di- C18-unsatd. PSE and C18-unsatd. AE5 PSE) C82H161O21P1 (representative ethoxylated phosphate diesters; di- C18-unsatd. AE5 PSE)

IUPAC Name:

Alcohols, C18-unsatd and ethoxylated C18-unsatd., phosphates (5 moles ethoxylation)

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Confirmatory Experiment 1: 50, 100, 150, 200, 300, 500 µg/plate (absence of S9) and 150, 300, 500, 750, 1000, 1500 µg/plate (presence of S9)
Confirmatory Experiment 2: 5, 15, 50, 150, 500, 1500, 5000 µg/plate (Presence and absence of S9)
The maximum concentration was 5000 µg/plate (the maximum recommended dose level).

Vehicle / solvent:

The test substance was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in house. Acetone was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Identity: Acetone, Supplier: Fisher Scientific, Batch number (purity): 1679335 (99.98%), Expiry: 11/2021

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: Identity: 2-Aminoanthracene (2AA), CAS No.: 613-13-8, Batch number: STBB1901M9, Purity: 97.5%, Expiry date: 08 October 2017, Solvent: DMSO, Concentration: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537 , 10 µg/plate for WP2uvrA

Details on test system and experimental conditions:

Bacteria
The five strains of bacteria used, and their mutations, are as follows:
Salmonella typhimurium
Strains - Genotype - Type of mutations indicated
TA1537 - his C 3076; rfa-; uvrB-: - frame shift
TA98 - his D 3052; rfa-; uvrB-; - R-factor
TA1535 - his G 46; rfa-; uvrB-: - base-pair substitution
TA100 - his G 46; rfa-; uvrB-;R-factor

Escherichia coli
Strain - Genotype - Type of mutations indicated
WP2uvrA - trp-; uvrA-: - base-pair substitution

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).

The bacteria used in the test were obtained from:
1) University of California, Berkeley, on culture discs, on 04 August 1995.
2) British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1865318 05/21) and incubated at 37 °C for approximately 10 h. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Sterility controls
The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2) A reproducible increase at one or more concentrations.
3) Biological relevance against in-house historical control ranges.
4) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5) Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test substance 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 making a definite judgment about test substance activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Key result

Species / strain:

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

Any other information on results incl. tables

Results

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test substance formulation was also shown to be sterile. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data were for concurrent untreated control plates performed on the same day as the Mutation Test. The vehicle (acetone) 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 maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method), although small reductions in revertant colony frequency were noted to TA1535 and TA1537 at 5000 µg/plate in both the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test substance being tested up to the maximum recommended dose level of 5000 µg/plate in Experiment 2. The test substance induced a much stronger toxic response in Experiment 2 after employing the pre-incubation modification with weakened bacterial background lawns and/or reductions in revertant colony frequency initially noted in the absence of S9‑mix from 500 µg/plate (TA1535), 1500 µg/plate (TA1537) and at 5000 µg/plate (TA100). In the presence S9-mix, weakened bacterial background lawns and/or reductions in revertant colony frequency were initially noted from 1500 µg/plate (TA1535) and at 5000 µg/plate (TA1537). No toxicity was noted toany of the remaining tester strainsat any test substance dose level in either the absence or presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9-mix and experimental methodology. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

 

Experiment 1 (plate incorporation)

The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. In both the absence and presence of S9, there were no biologically relevant increases in the frequency of revertant colonies at any test substance dose level. Small, statistically significant increases in TA100 revertant colony frequency were observed at 5 and 15 µg/plate in the absence of S9-mix. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the mean maximum fold increase was only 1.2 times the concurrent vehicle control. In light of these results, Experiment 2 was conducted using the pre-incubation method.

Table 1: Test results: Experiment 1 – without metabolic activation (plate incorporation)

Test Period		From: 21 June 2017					To: 24 June 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	103 106 108	(106) 2.5#	9 10 17	(12) 4.4	36 47 39		(41) 5.7	26 26 26	(26) 0.0	17 13 11	(14) 3.1
	1.5 µg	102 107 118	(109) 8.2	7 16 10	(11) 4.6	37 36 42		(38) 3.2	17 28 25	(23) 5.7	11 14 16	(14) 2.5
	5 µg	118 128 133	** (126) 7.6	11 11 17	(13) 3.5	40 31 36		(36) 4.5	17 19 30	(22) 7.0	12 13 13	(13) 0.6
	15 µg	129 120 124	* (124) 4.5	10 11 18	(13) 4.4	44 43 41		(43) 1.5	26 34 28	(29) 4.2	12 14 11	(12) 1.5
	50 µg	120 119 119	(119) 0.6	11 10 17	(13) 3.8	34 34 35		(34) 0.6	28 23 27	(26) 2.6	16 13 12	(14) 2.1
	150 µg	119 119 106	(115) 7.5	11 16 13	(13) 2.5	39 36 34		(36) 2.5	32 25 22	(26) 5.1	8 11 13	(11) 2.5
	500 µg	129 118 114	(120) 7.8	7 8 16	(10) 4.9	36 39 34		(36) 2.5	22 15 24	(20) 4.7	6 13 15	(11) 4.7
	1500 µg	103 114 116	(111) 7.0	14 17 10	(14) 3.5	36 31 31		(33) 2.9	15 25 27	(22) 6.4	11 8 10	(10) 1.5
	5000 µg	78 98 96	(91) 11.0	6 5 7	(6) 1.0	33 29 33		(32) 2.3	15 23 16	(18) 4.4	7 1 3	(4) 3.1
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		595 593 594	(594) 1.0	424 475 494	(464) 36.2	525 363 468		(452) 82.2	282 275 308	(288) 17.4	412 417 292	(374) 70.8

ENNG : N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO : 4-Nitroquinoline-1-oxide

9AA : 9-Aminoacridine

* : p≤0.05

** : p≤0.01

# : Standard deviation

Table 2: Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 21 June 2017					To: 24 June 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	121 111 127	(120) 8.1#	15 16 8	(13) 4.4	40 40 42		(41) 1.2	28 28 24	(27) 2.3	9 10 13	(11) 2.1
	1.5 µg	108 98 106	(104) 5.3	16 7 17	(13) 5.5	42 48 44		(45) 3.1	24 25 16	(22) 4.9	15 14 9	(13) 3.2
	5 µg	115 133 114	(121) 10.7	16 16 14	(15) 1.2	42 37 37		(39) 2.9	22 19 21	(21) 1.5	17 13 14	(15) 2.1
	15 µg	116 131 128	(125) 7.9	5 16 18	(13) 7.0	38 39 45		(41) 3.8	31 18 28	(26) 6.8	11 13 14	(13) 1.5
	50 µg	126 120 122	(123) 3.1	10 16 16	(14) 3.5	49 34 46		(43) 7.9	29 26 26	(27) 1.7	15 14 11	(13) 2.1
	150 µg	120 110 115	(115) 5.0	9 11 21	(14) 6.4	43 47 43		(44) 2.3	29 20 19	(23) 5.5	9 8 11	(9) 1.5
	500 µg	117 127 122	(122) 5.0	9 12 11	(11) 1.5	38 43 42		(41) 2.6	29 25 19	(24) 5.0	13 11 12	(12) 1.0
	1500 µg	123 144 123	(130) 12.1	11 10 11	(11) 0.6	36 41 40		(39) 2.6	25 24 20	(23) 2.6	6 5 6	(6) 0.6
	5000 µg	92 96 101	(96) 4.5	4 7 10	(7) 3.0	46 29 47		(41) 10.1	22 20 18	(20) 2.0	2 5 2	(3) 1.7
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1291 1401 1421	(1371) 70.0	256 247 265	(256) 9.0	218 246 222		(229) 15.1	206 202 195	(201) 5.6	380 432 376	(396) 31.2

BP : Benzo(a)pyrene

2AA : 2-Aminoanthracene

# : Standard deviation

Experiment 2 (pre-incubation)

The maximum dose level of the test substance in the second experiment was the same as for Experiment 1 (5000 µg/plate). In the absence of S9, there were statistically significant increases in the mean number of revertant colonies at 150, 500, 1500 and 5000 µg/plate with strain TA1535.The increases at 500, 1500 and 5000 µg/plate were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore these responses would be due to additional histidine being available to His^- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.The revertant counts noted at 150 µg/plate were in excess of two fold the concurrent vehicle control and were also above the in-house vehicle and untreated historical values.In the presence of S9, there were statistically significant increases in the mean number of revertant colonies at 500, 1500 and 5000 µg/plate with strain TA1535. The counts at 1500 and 5000 µg/plate were considered to have no biological relevance as weakened bacterial background lawns were also noted. The mean values at 500 µg/plate were in excess of twofold the concurrent vehicle control and also in excess of the maximum historical control value for the strain, therefore the results for TA1535 were investigated in further experiments. The increases observed with TA1535 were considered to be of no biological relevance, as there was no clear dose-response relationship and, more importantly, the results could not be reproduced in two confirmatory tests discussed below (Experiments 3 and 4), one of which contained extra intermediate test substance concentration levels and one of which was a direct repeat of Experiment 2, in an attempt to qualify the response. There are no obvious reasons for these increases noted with the TA1535 culture. Many different sized colonies were noted at the statistically significant dose levels in this tester strain, which may confirm the possibility that there may have been underlying contamination present which was indistinguishable from the revertant colonies; however, the culture was considered valid on the day of scoring as the untreated and vehicle control counts were within the in-house untreated/vehicle historical control data. Furthermore, the responses noted for TA1535 in Experiment 2 were considered false due to the fact that the test substance exhibited toxicity under certain circumstances and exposure conditions (a much stronger toxic response was noted after performing the pre‑incubation modification), therefore the increases in revertant colony frequency noted at 150 µg/plate (absence of S9) and 500 µg/plate (presence of S9) may have been an artefact resulting from a modest level of toxicity to the tester strain at the upper test substance dose levels. Even though weakened background lawns were not noted in the original test there may have been enough weakening (toxicity) to induce a ‘false’ response. A minor increase was also noted for TA100 at 5 µg/plate in the presence of S9-mix, however these increases were within the in-house historical untreated/vehicle control range for the tester strain.

Table 3: Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 03 July 2017					To: 06 July 2017
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	82 87 87	(85) 2.9#	22 12 13	(16) 5.5	23 13 26		(21) 6.8	19 14 29	(21) 7.6	12 18 23	(18) 5.5
	5 µg	76 73 67	(72) 4.6	27 23 12	(21) 7.8	21 22 13		(19) 4.9	11 15 22	(16) 5.6	11 17 18	(15) 3.8
	15 µg	71 79 77	(76) 4.2	23 23 28	(25) 2.9	20 23 18		(20) 2.5	22 22 18	(21) 2.3	11 15 21	(16) 5.0
	50 µg	79 81 98	(86) 10.4	21 15 21	(19) 3.5	31 33 19		(28) 7.6	19 10 13	(14) 4.6	14 8 18	(13) 5.0
	150 µg	111 73 72	(85) 22.2	43 47 41	*** (44) 3.1	33 25 25		(28) 4.6	16 6 12	(11) 5.0	5 5 3	(4) 1.2
	500 µg	56 58 46	(53) 6.4	82 S 107 S 102 S	*** (97) 13.2	20 19 23		(21) 2.1	12 13 10	(12) 1.5	16 12 7	(12) 4.5
	1500 µg	60 45 54	(53) 7.5	171 S 222 S 213 S	*** (202) 27.2	26 26 18		(23) 4.6	17 19 14	(17) 2.5	5 8 9	(7) 2.1
	5000 µg	25 38 35	(33) 6.8	242 S 231 S 212 S	*** (228) 15.2	22 33 23		(26) 6.1	15 21 17	(18) 3.1	3 S 2 S 8 S	(4) 3.2
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		810 749 864	(808) 57.5	1117 894 958	(990) 114.8	684 662 678		(675) 11.4	247 271 281	(266) 17.5	457 278 458	(398) 103.6

ENNG : N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO : 4-Nitroquinoline-1-oxide

9AA : 9-Aminoacridine

S : Sparse bacterial background lawn

*** : p≤0.001

# : Standard deviation

Table 4: Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 03 July 2017					To: 06 July 2017
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Acetone)	74 63 83	(73) 10.0#	26 24 31	(27) 3.6	27 32 24		(28) 4.0	17 19 19	(18) 1.2	18 22 22	(21) 2.3
	5 µg	84 104 117	* (102) 16.6	35 29 31	(32) 3.1	25 30 30		(28) 2.9	20 21 29	(23) 4.9	26 21 22	(23) 2.6
	15 µg	80 81 82	(81) 1.0	33 42 40	(38) 4.7	26 25 23		(25) 1.5	23 20 27	(23) 3.5	21 25 15	(20) 5.0
	50 µg	100 84 67	(84) 16.5	37 43 22	(34) 10.8	27 18 15		(20) 6.2	20 25 23	(23) 2.5	26 12 26	(21) 8.1
	150 µg	76 78 71	(75) 3.6	25 33 51	(36) 13.3	28 26 27		(27) 1.0	23 19 25	(22) 3.1	13 20 8	(14) 6.0
	500 µg	80 73 64	(72) 8.0	74 35 69	** (59) 21.2	28 29 23		(27) 3.2	28 17 21	(22) 5.6	22 9 19	(17) 6.8
	1500 µg	68 61 78	(69) 8.5	71 S 55 S 57 S	** (61) 8.7	20 25 19		(21) 3.2	20 19 18	(19) 1.0	17 19 20	(19) 1.5
	5000 µg	54 76 52	(61) 13.3	90 S 93 S 123 S	*** (102) 18.2	32 40 20		(31) 10.1	14 15 15	(15) 0.6	7 12 4	(8) 4.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1282 1284 1364	(1310) 46.8	260 274 252	(262) 11.1	114 91 120		(108) 15.3	235 199 199	(211) 20.8	415 350 387	(384) 32.6

2AA : 2-Aminoanthracene

BP : Benzo(a)pyrene

S : Sparse bacterial background lawn

* : p≤0.05

** : p≤0.01

*** : p≤0.001

# : Standard deviation

Experiment 3 (Confirmatory Test 1, (pre-incubation))

The dose range for this experiment was determined by the results of Experiment 2 in tester strain TA1535 (with and without S9-mix). Intermediate dose levels were included in an effort to confirm and/or enhance the results obtained previously in Experiment 2. There were no biologically relevant increases in revertant colony frequency observed at any of the dose levels tested.

Table 5: Test Results: Experiment 3 – (Confirmatory Test 1 – Pre-Incubation) With Metabolic Activation

Test Period		From: 10 July 2017		To: 13 July 2017
	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strain
		Without S9-mix		With S9-mix
		TA1535			TA1535
	Solvent Control (Acetone)	10 7 7	(8) 1.7#		11 13 11	(12) 1.2
	50 µg	7 8 8	(8) 0.6		N/T
	100 µg	11 8 13	(11) 2.5		N/T
	150 µg	10 10 14	(11) 2.3		13 14 18	(15) 2.6
	200 µg	16 12 9	(12) 3.5		N/T
	300 µg	9 9 9	(9) 0.0		11 15 17	(14) 3.1
	500 µg	11 7 14	(11) 3.5		15 14 18	(16) 2.1
	750 µg	N/T			18 13 16	(16) 2.5
	1000 µg	N/T			14 15 18	(16) 2.1
	1500 µg	N/T			17 17 12	(15) 2.9
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	ENNG			2AA
		5 µg			2 µg
		438 383 470	(430) 44.0		205 216 241	(221) 18.4

ENNG : N-ethyl-N'-nitro-N-nitrosoguanidine

2AA : 2-Aminoanthracene

N/T : Not tested at this dose level

# : Standard deviation

Experiment 4 (Confirmatory Test 2, pre-incubation))

A second confirmatory test was performed in TA1535 (with and without S9-mix) in an effort to attain reproducibility between two conflicting results employing the same dose range as Experiment 2. Again, no biologically relevant increases in revertant colony frequency were observed at any dose level tested. Statistical values were noted at 1500 and 5000 µg/plate (absence and presence of S9), however these counts were considered to have no biological relevance as weakened bacterial background lawns were also noted.Small, statistically significant increases were noted at 500 µg/plate (presence of S9), however these responses did not achieve a twofold increase over theconcurrent vehicle control and 2 out of 3 individual revertant counts (and the mean value)were inside the 2016 in-house vehicle and untreated historical values.

 

Table 6: Test Results: Experiment 4 – (Confirmatory Test 2 – Pre-Incubation) With Metabolic Activation

Test Period		From: 17 July 2017		To: 20 July 2017
	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strain
		Without S9-mix		With S9-mix
		TA1535			TA1535
	Solvent Control (Acetone)	24 22 23	(23) 1.0#		24 23 29	(25) 3.2
	5 µg	20 22 25	(22) 2.5		28 29 28	(28) 0.6
	15 µg	22 12 14	(16) 5.3		26 29 22	(26) 3.5
	50 µg	17 22 15	(18) 3.6		29 24 28	(27) 2.6
	150 µg	24 20 24	(23) 2.3		26 24 28	(26) 2.0
	500 µg	27 S 25 S 26 S	(26) 1.0		29 36 45	* (37) 8.0
	1500 µg	46 S 48 S 54 S	*** (49) 4.2		52 S 57 S 44 S	*** (51) 6.6
	5000 µg	85 S 86 S 69 S	*** (80) 9.5		60 S 62 S 47 S	*** (56) 8.1
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	ENNG			2AA
		2 µg			10 µg
		1455 1468 1251	(1391) 121.7		295 180 298	(258) 67.3

ENNG : N-ethyl-N'-nitro-N-nitrosoguanidine

2AA : 2-Aminoanthracene

S : Sparse bacterial background lawn

* : p≤0.05

*** : p≤0.001

# : Standard deviation

Conclusion

No biologically relevant, reproducible or dose-related increases in revertant colony numbers were observed in any strain tested, after exposure to test substance at up to 5000 µg/plate. It was, therefore, concluded that test substance was non-mutagenic at concentrations up to 5000 μg/plate, in the absence or presence of metabolic activation under the conditions of this test.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was considered to be non-mutagenic with and without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the mutagenic potential of the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE', using bacterial reverse mutation assay (Ames test), according to OECD Guideline 471, EU Method B13/14 and the USA, EPA OCSPP harmonized guideline, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels (i.e., 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate), in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (i.e., 10% liver S9 in standard co-factors). The doses for Experiment 1 (plate incorporation method) was predetermined and ranged from 1.5 to 5000 µg/plate. The maximum dose level of the test substance in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended (5 to 5000 µg/plate) following the results of Experiment 1. Seven test substance concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test substance following the change in test methodology. Two further experiments (Confirmatory Test 1 and Confirmatory Test 2) were performed using the pre-incubation method, in triplicate, in order to assess the reproducibility of non-dose related increases in TA1535 revertant colony frequency, seen in both the absence and presence of S9, in Experiment 2.

The vehicle (acetone) 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. In Experiment 1, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), although small reductions in revertant colony frequency were noted to TA1535 and TA1537 at 5000 µg/plate in both the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test substance being tested up to the maximum recommended dose level of 5000 µg/plate in Experiment 2.The test substance induced a much stronger toxic response in Experiment 2. Only weakened bacterial background lawns and/or reductions in revertant colony frequency was noted with TA1535 at 500 µg/plate (TA1535),1500 µg/plate (TA1537) and 5000 µg/plate (TA100) in the absence of S9 mix and at 1500 µg/plate (TA1535) and 5000 µg/plate (TA1537), in the presence of S9 mix, which were not considered to be biologically relevant. These responses were considered to be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies. Further, the revertant counts noted at 150 µg/plate (without S9) and 500 µg/plate (with S9) were in excess of twofold the concurrent vehicle control and were also above the in-house vehicle and/or untreated historical values for the strain TA1535. However, these results could not be reproduced in two confirmatory tests (Experiments 3 and 4), one of which contained extra intermediate test substance concentration levels and one of which was a direct repeat of Experiment 2, in an attempt to qualify the response. Therefore, the increases observed in TA 1535 were considered to be of no biological relevance. No toxicity was noted to any of the remaining tester strains at any test substance dose level in either the absence or presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test substance varied slightly between strain type, exposures with or without S9-mix and experimental methodology. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation (Envigo, 2017).