Reaction mass of orthophosphoric acid and potassium dihexadecyl phosphate and dipotassium hexadecyl phosphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From August 01, 2017 to October 30, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of mono- and di- hexadecyl phosphate esters, potassium salts and phosphoric acid

Molecular formula:

C16H34O4P1K1 (representative: mono- C16 PSE, K+)

IUPAC Name:

Reaction mass of mono- and di- hexadecyl phosphate esters, potassium salts and phosphoric acid

Test material form:

solid: particulate/powder

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:

Under study conditions, the test substance was considered to be non clastogenic in the chromosomal aberration assay, with and without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the clastogenicity of the test substance, 'mono- and di- C16 PSE, K+ and H3PO4' (purity: 100%) using human lymphocytes, according to the OECD Guideline 473 (Chromosome Aberration test) and Japanese Guidelines, 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 study conditions, the test substance was considered to be non-clastogenic to human lymphocytes in the chromosomal aberration assay, with and without metabolic activation (Envigo, 2017).