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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on results of the in vitro studies of the test substance as well as read across substances, the test substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', is considered to be non-genotoxic.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Ames test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 14, 2017 to July 14, 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)
Version / remarks:
OECD guidelines for testing of chemicals no. 471 (1997) "Bacterial Reverse Mutation Test".
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008.
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
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:
Eight concentrations of the test substance (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method in experiment 1. Experiment 2 was performed using pre-incubation method in the presence and absence of metabolic activation. The test substance concentrations were: Salmonella strains TA100 and TA1535 (absence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate. Salmonella strain TA1535 (presence of S9): 5, 15, 50, 150, 500, 1500, 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. 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) and consequently the same maximum dose level was initially used in the second mutation test. However, after incorporating the pre-incubation modification in the second mutation test, the test substance induced toxicity as weakened bacterial background lawns to the extent where a two of the strains required repeat analysis employing an amended test substance dose range. Therefore, depending on bacterial strain type and presence or absence of S9-mix, the maximum recommended dose level (5000 µg/plate) or the toxic limit of the test substance was employed in the second mutation test.
Vehicle / solvent:
Identity: Tetrahydrofuran
Batch number (purity): 1707545 (99.9%)

The test substance was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 10 minutes at 40 °C. No correction was required for purity. Tetrahydrofuran is toxic to the bacterial cells at and above 50 uL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 uL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran, Batch no. - 1707545, Purity- 99.9%, Expiry: 02/2019
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
not specified
Details on test system and experimental conditions:
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed at 5000 μg/plate because of test item precipitation. A number of manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983), Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 - 7 to 40
TA100 - 60 to 200
TA1537 - 2 to 30
TA98 - 8 to 60
WP2uvrA - 10 to 60

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.
Evaluation criteria:
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:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Results

The vehicle (tetrahydrofuran) 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) and consequently the same maximum dose level was initially used in the second mutation test. However, after incorporating the pre-incubation modification in the second mutation test, the test substance induced toxicity as weakened bacterial background lawns to the extent where a two of the strains required repeat analysis employing an amended test substance dose range. Therefore, depending on bacterial strain type and presence or absence of S9-mix, the maximum recommended dose level (5000 µg/plate) or the toxic limit of the test substance was employed in the second mutation test. Results from the second mutation test showed that the test substance induced a toxic response employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9-mix from 150 µg/plate (TA1535), 500 µg/plate (TA100) and 1500 µg/plate (TA98 and TA1537). In the presence S9-mix, weakened bacterial background lawns were initially noted from 500 µg/plate (TA1535) and 1500 µg/plate (TA100). No toxicity was noted to Escherichia coli strain WP2uvrA at any test substance dose level in either the absence or presence of S9-mix or Salmonella strains TA98 and TA1537 dosed in the 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.

A test substance precipitate (particulate in appearance) was noted at and above 1500 mg/plate, this observation did not prevent the scoring of revertant colonies. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (preincubation method). A small, statistically significant increase in TA98 revertant colony frequency was observed in the absence of S9-mix at 150 µg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility and the fold increase was only 1.4 times the concurrent vehicle control.

Further statistically significant increases were observed in Experiment 2 (TA100 dosed in the presence of S9 at 1500 µg/plate and TA1535 at 150 and 500 µg/plate in the absence of S9 and 500, 1500 and 5000 µg/plate in the presence of S9). These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response 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.

Conclusions:
Under the study conditions, the test substance was found to be non-mutagenic with and without metabolic activation.
Executive summary:

A study was conducted to determine mutagenic potential of the test substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE' by Ames test according to OECD 471, in compliance with GLP. 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) and consequently the same maximum dose level was initially used in the second mutation test. However, after incorporating the pre-incubation modification in the second mutation test, the test substance induced toxicity as weakened bacterial background lawns to the extent, where a two of the strains required repeat analysis employing an amended test substance dose range. Therefore, depending on bacterial strain type and presence or absence of S9-mix, the maximum recommended dose level (5000 µg/plate) or the toxic limit of the test substance was employed in the second mutation test. Results from the second mutation test showed that the test substance induced a toxic response employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9-mix from 150 µg/plate (TA1535), 500 µg/plate (TA100) and 1500 µg/plate (TA98 and TA1537). In the presence S9-mix, weakened bacterial background lawns were initially noted from 500 µg/plate (TA1535) and 1500 µg/plate (TA100). No toxicity was noted to Escherichia coli strain WP2uvrA at any test substance dose level in either the absence or presence of S9-mix or Salmonella strains TA98 and TA1537 dosed in the 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. A test substance precipitate (particulate in appearance) was noted at and above 1500 mg/plate, this observation did not prevent the scoring of revertant colonies. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). A small, statistically significant increase in TA98 revertant colony frequency was observed in the absence of S9-mix at 150 µg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility and the fold increase was only 1.4 times the concurrent vehicle control. Further statistically significant increases were observed in Experiment 2 (TA100 dosed in the presence of S9 at 1500 µg/plate and TA1535 at 150 and 500 µg/plate in the absence of S9 and 500, 1500 and 5000 µg/plate in the presence of S9). These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response 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 vehicle (tetrahydrofuran) 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. Under the study conditions, the test substance was considered to be non-mutagenic in the Ames test, with and without metabolic activation (Envigo, 2017).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 02, 2017 to August 08, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 test 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 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.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 test 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 test substance was determined to be non-mutagenic in the mouse lymphoma assay, with and without metabolic activation (Envigo, 2017).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
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 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
n.a.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
+S9: 313, 625, 1250, 2500, 5000 µg/mL
-S9: 1.25, 2.5, 5, 10, 20, 39, 78 µg/mL
Vehicle / solvent:
1% ethanol
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
Remarks:
1% ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (10-40 µg/mL), cyclophosphamide (20-60 µg/mL)
Details on test system and experimental conditions:
Method of application: in medium
Evaluation criteria:
According to Guideline.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Based on the results of the read across study, the test substance, is considered to be non-clastogenic with and without metabolic activation.
Executive summary:

An in vitro study was conducted to determine theclastogenic potential of the read across substance, 'C16 -18 AE1-2.5' (purity: >98%),using Chinese hamster ovary cells,according to OECD Guideline 473, in compliance with GLP. Chinese hamster ovary cells (CHO) were exposed to 0, 313, 625, 1250, 2500 and 5000 μg/mL test concentrations in the presence (+S9) and 1.25, 2.5, 5, 10, 20, 39 and 78 μg/mL test concentrations in the absence (-S9) of metabolic activation. Positive and vehicle (1% ethanol) control cultures were included in each assay. No increases in the number of chromosome aberrations in the presence or absence of metabolic activation were seen at any concentration tested. Appropriate reference mutagens used as positive controls, showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9 -mix. Under the study conditions, the read across substance was determined to be non-clastogenic in the chromosomal aberration study, with and without metabolic activation (Innes, 1995). Based on the results of the read across study, the test substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', is considered to be non-clastogenic with and without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From August 01, 2017 to October 30, 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 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, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL
Main experiment: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125 µg/mL
The test substance was insoluble in Dimethyl sulphoxide, Acetone and Tetrahydrofuran at 200, 100 and 50 mg/mL. The test substance was insoluble in Minimal Essential Medium (MEM) at 20 mg/mL but was partially soluble/ suspendable in MEM at 10 mg/mL in solubility checks performed in-house. Therefore 1000 µg/mL was considered to be the maximum achievable dose level due to formulation difficulties. The selection of the maximum dose level for the main experiment was based on the lowest precipitating dose level and was 125 µg/mL for the 4(20)-h exposure groups and for the continuous exposure group.
Vehicle / solvent:
The test substance was insoluble in Dimethyl sulphoxide, Acetone and Tetrahydrofuran at 200, 100 and 50 mg/mL. The test substance was insoluble in Minimal Essential Medium (MEM) at 20 mg/mL but was partially soluble/ suspendable in MEM at 10 mg/mL in solubility checks performed in-house. Therefore MEM was used as vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Identity: Minimal Essential Medium, Supplier: Sigma, Batch number: RNBF9655, Expiry Date: September 2018, Purity: Treated as 100%
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: male, aged 28 years
Main experiment: male, aged 26 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 37ºC 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). Batch Nos. PB/NF S9 30/6/17 and 20/8/17 were used in this study. Prior to use each batch of S9 is tested for its capability to activate known mutagens in the Ames test. 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:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Tested concentrations: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: no mutagenic potential

Results

Preliminary toxicity test

The dose range for the preliminary toxicity test was 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/mL.The maximum dose was the maximum achievable dose level due to formulation difficulties. A precipitate of the test substance was observed in the parallel blood-free cultures at the end of the exposure, at and above 31.25 µg/mL in the 4(20)-h exposure group in the absence of S9, at and above 125 µg/mL in the presence of S9, and at and above 62.5 µg/mL in the continuous exposure group. Precipitate from the test substance was also noted on the slides at 1000 µg/mL in all three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1000 µg/mL in all three exposure groups. The maximum dose selected for mitotic index analysis was limited to 125 µg/mL based on the precipitate observations.The test substance induced no evidence of toxicity in any of the exposure groups. The selection of the maximum dose level for the main experiment was based on the lowest precipitating dose level and was 125 µg/mL for the 4(20)-h exposure groups and for the continuous exposure group.

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

8.35

100

10.55

100

16.25

100

3.91

-

-

-

-

-

-

7.81

6.65

80

-

-

-

-

15.63

8.10

97

7.55

72

17.55

108

31.25

17.05 P

204

6.95

66

14.35

88

62.5

- P

-

8.80

83

13.30 P

82

125

8.35 P

100

9.90 P

94

13.95 P

86

250

- P

-

- P

-

- P

-

500

- P

-

- P

-

- P

-

1000

- P

-

- P

-

- P

-

- = Not assessed for mitotic index

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

 = Precipitate observed on the slides  

Chromosome aberration test – main experiment

The qualitative assessment of the slides determined that there was no marked toxicity as in the preliminary toxicity test and that there were metaphases suitable for scoring present up to 125 µg/mL in all three exposure groups. Precipitate observations were made at the end of exposure in blood-free cultures and precipitate was noted at and above 62.5 µg/mL in the absence of S9 and at and above 31.25 µg/mL in the presence of S9. The mitotic index data for the Main Experiment confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in any of the three exposure groups. The maximum dose level selected for metaphase analysis was the lowest precipitating dose level in each of the exposure groups and was 62.5 µg/mL in the absence of S9 and 31.25 µg/mL in the presence of S9.

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

9.75

11.70

10.73

100

5.95

5.05

5.50

100

3.91

-

-

-

-

-

-

-

-

7.81

-

-

-

-

5.25

6.20

5.73

104

15.63

9.75

10.30

10.03

93

7.25

4.35

5.80

105

31.25

11.15

9.75

10.45

97

4.95 P

5.20 P

5.08

92

62.5

12.05 P

11.05 P

11.55

108

- P

- P

-

-

125

- P

- P

-

-

- P

- P

-

-

MMC 0.4

- P

- P

-

-

NA

NA

NA

NA

CP 2

NA

NA

NA

NA

4.25

3.35

3.80

69

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

14.20

13.65

13.93

100

3.91

-

-

-

-

7.81

-

-

-

-

15.63

10.90

12.45

11.68

84

31.25

9.95

10.30

10.13

73

62.5

14.35 P

18.10 P

16.23

117

125

- P

- P

-

-

MMC 0.1

7.75

5.70

6.73

48

Table 4: Main experiment (4-h treatment, 24 h Harvest) without metabolic treatment

Treatment Group

Replicate

Mitotic Index (%)

Number of Cells Scored

Number of Aberrations

Total Number of Aberrations

Frequency of Aberrant Cells (%)

Gaps

Chromatid

Chromosome

Others

Breaks

Exchanges

Breaks

Exchanges

X

(+ Gaps)

(-Gaps)

(+Gaps)

(-Gaps)

Vehicle Control (MEM)

A

9.75

150

0

0

0

0

0

0

0

0

0

0

B

11.70

150

0

0

0

0

0

0

0

0

0

0

Total

21.45

300

0

0

0

0

0

0

0

0

0

0

(100)

(0.0)

(0.0)

A

9.75

150

1

0

0

0

0

0

1

0

1

0

15.63

B

10.30

150

0

0

0

0

0

0

0

0

0

0

µg/mL

Total

20.05

300

1

0

0

0

0

0

1

0

1

0

(93)

(0.3)

(0.0)

A

11.15

150

0

0

0

0

0

0

0

0

0

0

31.25

B

9.75

150

0

0

0

0

0

0

0

0

0

0

µg/mL

Total

20.90

300

0

0

0

0

0

0

0

0

0

0

(97)

(0.0)

(0.0)

A

12.05

150

0

0

0

0

0

0

0

0

0

0

62.5

B

11.05

150

0

0

0

0

0

0

0

0

0

0

µg/mL

Total

23.10

300

0

0

0

0

0

0

0

0

0

0

(108)

(0.0)

(0.0)

Positive Control

A

5.90

96a

4

8

9

2

0

0

23

19

22

18

MMC 0.4

B

7.60

46a

0

10

11

1

0

0

22

22

15

15

µg/mL

Total

13.50

142

4

18

20

3

0

0

45

41

37

33***

(63)

(26.1)

(23.2)

MMC - Mitomycin C

a - Slide evaluation terminated when at least 15 cells with aberrations (excluding gaps) had been observed

*** - P < 0.001

MEM - Minimal Essential Medium

Table 5: Main experiment (4-h treatment, 24 h Harvest) with metabolic treatment

Treatment Group

Replicate

Mitotic Index (%)

Number of Cells Scored

Number of Aberrations

Total Number of Aberrations

Frequency of Aberrant Cells (%)

Gaps

Chromatid

Chromosome

Others

Breaks

Exchanges

Breaks

Exchanges

X

(+ Gaps)

(-Gaps)

(+Gaps)

(-Gaps)

Vehicle Control (MEM)

A

5.95

150

0

5

0

0

0

0

5

5

4

4

B

5.05

150

0

0

0

0

0

0

0

0

0

0

Total

11.00

300

0

5

0

0

0

0

5

5

4

4

(100)

(1.3)

(1.3)

A

5.25

150

0

0

0

0

0

0

0

0

0

0

7.81

B

6.20

150

0

3

0

1

0

0

4

4

4

4

µg/mL

Total

11.45

300

0

3

0

1

0

0

4

4

4

4

(104)

(1.3)

(1.3)

A

7.25

150

0

0

0

0

0

0

0

0

0

0

15.63

B

4.35

150

0

0

0

0

0

0

0

0

0

0

µg/mL

Total

11.60

300

0

0

0

0

0

0

0

0

0

0

(105)

(0.0)

(0.0)

A

4.95

150

0

0

0

0

0

0

0

0

0

0

31.25

B

5.20

150

0

0

0

0

0

0

0

0

0

0

µg/mL

Total

10.15

300

0

0

0

0

0

0

0

0

0

0

(92)

(0.0)

(0.0)

Positive Control

A

4.25

104a

0

17

2

0

0

0

19

19

15

15

CP 2

B

3.35

109a

1

11

5

1

0

0

18

17

16

15

µg/mL

Total

7.60

213

1

28

7

1

0

0

37

36

31

30***

(69)

(14.6)

(14.1)

Table 6: Main experiment (24-h treatment) without metabolic treatment

Treatment Group

Replicate

Mitotic Index (%)

Number of Cells Scored

Number of Aberrations

Total Number of Aberrations

Frequency of Aberrant Cells (%)

Gaps

Chromatid

Chromosome

Others

Breaks

Exchanges

Breaks

Exchanges

X

(+ Gaps)

(-Gaps)

(+Gaps)

(-Gaps)

Vehicle Control (MEM)

A

14.20

150

1

0

0

1

0

0

2

1

2

1

B

13.65

150

0

0

0

0

0

0

0

0

0

0

Total

27.85

300

1

0

0

1

0

0

2

1

2

1

(100)

(0.7)

(0.3)

A

10.90

150

0

0

0

0

0

0

0

0

0

0

15.63

B

12.45

150

0

0

0

0

0

0

0

0

0

0

µg/mL

Total

23.35

300

0

0

0

0

0

0

0

0

0

0

(84)

(0.0)

(0.0)

A

9.95

150

0

1

0

0

0

0

1

1

1

1

31.25

B

10.30

150

0

0

0

0

0

0

0

0

0

0

µg/mL

Total

20.25

300

0

1

0

0

0

0

1

1

1

1

(73)

(0.3)

(0.3)

A

14.35

150

0

0

0

2

0

0

2

2

2

2

62.5

B

18.10

150

0

0

0

0

0

0

0

0

0

0

µg/mL

Total

32.45

300

0

0

0

2

0

0

2

2

2

2

(117)

(0.7)

(0.7)

Positive Control

A

7.75

45a

4

24

1

3

0

0

32

28

19

18

MMC 0.1

B

5.70

93a

2

14

1

2

0

0

19

17

15

15

µg/mL

Total

13.45

138

6

38

2

5

0

0

51

45

34

33***

(48)

(24.6)

(23.9)

Table 7: 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

7.81

-

0

0

15.63

0

0

0

31.25

0

0

0

62.5

0

0

-

MMC 0.4

0

NA

NA

MMC 0.1

NA

NA

0

CP 5

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. Thetest substancedid not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.


Conclusions:
Based on the results of the read across study, the test substance, is considered to be non-clastogenic with and without metabolic activation.
Executive summary:

An in vitro study was conducted to determine the clastogenicity of the read across substance, 'mono- and di- C16 PSE, K+ and H3PO4', usinghuman lymphocytes, according to OECD Guideline 473 (Chromosome Aberration test), in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at doses 0, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/mL, together with vehicle and positive controls. In this study, three exposure conditions were investigated: 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, 4 h exposure in the absence of metabolic activation (S9) with a 20-h expression period and a 24-h exposure in the absence of metabolic activation. 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 by precipitate. All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substances (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 non-toxic 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 the lowest precipitating dose level. Under the study conditions, the reada across substance was considered to be non-clastogenic in the chromosomal aberration test, with and without metabolic activation (Envigo, 2017). Based on the results of the read across study, the test substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', is considered to be non-clastogenic with and without metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitrobacterial reverse mutation (Ames) test:A study was conducted to determine mutagenic potential of the test substance, ‘mono- and di- C16-18 PSE and C16-18 AE10 PSE’ by Ames test according to OECD 471, in compliance with GLP. 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) and consequently the same maximum dose level was initially used in the second mutation test. However, after incorporating the pre-incubation modification in the second mutation test, the test substance induced toxicity as weakened bacterial background lawns to the extent, where a two of the strains required repeat analysis employing an amended test substance dose range. Therefore, depending on bacterial strain type and presence or absence of S9-mix, the maximum recommended dose level (5000 µg/plate) or the toxic limit of the test substance was employed in the second mutation test. Results from the second mutation test showed that the test substance induced a toxic response employing the pre-incubation modification with weakened bacterial background lawns initially noted in the absence of S9-mix from 150 µg/plate (TA1535), 500 µg/plate (TA100) and 1500 µg/plate (TA98 and TA1537). In the presence S9-mix, weakened bacterial background lawns were initially noted from 500 µg/plate (TA1535) and 1500 µg/plate (TA100). No toxicity was noted to Escherichia coli strain WP2uvrA at any test substance dose level in either the absence or presence of S9-mix or Salmonella strains TA98 and TA1537 dosed in the 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. A test substance precipitate (particulate in appearance) was noted at and above 1500 mg/plate, this observation did not prevent the scoring of revertant colonies. There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation (S9-mix) in Experiment 2 (preincubation method). A small, statistically significant increase in TA98 revertant colony frequency was observed in the absence of S9-mix at 150 µg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility and the fold increase was only 1.4 times the concurrent vehicle control. Further statistically significant increases were observed in Experiment 2 (TA100 dosed in the presence of S9 at 1500 µg/plate and TA1535 at 150 and 500 µg/plate in the absence of S9 and 500, 1500 and 5000 µg/plate in the presence of S9). These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response 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 vehicle (tetrahydrofuran) 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. 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 vitromammalian cell gene mutation test:A study was conducted to determine the mutagenic potential of the test 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 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. 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 test 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 test substance was determined to be non-mutagenic in the mouse lymphoma assay, with and without metabolic activation (Envigo, 2017).

In vitromammalian cytogenicity study: 

In absence of chromosomal aberration study with the test substance, the endpoint can be assessed based on read across to 'mono- and di- C18-unsatd PSE and C18-unsatd AE5 PSE' as well as based on studies available on the main constituents, which can be categorised as phosphate esters (PSE), ethoxylated phosphate ester (AE PSE) and/or free ethoxylated alcohol (AE). As, no specific study is available for AE PSE constituent, then the read across has been based on studies available on PSE and AE only and a worst case value is considered further for hazard/risk assessment.

Read across study: A study was conducted to determine the clastogenicity of the read across 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 read across substance was considered to be non-clastogenic to human lymphocytes. Under the study conditions, the read across substance was determined to be non-clastogenic in the chromosomal aberration test, with and without metabolic activation (Envigo, 2018).

Constituent PSE - read across study: A study was conducted to determine the clastogenicity of the read across substance, ‘mono- and di- C16 PSE, K+ and H3PO4’, using human lymphocytes, according to OECD Guideline 473 (Chromosome Aberration test), in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at doses 0, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/mL, together with vehicle and positive controls. In this study, three exposure conditions were investigated: 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, 4 h exposure in the absence of metabolic activation (S9) with a 20-h expression period and a 24-h exposure in the absence of metabolic activation. 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 by precipitate. All vehicle (Minimal Essential Medium) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control substances (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 read across substance was non-toxic 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 the lowest precipitating dose level. Under the study conditions, the read across substance was considered to be non-clastogenic in the chromosomal aberration test, with and without metabolic activation (Envigo, 2017).

Constituent AE - read across study:A study was conducted to determine the clastogenic potential of the read across substance, C16 -18 AE (1-2.5EO) (purity: >98%), using Chinese hamster ovary cells, according to OECD Guideline 473, in compliance with GLP. Chinese hamster ovary cells (CHO) were exposed to 0, 313, 625, 1250, 2500 and 5000 μg/mL test concentrations in the presence (+S9) and 1.25, 2.5, 5, 10, 20, 39 and 78 μg/mL test concentrations in the absence (-S9) of metabolic activation. Positive and vehicle (1% ethanol) control cultures were included in each assay. No increases in the number of chromosome aberrations in the presence or absence of metabolic activation were seen at any concentration tested. Appropriate reference mutagens used as positive controls, showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9 -mix. Under the study conditions, the read across substance was determined to be non-clastogenic in the chromosomal aberration study, with and without metabolic activation (Innes, 1995).

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 availablein vitroand in vivo genotoxicity assays. Most of the studies were performed in accordance with GLP and following OECD guideline methodologies. The remainingin vitroand in vivo studies were well documented and conducted. The structure of alcohol ethoxylates are not of concern for potential genotoxicity (HERA, 2009). Based on the results of the read across studies for PSE and AE constituents, the test substance, ‘mono- and di- C16-18 PSE and C16-18 AE10 PSE’,is considered to be non-clastogenic with and without metabolic activation.

Overall, based on the available studies, the test substance, ‘mono- and d- C16 -18 PSE and C16 -18 AE10 PSE’, is considered to be non-genotoxic.

Justification for classification or non-classification

Based on the negative results in the in vitro Ames and mouse lymphoma studies with the test substance as well as chromosome aberration studies available with read across substances, the test substance, 'mono- and di- C16-18 PSE and C16-18 AE10 PSE', is concluded not to warrant classification for genotoxicity, according to the EU CLP criteria (Regulation 1272/2008/EC).​​