Reaction mass of hexadecyl dihydrogen phosphate and cetyl alcohol (mono- C16 PSE and C16-OH)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available weight of evidence information from the in vitro/in vivo read across studies of the main constituents as well as in vitro mammalian cell gene mutation assay, the test substance, ‘mono- C16 PSE and C16-OH’ 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:

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

Adequacy of study:

weight of evidence

Study period:

From June 08, 2017 to June 22, 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 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

GLP compliance:

yes (incl. QA statement)

Remarks:

no analytical veryfication

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:

S9 fraction (10% liver in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1: All strains (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Experiment 2: The dose range used was determined by the results of Experiment 1. All strains (with and without S9-mix): 15, 50, 150, 500, 1500, 5000 μg/plate.

Vehicle / solvent:

Identity: Sterile distilled water
Supplier: Aguettant
Batch number (purity): 3012436 (N/A)
Exp: 10/2018

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

Preparation of the test substance formulations:
The test substance was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer and sonication for 20 minutes at 40 °C on the day of each experiment. No correction for purity was required.

EXPERIMENT 1: direct plate incorporation method,
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain.

Without Metabolic Activation:
0.1 mL of the appropriate concentration of test substance, solvent vehicle or appropriate positive control was added together with 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer to 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test substance, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation:
The procedure was the same as for without of metabolic activation except that following the addition of the test substance formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring:
All of the plates were incubated at 37 ± 3 °C for approximately 48 h 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 (presence of S9-mix only) because of a patchy test substance precipitation.

EXPERIMENT 2:

Without Metabolic Activation:
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test subsatnce formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation:
The procedure was the same as described above except that following the addition of the test substance formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring:
All of the plates were incubated at 37 ± 3 °C for approximately 48 h 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 and above 1500 μg/plate (presence and absence of S9-mix only) because of test substance precipitation. Several further manual counts were also required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Rationale for test conditions:

The test substance was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test substance formed the best doseable suspension in sterile distilled water, therefore, this solvent was selected as the vehicle.

Evaluation criteria:

There are several criteria for determining positive result. Any one or all of the following can be used to determine overall result of the study:
1. 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. Statystical analysis of data as determined by UKEMS (Mahon et al. 1989).
5. Fold increase greater than two times the concurent solvent control for any tester strain (especially if acompanied by 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, is 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:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- 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 used in the second mutation test. Similarly, 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 second mutation test (pre-incubation method).
- A test substance precipitate (particulate in appearance) was noted at and above 1500 μg/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 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).
- The vehicle (sterile distilled water) control plates gave counts of revertant colonies generally 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.

Conclusions:

Bsaed on the results of the read across study, a similar absence of genotoxicity potential in Ames test can be expected for the test substance, with and without metabolic activation.

Executive summary:

A study was conducted to determine the genotoxic potential of read across substance, 'mono- and di- C16 PSE, K+ and H3PO4' (purity: 100%), using Ames test, according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of 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 metabolic activation (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and ranged from 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test substance concentrations per bacterial strain 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. The vehicle (sterile distilled water) control plates gave counts of revertant colonies generally 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. 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 second mutation test (pre-incubation method). A test substance precipitate (particulate in appearance) was noted at and above 1500 μg/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) and 2 (pre-incubation method). Under the study conditions, the read across substance was determined to be non-genotoxic in the Ames test, with and without metabolic activation (Envigo, 2017). Bsaed on the results of the read across study, a similar absence of genotoxicity potential in Ames test can be expected for the test substance, 'mono- C16 PSE and C16-OH'.

Reference 2

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 with acceptable restrictions

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	96^a	4	8	9	2	0	0	23	19	22	18
MMC 0.4	B	7.60	46^a	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	104^a	0	17	2	0	0	0	19	19	15	15
CP 2	B	3.35	109^a	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	45^a	4	24	1	3	0	0	32	28	19	18
MMC 0.1	B	5.70	93^a	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 in the chromosomal aberration assay, with and without metabolic activation.

Executive summary:

A vitro study was conducted to determine the clastogenicity of the read across substance, mono- and di- C16 PSE, K+, H3PO4 (Purity: 100%), according to the OECD Guideline 473, using Chromosome Aberration test in human lymphocytes, in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the read across 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 to human lymphocytes in the Chromosomal aberration assay, with and without metabolic activation (Envigo, 2017). Based on the results of the read across study, a similar absence of genotoxicity in the chromosomal aberration assay can be expected for the test substance, 'mono- C16 PSE and C16-OH'.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 26, 2017 to June 27, 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)

Version / remarks:

adopted 29 July 2016,

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MITI/MHW guidelines for testing of new chemical substances.

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 gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Group 1 (4-h without S9): 1.22, 2.44, 4.88, 9.75, 19.5 and 39 μg/mL
Group 2 (4-h with S9 (2%)): 0.61, 1.22, 2.44, 4.88, 9.75 and 19.5 μg/mL
Group 3 (24-h without S9): 1.22, 2.44, 4.88, 9.75, 19.5 and 39 μg/mL

The dose range of test substance used in the main test was selected following the results of a preliminary toxicity test. A precipitate of the test substance was observed at and above 39 μg/mL in all three exposure groups. The dose levels plated for viability and expression of mutant colonies were as follows:

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

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 Burroughs 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 approximately 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:
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%.

Test substance preparation:
Following solubility checks performed in-house, the test substance was accurately weighed and formulated in DMSO prior to serial dilutions being prepared. The test substance is a UVCB with a purity value of 100%; therefore the maximum recommended dose level was initially set at 5000 μg/mL. However the maximum achievable dose level was 2500 μg/mL due to formulation problems at 5000 μg/mL. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in Mutation Frequency (MF) above the concurrent background exceeds the Global Evaluation Factor (GEF) and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Remarks:

DMSO

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

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

Executive summary:

An in vitro study was conducted to determine the genotoxic potential of the test substance, 'mono- C16 PSE and C16-OH', using the thymidine kinase gene according to OECD Guideline 490, in compliance with GLP. This study was performed to investigate the potential of the test substance to induce mutations at the thymidine kinase locus (TK1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y TK+/- cells. One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at 8 dose levels (ranging from 0.61 to 78 μg/mL) in duplicate, together with vehicle (DMSO), 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 dose range of test substance used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies in the preliminary test ranged from 2.44 to 625 μg/mL in the three exposure groups (i.e., 4 h with and without S9 and 24 h without S9). A precipitate of the test substance was observed at and above 39 μg/mL in all three exposure groups. Therefore, the maximum dose level used for the main test was limited by precipitate.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. There was evidence of moderate toxicity following exposure to the test substance in the 4 h and 24 h exposure in the absence of metabolic, as indicated by the % RSG (relative suspension growth) and RTG (relative total growth) values. There was no evidence of reductions in viability (%V) in either of the three exposure groups, therefore indicating that residual toxicity had not occurred. The dose levels of 0.61 and 78 μg/mL in both the 4 and 24 h exposure –S9 were not plated out for 5-TFT resistance and viability due to excessive toxicity. The dose levels of 39 and 78 μg/mL in the 4 h +S9 exposure again were not plated out for 5-TFT resistance and viability due to excessive toxicity. 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 x 10-6 per viable cell in either of the three exposure groups. The GEF (Global Evaluation Factor) value of the test substance dose levels were not exceeded in any of the three exposure groups, including dose levels beyond the acceptable level of toxicity in the 24 h exposure. Under the study conditions, the test substance was considered not to induce mutations in the mouse lymphoma thymidine kinase locus assay, with and without metabolic activation in the absence and presence of metabolic activation (Envigo, 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In absence of bacterial reversion mutation (Ames) and chromosomal aberration assays with the test substance, these endpoints have been assessed based on studies for substances representative of the two main constituents, which can be categorised as phosphate esters (PSE) and alcohol. The results are presented below:

Ames test:

Constituent 1: PSE – read across study:

A study was conducted to determine the genotoxic potential of read across substance, ‘mono- and di- C16 PSE, K+ and H3PO4’ (purity: 100%), using Ames test, according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of 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 metabolic activation (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and ranged from 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test substance formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test substance concentrations per bacterial strain 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. The vehicle (sterile distilled water) control plates gave counts of revertant colonies generally 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. 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 second mutation test (pre-incubation method). A test substance precipitate (particulate in appearance) was noted at and above 1500 μg/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) and 2 (pre-incubation method). Under the study conditions, the read across substance was determined to be non-genotoxic in the Ames test, with and without metabolic activation (Envigo, 2017).

Constituent 2: Alcohol

A study was conducted to determine the genotoxic potential of hexadecan-1-ol (purity not specified), according to the OECD Guideline 471, using Ames test, in compliance with GLP. Two plate incorporation assays were performed, each in triplicate. Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 were incubated (at 37ºC) with the test substance at concentrations 50, 150, 500, 1500 and 5000 µg/plate for 48 h, with and without metabolic activation. Aroclor 1254 induced rat liver S9 was used as the metabolic activation system. No increase in reverse mutation rate in any strain at dose levels up to 5000 µg/plate was observed. Respective positive (without S9: N-ethyl-N’-nitrosoguanidine (TA100, TA1535), 9-aminoacridine (TA1537), 4-nitro-o-phenylene diamine (TA 1538), 4-nitroquinoline-1-oxide (TA98); with S9: 2-aminoanthracene) and negative (acetone) controls gave appropriate responses. Precipitation was noted at 5000 µg/plate, however, the study author concluded that this did not interfere with counting revertant colonies and the results. There was no evidence of cytotoxicity up to 5000 µg/plate with or without metabolic activation. There were no statistically significant differences between test and control plates observed. The study has met the validity criteria. Under the study conditions, the read across substance was determined to be non-mutagenic in the Ames test, with and without metabolic activation (OECD SIDS, 2006).

Mammalian cytogenicity assay:

Constituent 1: PSE – read across study:

A vitro study was conducted to determine the clastogenicity of the read across substance, 'mono- and di- C16 PSE, K+ and H3PO4' (purity: 100%), according to the OECD Guideline 473, using Chromosome Aberration test in human lymphocytes, in compliance with GLP. Duplicate cultures of human lymphocytes, treated with the read across 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 to human lymphocytes in the Chromosomal aberration assay, with and without metabolic activation (Envigo, 2017).

Constituent 2: Alcohol – read across study:

Anin vivostudy was conducted to determine the genotoxic potential of 1-dodecanol (purity not specified) in mice, according to OECD Guideline 474 (Micronucleus test), in compliance with GLP. In this study, 12 mice (6 per sex) per dose, were administered single dose of each, read across substance (5000 mg/kg bw dodecanol in arachis oil), positive control (Cyclophosphamide 20 mg/kg bw) and solvent control (Arachis oil) substances at 10 mL/kg bw dose, by oral gavage. The maximum tolerated dose (MTD) of 5000 mg/kg bw was determined in a screening test. Samples of blood were collected and analysed 24, 48 and 72 h after treatment period. Two slides were prepared for each animal and 1000 polychromatic erythrocytes (PCE) scored. A statistically significant (p<0.05) increase in PCE compared to normochromatic erythrocytes (NCE) was examined by Kastenbaum & Bowman method. Clinical examination was performed daily. No mortality was observed during the study. Only reported clinical sign was piloerection in all animals. There was no increase in the incidence of micronucleated cells in the test group. The incidence of micronuclei in the control group was within historical control ranges. The positive control group produced an appropriate increase in numbers of micronucleated cells. No statistically significant increase in micronucleated polychromatic erythrocytes in mice was observed at any time interval after treatment (24, 48 or 72 h) at dose levels up to 5000 mg/kg bw when compared to vehicle controls (PCE/NCE ratio), whereas the positive control group did produce a statistically significant increase. The study had met all the validity criteria required for the test. Under the study conditions, the read across substance was concluded to be non-clastogenic in the micronuleus assay in mice (OECD SIDS, 2006). Based on the result of the read across study, similar absence of genotoxicity inin vivomicronucleus test can be expected for the test substance, ‘mono- C16 PSE and C16-OH’.

Mammalian cell gene mutation assay:

A study was conducted to determine the genotoxic potential of the test substance, 'mono- C16 PSE + C16-OH', with thein vitro mammalian cell gene mutation tests using the thymidine kinase gene according to OECD Guideline 490, in compliance with GLP. This study was performed to investigate the potential of the test substance to induce mutations at the thymidine kinase locus (Tk1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y Tk+/- cells. The dose range of test substance used in the main test was selected following the results of a preliminary toxicity test. A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 h exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24 h exposure period without S9. The dose range was set at 2.44 to 625 μg/mL in all three exposure groups. A precipitate of the test substance was observed at and above 39 μg/mL in all three exposure groups. One main mutagenicity test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at 8 dose levels in duplicate, together with vehicle (DMSO), 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. There was evidence of moderate toxicity following exposure to the test substance in the 4 h and 24 h exposure in the absence of metabolic, as indicated by the %RSG and RTG values. There was no evidence of reductions in viability (%V) in either of the three exposure groups, therefore indicating that residual toxicity had not occurred. The dose levels of 0.61 and 78 μg/mL in both the 4 and 24 h exposure –S9 were not plated out for 5-TFT resistance and viability due to excessive toxicity. The dose levels of 39 and 78 μg/mL in the 4 h +S9 exposure again were not plated out for 5-TFT resistance and viability due to excessive toxicity. The test system was operating satisfactorily, and that the metabolic activation system was functional, based on the vehicle control and positive control mutant frequency. The test substance did not induce any toxicologically significant increases in the mutant frequency x 10-6per viable cell in either of the three exposure groups. The GEF value of the test substance dose levels were not exceeded in any of the three exposure groups, including dose levels beyond the acceptable level of toxicity in the 24 h exposure. The test substance did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Under the study conditions, the test substance was determined to be non-mutagenic with and without metabolic activation (Envigo, 2017).

Overall, based on the available weight of evidence from the in vitro and in vivo read across studies on the main constituents as well as the results of the in vitro mammalian cell gene mutation assay, the test substance, ‘mono- C16 PSE and C16-OH’ is considered to be non-genotoxic.

Justification for classification or non-classification

Based on the available weight of evidence information from the in vitro and in vivo read across studies of the main constituents as well as the results of the in vitro mammalian cell gene mutation assay, the test substance, ‘mono- C16 PSE and C16-OH’ does not warrant a classification for genotoxicity according to the EU CLP criteria (Regulation 1272/2008/EC).​​