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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on results of the in vitro studies on the test substance as well as read across substances, the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE', is considered to be non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From August 04, 2004 to September 08, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Lot no.: P-2903
Appearance: viscous liquid

Target gene:

Histidine

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

- In the preliminary assay, the solubility of the test substance was assessed in different solvents. The maximum dose tested was 5000 μg per plate.
- In the main mutagenicity assay, the dose levels tested were up to 5000 μg per plate (based on solubility and precipitaton results from the preliminary assay).

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

- Dose levels up to 5000 µg/L of test substance along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test substance, vehicle control and positive controls were plated in triplicate.
- The plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Rationale for test conditions:

Solubility and precipitation of the test substance (preliminary test)

Evaluation criteria:

- Scoring:
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.
- Evaluation:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Key result

Species / strain:

other: S. typhymurium TA98, TA100, TA1535 and TA1537 + E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No positive mutagenic response was observed with any of the tester strains in either the presence or absence of metabolic activation (S9-Mix). The negative and positive controls gave results within the expected range; hence the experiment was considered valid.

Conclusions:

Under the study conditions, the test substance was determined to be non-mutagenic with or 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, in compliance with GLP. Salmonella typhimurium strains TA 98, TA100, TA 1535 and TA 1537 as well as Escherichia coli strain WP2 our A were used in this experiment. The assay was performed in two phases, using the plate incorporation method. In the preliminary assay, the solubility of the test substance was assessed in different solvents. The maximum dose tested was 5000 μg per plate. In the main mutagenicity assay, the dose levels tested were up to 5000 μg per plate (based on solubility and precipitation results from the preliminary assay). Dose levels up to 5000 µg/L of test substance along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test substance, vehicle control (DMSO) and positive controls were plated in triplicate. The plates were inverted and incubated for 48 to 72 h at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted. No positive mutagenic response was observed with any of the tester strains in either the presence or absence of metabolic activation (S9-Mix). The negative and positive controls gave results within the expected range; hence the experiment was considered valid. Under the study conditions, the test substance was determined to be non-mutagenic in Ames test, with or without metabolic activation (BioReliance, 2004).

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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.

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.

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

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.

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).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From June 02, 2017 to August 08, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Target gene:

Thymidine kinase heterozygote system, TK +/- to TK -/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.

Test concentrations with justification for top dose:

Preliminary test
0, 2.44, 4.88, 9.77, 19.53, 39.06, 78.13, 456.25, 312.5, 625 µg/mL, 4-h exposure -/+S9 and 24-h exposure -S9

Main test
0, 4.88, 9.75, 19.5, 39, 78, 104, 130, 156 µg/mL, 4-h exposure, -/+S9
0, 0.63, 1.25, 2.5, 5, 10, 20, 40 µg/mL, 24-h exposure, -S9

Vehicle / solvent:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burrough Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 h and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Microsomal Enzyme Fraction
Lot No. PB/NF S9 31/03/17 was used in this study, and was pre-prepared in house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0. 20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Results

Preliminary Cytotoxicity Test

The dose range of the test substance used in the preliminary toxicity test was 2.44 to 625 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (mg/mL)	% RSG (-S9) 4-h Exposure	% RSG (+S9) 4-h Exposure	% RSG (-S9) 24-h Exposure
0	100	100	100
2.44	95	104	66
4.88	93	92	27
9.77	88	95	29
19.53	83	109	20
39.06	88	98	7
78.13	60	67	0
156.25	0	0	0
312.5	0	0	0
625	0	0	0

There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test substance in all of the three exposure groups when compared to the concurrent vehicle control groups. However, the reductions were only observed at dose levels at and beyond the onset of test substance precipitate observed at 156.25 µg/mL in the 4-h exposure groups at the end of the exposure periods. Therefore,the maximum dose levels in the subsequent Mutagenicity Test were limited by a combination of test substance‑induced toxicity and test substance precipitate in the 4-h exposure groups, and test substance‑induced toxicity in the 24-h exposure group.

 

Mutagenicity Test

There was evidence of marked dose related toxicity following exposure to the test substance in all three of the exposure groups, as indicated by the %RSG and RTG values. There was also evidence of significant reductions in viability (%V) in the 4-h exposure groups, indicating that residual toxicity had occurred. In the 4-h exposure group in the presence of metabolic activation, toxicity was only observed at dose levels beyond the onset of test substance precipitate. Based on the RTG values and / or %RSG values, optimum levels of toxicity were considered to have been achieved in the 24‑h exposure group. Whilst optimum levels of toxicity were not achieved in the 4-h exposure group in the absence of metabolic activation due to the very steep toxicity curve of the test substance, a dose level that marginally exceeded the upper limit of acceptable toxicity was plated for viability and 5-TFT resistance as sufficient numbers of cells were available. Acceptable levels of toxicity were seen with the positive control substances. Precipitate of the test substance was observed at and above 130 µg/mL in the 4-h exposure group in the absence of metabolic activation, and at and above 78 µg/mL in the 4-h exposure group in the presence of metabolic activation at the end of the exposure periods. The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. The test substance did not induce any toxicologically significant or dose related increases in the mutant frequency x 10^-6per viable cell at any of the dose levels, in any of the three exposure groups.The test substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Main Experiment results

Concentration (µg/mL)			4-H-S9					Concentration (µg/mL)			4-H+S9
			%RSG	RTG	MF§						%RSG	RTG	MF§
0			100	1.00	156.17			0			100	1.00	150.84
4.88			92	0.85	179.00			4.88			126	1.24	147.25
9.75			95	1.13	153.20			9.75			119	1.23	106.59
19.5			94	1.06	152.51			19.5			103	1.09	87.25
39			93	0.93	152.04			39			97	0.87	149.70
78			65	0.80	120.96			78			66	0.74	131.16
104		X	5	0.04	236.50			104		X	2	0.01	270.56
130		Ø	0					130		Ø	0
156		Ø	0					156		Ø	0
MF threshold for a positive response = 282.17								MF threshold for a positive response = 276.84
Positive control								Positive control
EMS								CP
400			72	0.62	1297.31			1.5			91	0.66	992.89

 

Concentration (µg/mL)		24-H-S9
		%RSG	RTG	MF§
0		100	1.00	151.90
0.63	Ø	90
1.25	Ø	89
2.5		80	1.16	113.87
5		68	1.00	141.53
10		45	0.77	108.92
20		26	0.50	147.44
30		12	0.29	83.77
40		11	0.29	116.25
MF threshold for a positive response = 277.90
Positive control
EMS
150		61	0.58	1829.03

%RSG =         Relative Suspension Growth

RTG     =         Relative Total Growth

CP       =         Cyclophosphamide

EMS    =         Ethylmethanesulphonate

MF§     =         5-TFT resistant mutants/10⁶ viable cells 2 d after exposure

Ø         =         Not plated surplus to requirements

X         =         Excluded due to toxicity or beyond onset of precipitate

Conclusions:

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

Executive summary:

A study was conducted to determine the mutagenic potential of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', using L5178Y TK +/- 3.7.2c mouse lymphoma cells, according to OECD Guideline 490, EU Method B.17 and US EPA OPPTS 870.5300, in compliance of GLP. In the main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at eight dose levels in duplicate (0, 4.88, 9.75, 19.5, 39, 78, 104, 130, 156 µg/mL), together with vehicle (acetone), and positive controls using 4 h exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 h exposure group in the absence of metabolic activation. The maximum dose levels in the subsequent Mutagenicity Test were limited by a combination of the substance-induced toxicity and the substance precipitate in the 4-h exposure groups, and test substance-induced toxicity in the 24-h exposure group.The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test substance did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.The read across substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it was considered to be non-mutagenic in this assay. Under study conditions, the read across substance was determined to be non-mutagenic in the mouse lymphoma assay, with and without metabolic activation (Envigo, 2017). Based on the results of the read across study, a similar behaviour is expected for the test substance, 'mono- and di- C18-unsatd. PSE and C18 -unsatd. AE5 PSE' in the mouse lymphoma assay.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 04, 2017 to April 10, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 h. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 h.
The details of the donors used are:
Preliminary toxicity test: male, aged 28 years
Main Experiment: male, aged 26 years

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate metabolizing system S9 fraction (20% (v/v))

Test concentrations with justification for top dose:

Preliminary test: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL
Main experiment: (a) 4(20)-h without S9: 0, 10, 20, 40, 60, 80, 120, 160 µg/mL (b) 4(20)-h with S9 (2%): 0, 10, 20, 40, 60, 80, 120, 160 µg/mL (c) 24-h without S9: 0, 2.5, 5, 10, 15, 20, 30, 40, 60, 80, 160 µg/mL
The dose levels used in the main experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on both precipitate and toxicity.

Vehicle / solvent:

Preliminary toxicity test and main test - 4(20)-h group without S9-mix only
Identity: DMSO
Supplier: Fisher Scientific
Batch number (purity): A0385951 (99.7%)

Main Test - 4(20)-h group with S9-mix only and 24-h exposure group
Identity: DMSO
Supplier: Fisher Scientific
Batch number (purity): A0362204 (99.7%)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 h. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 h.

The details of the donors used are:
Preliminary Toxicity Test (4(20)-h exposure groups): Female, aged 25 years
Preliminary Toxicity Test (24-h exposure group): Male, aged 32 years
Main Experiment (4(20)-h exposure group without S9-mix): Female, aged 25 years
Main Experiment (4(20)-h exposure group with S9-mix): Female, aged 24 years
Main Experiment (24-h exposure group): Male, aged 32 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No’s. PB/NF S9 01/10/17 (Preliminary Toxicity Test) and PB/NF S9 14/12/17 (main test) were used in this study. The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Evaluation criteria:

Data Evaluation
The following criteria were used to determine a valid assay:
1) The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
2) All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
3) The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
4) The required number of cells and concentrations were analyzed.

Statistics:

Statistical Analysis
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989). A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: non-mutagenic potential

Results

Preliminary toxicity test

The dose range for the Preliminary Toxicity Test was 19.53 to 5000 µg/mL. The maximum dose was the maximum recommended dose level. A precipitate of the test substance was observed in the parallel blood-free cultures at the end of the exposure, at and above 156.25 µg/mL, in the 4(20)-h exposure groups and at and above 78.13 µg/mL in the 24-h continuous exposure group. Hemolysis was observed following exposure to the test substance at and above 312.5µg/mL in the 4(20)-h exposure group in the absence of metabolic activation (S9), at 5000 µg/mL in the 4(20)-h exposure group in the presence of S9 and at and above 1250 µg/mL in the 24-h continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2500 µg/mL in the 4(20)-h exposure in the absence of S9, up to 1250 µg/mL in the 4(20)-h exposure in the presence of S9 and up to 78.13 µg/mL in the 24‑h continuous exposure. The test substance induced extreme evidence of toxicity in all of the exposure groups, particularly in the 24-h continuous exposure group. The selection of the maximum dose level for the Main Experiment for the 4(20)-h exposure groups was based on both the lowest precipitating dose level and toxicity because the onset of both coincided. The selection of the maximum dose level for the 24-h continuous exposure group was based on toxicity only.

Table 1: Mitotic Index - Preliminary toxicity test

Dose Level (µg/mL)	4(20)-H Without S9		4(20)-H With S9		24-H Without S9
	Mitotic Index	% of Control	Mitotic Index	% of Control	Mitotic Index	% of Control
0	3.90	100	7.20	100	4.35	100
19.53	-	-	-	-	0.95	22
39.06	3.65	94	4.45	62	0.45	10
78.13	3.45	88	1.85	26	- P	-
156.25	0.85 P	22	1.35 P	19	NM P	-
312.5	- P H	-	- P	-	NM P	-
625	- P H	-	- P	-	NM P	-
1250	- P H	-	- P	-	NM P H	-
2500	- P H	-	NM P	-	NM P H	-
5000	NMP H	-	NM P H	-	NM P H	-

- = Not assessed for mitotic index

NM = No metaphases or insufficient metaphases suitable for scoring

P = Precipitate observed at end of exposure period in blood-free cultures

H = Hemolysis observed at the end of exposure in blood cultures

Chromosome aberration test – main experiment

The study underwent multiple Main Experiment repeats due to technical issues. Consequently, each exposure group was performed on separate days however, the repeat experiments have been considered together for the purposes of reporting. The qualitative assessment of the slides determined that the precipitate and the toxicity, depending on the exposure group, was similar to that observed in the Preliminary Toxicity Test. The qualitative assessment of the slides also determined and that there were metaphases suitable for scoring present up to the maximum dose level of test substance in the the 4(20)-h exposure groups. However, in the 24-h continuous exposure group, limited numbers of metaphases were present up to 80 µg/mL. Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 120 µg/mL in the 4(20)-h exposure groups in the presence and absence of metabolic S9 and at and above 80 µg/mL in the 24-h continuous exposure group. No hemolysis was observed in any of the exposure groups tested.

Table 2: Mitotic Index – Main experiment(4(20)-h Exposure Groups)

Dose Level (mg/mL)	4(20)-H Without S9				4(20)-H With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	7.30	6.30	6.80	100	2.80	2.30	2.55	100
10	-	-	-	-	-	-	-	-
20	-	-	-	-	-	-	-	-
40	-	-	-	-	-	-	-	-
60	2.65	2.95	2.80	41	2.40	3.20	2.80	110
80	2.50	3.10	2.80	41	2.35	2.90	2.63	103
120	1.50 P	4.60 P	3.05	45	2.45 P	2.95 P	2.70	106
160	- P	- P	-	-	- P	- P	-	-
MMC 0.2	2.60	3.35	2.98	44	NA	NA	NA	NA
CP 4	NA	NA	NA	NA	0.70	1.60	1.15	45

MMC = Mitomycin C

CP = Cyclophosphamide

P = Precipitate observed at end of exposure period in blood-free cultures

NA = Not applicable

- = Not assessed for mitotic index

Table 3: Mitotic Index – Main experiment (24-h Exposure Group)

Dose Level (µg/mL)	24-H Without S9
	A	B	Mean	% of Control
0	1.30	3.95	2.63	100
2.5	-	-	-	-
5	-	-	-	-
10	1.55	1.90	1.73	66
15	1.45	1.05	1.25	48
20	0.80	0.90	0.85	32
30	0.95	1.55	1.25	48
40	FM	FM	-	-
60	FM	FM	-	-
80	FM P	FM P	-	-
160	NM P	NM P	-	-
MMC 0.2	1.45	1.30	1.38	52

MMC = Mitomycin C

P = Precipitate at end of exposure period in blood-free cultures

- = Not assessed for mitotic index

FM = Insufficient numbers of metaphases suitable for scoring

NM = No metaphases or insufficient metaphases suitable for scoring

In the 4(20)-h exposure group in the absence of S9, 59%, 59% and 55% mitotic inhibition was achieved at 60, 80 and 120 µg/mL, respectively. Above this dose level, there were too few metaphases available for analysis. Therefore, the maximum dose level selected for metaphase analysis was 120 µg/mL because all of the dose levels selected achieved optimum toxicity as defined by the OECD 473 test guideline (55±5%) and it coincided with the lowest precipitating dose level. In the presence of S9, no dose-related inhibition of mitotic index was observed and metaphases were noted up to the maximum dose level of 160 µg/mL. Therefore, the maximum dose level selected for metaphase analysis was the lowest precipitating dose level (120 µg/mL). In the 24-h continuous exposure group, an inhibition of mitotic index of 34%, 52%, 68% and 52% was noted at 10, 15, 20 and 30 µg/mL, respectively. Above this dose level, there were few metaphases available up to 80 µg/mL with no scorable ones observed at 160 µg/mL. Consequently, the maximum dose level selected for metaphase analysis was 30 µg/mL because two of the four dose levels selected achieved optimum toxicity as defined by the OECD 473 test guideline (55±5%), including the maximum selected dose level. Additional results tables document is attached in background material section of IUCLID.

Table 4: Mean Frequency of Polyploid Cells (%)

Dose Level (µg/mL)	Exposure Group
	4(20)-H Without S9	4(20)-H With S9	24-H Without S9
0	0	0	0
10	NA	NA	0
15	NA	NA	0
20	NS	NA	0
30	NA	NA	0.3
60	0	0	NA
80	0	0	NA
120	0	0	NA
MMC 0.2	0	NA	0
CP 4	NA	0	NA

Validity of assay

The assay was considered valid as it met all of the following criteria:

1) The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.

2) All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.

3) The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.

4) The required number of cells and concentrations were analyzed.

The test substance did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation. The test substancedid not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

Conclusions:

Under the study conditions, the test substance was determined to be non-clastogenic in human lymphocytes, with and without metabolic activation.

Executive summary:

A study was conducted to determine the clastogenicity of the test substance, 'mono- and di- C18-unsatd PSE and C18-unsatd AE5 PSE', using Chromosome Aberration test in human lymphocytes , according to the OECD Guideline 473, in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated: (a) 4 h exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-h expression period and test concentrations of 0, 10, 20, 40, 60, 80, 120, 160 μg/mL, (b) 4 h exposure in the absence of metabolic activation (S9), with a 20-h expression period and at test concentrations of 0, 10, 20, 40, 60, 80, 120, 160 μg/mL and (c) a 24-h exposure in the absence of metabolic activation, and at test concentrations of 0, 2.5, 5, 10, 15, 20, 30, 40, 60, 80, 160 μg/mL. The study underwent multiple preliminary toxicity tests and main experiment repeats due to technical issues. Consequently, each exposure group was performed on a separate day, however, the repeat experiments (for both the preliminary toxicity test and main experiment) have been considered together, where possible, for the purposes of reporting. The dose levels used in the main experiment (mentioned above) were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on both precipitate and toxicity. All vehicle (dimethyl sulphoxide (DMSO)) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substance (Mitomycin C and Cyclophosphamide) induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance was toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was either the lowest precipitating dose level or induced 55 ± 5% mitotic inhibition, depending on exposure group and presence or absence of metabolic activation. Based on the study results, the test substance was considered to be non-clastogenic to human lymphocytes in vitro. Under the study conditions, the test substance was determined to be non-clastogenic in the chromosomal aberration test, with and without metabolic activation (Envigo, 2018).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro bacterial reverse mutation (Ames) test:

Study 1: 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, in compliance with GLP. Salmonella typhimurium strains TA 98, TA100, TA 1535 and TA 1537 as well as Escherichia coli strain WP2 our A were used in this experiment. The assay was performed in two phases, using the plate incorporation method. In the preliminary assay, the solubility of the test substance was assessed in different solvents. The maximum dose tested was 5000 μg per plate. In the main mutagenicity assay, the dose levels tested were up to 5000 μg per plate (based on solubility and precipitation results from the preliminary assay). Dose levels up to 5000 µg/L of test substance along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test substance, vehicle control (DMSO) and positive controls were plated in triplicate. The plates were inverted and incubated for 48 to 72 h at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted. No positive mutagenic response was observed with any of the tester strains in either the presence or absence of metabolic activation (S9-Mix). The negative and positive controls gave results within the expected range; hence the experiment was considered valid. Under the study conditions, the test substance was determined to be non-mutagenic in Ames test, with or without metabolic activation (BioReliance, 2004).

Study 2:

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).

In vitro mammalian cytogenicity study: 

A study was conducted to determine the clastogenicity of the test substance, 'mono- and di- C18-unsatd PSE and C18-unsatd AE5 PSE', using Chromosome Aberration test in human lymphocytes , according to the OECD Guideline 473, in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated: (a) 4 h exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-h expression period and test concentrations of 0, 10, 20, 40, 60, 80, 120, 160 μg/mL, (b) 4 h exposure in the absence of metabolic activation (S9), with a 20-h expression period and at test concentrations of 0, 10, 20, 40, 60, 80, 120, 160 μg/mL and (c) a 24-h exposure in the absence of metabolic activation, and at test concentrations of 0, 2.5, 5, 10, 15, 20, 30, 40, 60, 80, 160 μg/mL. The study underwent multiple preliminary toxicity tests and main experiment repeats due to technical issues. Consequently, each exposure group was performed on a separate day, however, the repeat experiments (for both the preliminary toxicity test and main experiment) have been considered together, where possible, for the purposes of reporting. The dose levels used in the main experiment (mentioned above) were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on both precipitate and toxicity. All vehicle (dimethyl sulphoxide (DMSO)) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substance (Mitomycin C and Cyclophosphamide) induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance was toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was either the lowest precipitating dose level or induced 55 ± 5% mitotic inhibition, depending on exposure group and presence or absence of metabolic activation. Based on the study results, the test substance was considered to be non-clastogenic to human lymphocytes in vitro. Under the study conditions, the test substance was determined to be non-clastogenic in the chromosomal aberration test, with and without metabolic activation (Envigo, 2018).

In vitro mammalian cell gene mutation test:

A study was conducted to determine the mutagenic potential of the read across substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', using L5178Y TK +/- 3.7.2c mouse lymphoma cells, according to OECD Guideline 490, EU Method B.17 and US EPA OPPTS 870.5300, in compliance of GLP. In the main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at eight dose levels in duplicate (0, 4.88, 9.75, 19.5, 39, 78, 104, 130, 156 µg/mL), together with vehicle (acetone), and positive controls using 4 h exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 h exposure group in the absence of metabolic activation. The maximum dose levels in the subsequent Mutagenicity Test were limited by a combination of the substance-induced toxicity and the substance precipitate in the 4-h exposure groups, and test substance-induced toxicity in the 24-h exposure group.The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test substance did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.The read across substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it was considered to be non-mutagenic in this assay. Under study conditions, the read across substance was determined to be non-mutagenic in the mouse lymphoma assay, with and without metabolic activation (Envigo, 2017).

Further, as per HERA 2009 review report on AEs, there was no indication of genetic toxicity of broad range of structurally different alcohol ethoxylates in all available in vitro and in vivo genotoxicity assays. Most of the studies were performed in accordance with GLP and following OECD guideline methodologies. The remaining in vitro and in vivo studies were well documented and conducted. The structure of alcohol ethoxylates are not of concern for potential genotoxicity (HERA, 2009).

Overall, based on the available studies, the test substance, ‘mono- and d- C18-unsatd. PSE and C18-unsatd. AE5 PSE’, is considered to be non-genotoxic.

Justification for classification or non-classification

Based on the negative results in the in vitro Ames and chromosomal aberration studies with the test substance as well as mouse lymphoma study with the read across substance, the test substance, 'mono- and di- C18-unsatd. PSE and C18-unsatd. AE5 PSE', does not warrant classification for genotoxicity according to the EU CLP criteria (Regulation 1272/2008/EC).​​