Fatty acids, C18-unsatd., phosphates

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): non-mutagenic with or without metabolic activation

In vivo cytogenicity assay (OECD 473): negative with or without metabolic activation

In vitro gene mutation assay (OECD 490): negative with or without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

In the pre - incubation assay in the tester strain TA100, the test item solutions were incubated for 29 minutes. The deviation had no effect on the study integrity.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0101891886
- Expiration date of the lot/batch: 02 November 2017
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Stable, maximum temperature: 60 °C
- Solubility and stability of the test substance in the solvent/vehicle: Dimethyl sulfoxide: Not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Dimethyl sulfoxide: Not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the purity/composition of the test compound. A solubility test was performed based on visual assessment. The test item was dissolved in dimethyl sulfoxide. Test item concentrations were used within 1.5 hours after preparation. Any residual volumes were discarded.
- Final dilution of a dissolved solid, stock liquid or gel: A solubility test was performed based on visual assessment. The test item was dissolved in dimethyl sulfoxide.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate.

Vehicle / solvent:

The vehicle of the test item was dimethyl sulfoxide (Merck, Darmstadt, Germany).

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO; Saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Preincubation period: 30 minutes
- Exposure duration:48±4 h

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a)The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b)The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a)The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b)In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment

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

Untreated negative 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

Untreated negative 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

Untreated negative 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:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence of S9-mix at the highest concentration (5000 ug/ml) tested.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
In conclusion, based on the results of this study it is concluded that Fatty acids, C18-unsatd., phosphates is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of Fatty acids, C18-unsatd., phosphates. and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli(E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 0101891886 of Fatty acids, C18-unsatd., phosphates. was a yellow liquid. The test item was dissolved in dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence of S9-mix at the highest concentration tested.  In all other tester strains, the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that Fatty acids, C18-unsatd., phosphates.is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.  

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 29, 2017 - November 3, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and in compliance with OECD guideline 473.

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

Specific details on test material used for the study:

Appearance: Yellow liquid
Batch: 0101891886
Purity/Composition: UVCB
Test item storage: At room temperature

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age).
- Suitability of cells: Peripheral human lymphocytes are recommended in international guideline (OECD).

The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016) are presented below:
Dose-range finding study: age 29, AGT = 14.2 h
First cytogenetic assay: age 25, AGT = 13.2 h
Cytogenetic assay 1A: age 35, AGT = 13.9 h
Cytogenetic assay 1B: age 31, AGT = 13.2 h
Second cytogenetic assay: age 25, AGT = 14.3 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) (Life technologies) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).
- Properly maintained: yes , All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 42 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 28.4 - 37.2°C).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg)

Test concentrations with justification for top dose:

Range finding assay:
Fatty acids, C18-unsatd., phosphates was tested in the absence and in the presence of 1.8% (v/v) S9-fraction. Fatty acids, C18-unsatd., phosphates concentrations tested in the range-finding assay were 39, 78, 156, 313, 625 and 1250 µg/ml culture medium

First cytogenetic assay:

3-hour exposure, 24 h fixation time
with and without S9-mix: 500, 600, 700, 800 µg/ml culture medium
no appropriate dose levels could be selected for scoring of chromosome aberrations.

The experiment was repeated in cytogenetic assay 1A. (3 h exposure time, 24 h fixation time):
Without S9-mix : 400, 500, 525, 550, 575, 600, 625 and 650 µg/ml culture medium
With S9-mix : 500, 600, 675, 700, 725, 750 and 775 µg/ml culture medium

An additional experiment was performed to obtain additional information about the cytotoxicity of the test item. Cytogenetic assay 1B:
With S9-mix : 500, 600, 700, 710, 720, 730, 740, 750 and 760 µg/ml culture medium (3 h exposure time, 24 h fixation time).

Based on both experiments the following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix : 400, 550 and 575 µg/ml culture medium (3 h exposure time, 24 h fixation time).
With S9-mix : 500, 700 and 725 µg/ml culture medium (3 h exposure time, 24 h fixation time).

Second cytogenetic assay:

To obtain more information about the possible clastogenicity of Fatty acids, C18-unsatd., phosphates, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Fatty acids, C18-unsatd., phosphates in the absence of S9-mix for 24 or 48 hours.

Without S9-mix : 10, 50, 100, 150, 200, 250, 300 and 350 µg/ml culture medium

Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix : 10, 150 and 250 µg/ml culture medium (24 h exposure time, 24 h fixation time).
10, 50 and 200 µg/ml culture medium (48 h exposure time, 48 h fixation time).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
As DMSO is compatible to the test system, it was selected for vehicle (solvent) of the study.

Untreated negative controls:

yes

Remarks:

Negative (solvent) control was DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO was run concurrently with treatment groups

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

SPINDLE INHIBITOR (cytogenetic assays): During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/ml medium) (Acros Organics, Geel, Belgium).

STAIN (for cytogenetic assays): The cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for
10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

NUMBER OF CELLS EVALUATED: Fifty six metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations (see study plan deviations). In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations were calculated. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analyzable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analyzed from those cultures with an inhibition of the mitotic index of 55 ± 5%, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

Evaluation criteria:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test,
one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

lymphocytes: human peripheral

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

The ability of Fatty acids, C18-unsatd., phosphates to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analyzed was selected based on the solubility of the test item in the culture medium (presence of S9-mix) or on toxicity, inhibition of the mitotic index of about 50% or greater. 

The scores for the number of aberrant cells (gaps included and excluded) and the number of the various types of chromosome aberrations at the various concentrations of Fatty acids, C18-unsatd., phosphates are presented in Table1,Table 2 and Table 3 -4. Duplicate cultures are indicated by A and B. 

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals (-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix Fatty acids, C18-unsatd., phosphates did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No biologically relevant effects of Fatty acids, C18-unsatd., phosphates on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Fatty acids, C18-unsatd., phosphates does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described. 

Table 1    
Chromosome Aberrations in Human Lymphocyte Cultures Treated with Fatty acids, C18-unsatd., phosphates in the Absence of S9-Mix in Cytogenetic Assay 1A (3 H Exposure Time, 24 H Fixation Time)

Conc	DMSO (1.0% v/v)				400 µg/ml				550 µg/ml				575 µg/ml				MMC-C 0.5 µg/ml
Culture	A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B
Mitotic Index (%)	100				80				61				43				66
No. of Cells scored	150  150300				150  150300				150  150300				150  150300				150  150300
No. of Cells with aberrations (+ gaps) a)	0	0		0	2	1		3	2	0		2	0		0	0	27	27		***^) 54
No. of Cells with aberrations (- gaps)	0	0		0	1	1		2	1	0		1	0		0	0	27	26		***^) 53
g’					1				1									1
g”
b’																	16	21
b”					1	1			1								6	6
m’
m”
exch.																	7	3
dic
d’
misc.														endo poly
total aberr (+ gaps)	0	0			2	1			2	0			0		0		29	31
total aberr (- gaps)	0	0			1	1			1	0			0		0		29	30

^a)    Abbreviations used for various types of aberrations are listed inAppendix 2.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
The numerical variations endoreduplication (endo) and polyploidy (poly) were not counted as an aberration.

*)   Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or
*** P < 0.001

  Table 2      
Chromosome Aberrations in Human Lymphocyte Cultures Treated with Fatty acids, C18-unsatd., phosphates in the Presence of S9-Mix in Cytogenetic Assay 1A (3 H Exposure Time, 24 H Fixation Time)

Conc	DMSO (1.0% v/v)				500 µg/ml				700 µg/ml				725 µg/ml				CP 10 µg/ml
Culture	A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B
Mitotic Index (%)	100				95				62				57				54
No. of Cells scored	150  150300				150  150300				145  150295				150  56  206				150  150300
No. of Cells with aberrations (+ gaps) a)	0	1		1	0	1		1	0	0		0	1	0		1	23	29		***^) 52
No. of Cells with aberrations (- gaps)	0	1		1	0	1		1	0	0		0	1	0		1	23	27		***^) 50
g’
g”																	2	4
b’						1							1				16	11
b”																	7	15
m’		1															1
m”
exch.																	3	5
dic
d’
misc.									poly endo		endo		endo		poly endo
total aberr (+ gaps)	0	1			0	1			0	0			1	0			29	35
total aberr (- gaps)	0	1			0	1			0	0			1	0			27	31

^a)    Abbreviations used for various types of aberrations are listed inAppendix 2.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
The numerical variations endoreduplication (endo) and polyploidy (poly) were not counted as an aberration.

*)   Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or
*** P < 0.001.

 

Table 3        
Chromosome Aberrations in Human Lymphocyte Cultures Treated with Fatty acids, C18-unsatd., phosphates in the Absence of S9-Mix in the Second Cytogenetic Assay
(24 H Exposure Time, 24 H Fixation Time)

Conc	DMSO (1.0% v/v)				10 µg/ml				150 µg/ml				250 µg/ml				MMC-C 0.2 µg/ml
Culture	A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B
Mitotic Index (%)	100				89				69				52				37
No. of Cells scored	150  150300				150  150300				150  150300				150  150300				150  150300
No. of Cells with aberrations (+ gaps) a)	0	0		0	0	0		0	0	0		0	1	0		1	24	22		***^) 46
No. of Cells with aberrations (- gaps)	0	0		0	0	0		0	0	0		0	1	0		1	24	19		***^) 43
g’																		3
g”
b’													1				23	13
b”																	5	5
m’
m”
exch.																	2	3
dic
d’
misc.
total aberr (+ gaps)	0	0			0	0			0	0			1	0			30	24
total aberr (- gaps)	0	0			0	0			0	0			1	0			30	21

^a)    Abbreviations used for various types of aberrations are listed inAppendix 2.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

*)   Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or
*** P < 0.001.

  

Table 4      
Chromosome Aberrations in Human Lymphocyte Cultures Treated with Fatty acids, C18-unsatd., phosphates in the Absence of S9-Mix in the Second Cytogenetic Assay
(48 H Exposure Time, 48 H Fixation Time)

Conc	DMSO (1.0% v/v)				10 µg/ml				50 µg/ml				200 µg/ml				MMC-C 0.1 µg/ml
Culture	A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B				A   B    A+B
Mitotic Index (%)	100				85				67				51				65
No. of Cells scored	150  150300				150  150300				150  150300				150  150300				150  150300
No. of Cells with aberrations (+ gaps) a)	0	1		1	1	0		1	0	0		0	2	0		2	52	57		***^) 109
No. of Cells with aberrations (- gaps)	0	0		0	1	0		1	0	0		0	1	0		1	48	53		***^) 101
g’		1											1				8	3
g”																		2
b’					1												42	41
b”																	6	9
m’
m”													1				6	3
exch.																	17	14
dic
d’
misc.
total aberr (+ gaps)	0	1			1	0			0	0			2	0			79	72
total aberr (- gaps)	0	0			1	0			0	0			1	0			71	67

^a)    Abbreviations used for various types of aberrations are listed inAppendix 2.
misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.

*)   Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or
*** P < 0.001.

Conclusions:

Fatty acids, C18-unsatd., phosphates is not clastogenic in human lymphocytes under the experimental conditions described.

Executive summary:

The objective of this study was to evaluate Fatty acids, C18-unsatd., phosphates for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

The possible clastogenicity of Fatty acids, C18-unsatd., phosphates was tested in two independent experiments.

The study procedures described are in compliance with the most recent OECD guideline. The test item was dissolved in dimethyl sulfoxide.

In the first cytogenetic assay, Fatty acids, C18-unsatd., phosphates was tested up to 575 and 725 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. In the absence of S9-mix, appropriate toxicity was reached at this dose level. In the presence of S9-mix, Fatty acids, C18-unsatd., phosphates precipitated in the culture medium at this dose level. 

In the second cytogenetic assay, Fatty acids, C18-unsatd., phosphates was tested up to 250 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 200 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Fatty acids, C18-unsatd., phosphates did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No biologically relevant effects of Fatty acids, C18-unsatd., phosphates on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Fatty acids, C18-unsatd., phosphates does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. 

In conclusion, this test is valid and Fatty acids, C18-unsatd., phosphates is not clastogenic in human lymphocytes under the experimental conditions described. 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 8, 2018 - March 11, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was regarded reliable without restriction since the study was conducted according to the OECD guideline 471 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Appearance: Yellow liquid
Batch: 0101891886
Purity/Composition: UVCB
Test item storage: At room temperature

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale: Recommended test system in international guidelines (e.g.OECD).
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 106 cells/mL.

Horse serum
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.

Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (LifeTechnologies).

Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).

Exposure medium
For 3 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure:
Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine (TFT) (Sigma).

Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).

Environmental conditions
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 66 - 99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.9 - 37.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.

- Properly maintained: yes

- Periodically checked for Mycoplasma contamination: yes

- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).

Test concentrations with justification for top dose:

The dose range finding test
-In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 78 to 1250 μg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

First mutagenicity test
Without S9-mix: 3.1, 6.3, 12.5, 25, 50, 80, 90, 100, 110, 120, 130, 140 and 150 μg/mL exposure medium.
With S9-mix: 6.3, 12.5, 25, 50, 100, 150, 175, 200, 220, 240, 260, 280 and 300 μg/mL exposure medium.

Second mutagenicity test
To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period.
Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing:
Without S9-mix: 3.8, 7.5, 15, 30, 50, 70, 90, 100, 110, 120, 130, 140 and 150 μg/mL exposure medium.

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide (SeccoSolv, Merck Darmstadt,Germany).

Untreated negative controls:

yes

Remarks:

The negative control was dimethyl sulfoxide, the vehicle of the test item.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment. followed by a recovery period of 2 days.

- Exposure duration: The cell cultures were exposed for 3 hours or 24 hours

- Expression time (cells in growth medium): 2 days

- Selection time (if incubation with a selection agent): 11 - 12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF CELLS EVALUATED: 10 000 cells

DETERMINATION OF CYTOTOXICITY

The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. This value obtained is the P(0), the zero term of the Poisson distribution:
P(0) = number of empty wells/total number of wells

The cloning efficiency (CE) is therefore:
CE = -ln P(0)/number of cells plated per well

The relative survival (RS) in each treatment group was determined by comparing cloning efficiencies in treatment and control cultures:
RS = [CE(test)/CE(controls)] x 100

The Relative Total Growth (RTG) was calculated as the product of the cumulative relative suspension growth (RSG) and the relative cloning efficiency for each culture:
RTG = RSG x RSday2/100

Suspension Growth (SG) =
[Day 0 cell count/ (1.25 x 105) *] x [Day 1 cell count/(1.25 x 105) *] x [Day 2 cell count]

Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100
RSday2 = CE day2 (test) / CE day2 (controls) x 100

The growth rate, as an indicator of optimally growing cultures, was calculated for the solvent control cultures:
Growth Rate (GR) for the 3 hours treatment= [Day 1 cell count/(1.25 x 105) *] x [Day 2 cell count/(1.25 x 105) *]

* Or appropriate cell concentration

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 (ref. 12).
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if: None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The relative suspension growth was 8 % at the test substance concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the concentration of 2024 ug/ml.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The relative suspension growth was 19 % at the test substance concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the concentration of 2024 ug/ml.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 78 to 1250 μg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

In the absence of S9-mix, the relative suspension growth was 75% at the test item concentration of 78 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 156 μg/mL and above. In the presence of S9-mix, the relative suspension growth was 80% at the test item concentration of 156 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 313 μg/mL and above.

The relative suspension growth was 60% at the test item concentration of 78 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentrations of 156 μg/mL and above.

Remarks on result:

other: strain/cell type: L5178Y/TK+/--3.7.2C mouse lymphoma cells

First mutagenicity test

Based on the results of the dose-range finding test, the following dose-ranges were selected for the first mutagenicity test:

Without S9-mix: 3.1, 6.3, 12.5, 25, 50, 80, 90, 100, 110, 120, 130, 140 and 150 μg/mL exposure medium.

With S9-mix: 6.3, 12.5, 25, 50, 100, 150, 175, 200, 220, 240, 260, 280 and 300 μg/mL exposure medium.

Evaluation of toxicity

In the absence of S9-mix, the dose levels of 3.1 to 110 μg/mL showed similar no cytotoxicity.

Therefore, the dose levels of 3.1, 80, 90 and 110 μg/mL were not regarded relevant for mutation frequency measurement.

The dose level of 150 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing.

 

In the presence of S9-mix, the dose levels of 6.3 to 100 μg/mL showed similar no cytotoxicity. Therefore, the dose level of 6.3 μg/mL was not regarded relevant for mutation frequency measurement. The dose levels of 240 to 300 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were:

Without S9-mix: 6.3, 12.5, 25, 50, 100, 120, 130 and 140 μg/mL exposure medium.

With S9-mix: 12.5, 25, 50, 100, 150, 175, 200 and 220 μg/mL exposure medium.

In the absence of S9-mix (Table 3), the relative total growth of the highest test item concentration was 2% compared to the total growth of the solvent controls.

In the presence of S9-mix, the relative total growth of the highest test item concentration was 23% compared to the total growth of the solvent controls.

Evaluation of the mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

 

Second mutagenicity test

To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period.

Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 3.8, 7.5, 15, 30, 50, 70, 90, 100, 110, 120, 130, 140 and 150 μg/mL exposure medium.

Evaluation of toxicity

The dose levels of 3.8 to 70 μg/mL showed similar no cytotoxicity. Therefore, the dose levels of 3.8, 15 and 50 μg/mL were not regarded relevant for mutation frequency measurement. The dose levels of 140 and 150 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

The dose levels selected to measure mutation frequencies at the TK-locus were: 7.5, 30, 70, 90, 100, 110, 120 and 130 μg/mL exposure medium.

The relative total growth of the highest test item was 10% compared to the total growth of the solvent controls.

Evaluation of mutagenicity

No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

 

Conclusions:

Fatty acids, C18-unsatd., phosphates. is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of Fatty acids, C18-unsatd., phosphates. by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.  The study procedures described in this report were based on the most recent OECD guideline. 

In the first experiment, the test item was tested up to concentrations of 140 µg/mL and 220 µg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 2% and 23% in the absence and presence of S9-mix, respectively.

In the second experiment, the test item was tested up to concentrations of 130 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 10%. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. 

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

In conclusion, Fatty acids, C18-unsatd., phosphates. is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames (OECD471)

 

The objective of this study was to determine the potential of Fatty acids, C18-unsatd.,phosphates. and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. The test item was dissolved in dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first mutation experiment, the test item was tested up to concentrations of 5000μg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 5000μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and a reduction of the bacterial background lawn, was observed in tester strain TA100 in the absence of S9-mix at the highest concentration tested. In all other tester strains, the bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that Fatty acids, C18-unsatd., phosphates. is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Cytogenicity Assay (OECD473)

 

The objective of this study was to evaluate Fatty acids, C18-unsatd., phosphates for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity of Fatty acids, C18-unsatd., phosphates was tested in two independent experiments. The test item was dissolved in dimethyl sulfoxide.

In the first cytogenetic assay, Fatty acids, C18-unsatd., phosphates was tested up to 575 and 725μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction, respectively. In the absence of S9-mix, appropriate toxicity was reached at this dose level. In the presence of S9-mix, Fatty acids, C18-unsatd., phosphates precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Fatty acids, C18-unsatd., phosphates was tested up to 250μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 200 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Fatty acids, C18-unsatd., phosphates did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No biologically relevant effects of Fatty acids, C18-unsatd., phosphates on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Fatty acids, C18-unsatd., phosphates does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

In conclusion, this test is valid and Fatty acids, C18-unsatd., phosphates is not clastogenic in human lymphocytes under the experimental conditions described in this report.

 

Mutagenicity Assay (OECD490)

 

The objective of this study was to evaluate the mutagenic potential of Fatty acids, C18-unsatd., phosphates. by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The vehicle of the test item was dimethyl sulfoxide.

In the first experiment, the test item was tested up to concentrations of 140 µg/mL and 220 µg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 2% and 23% in the absence and presence of S9-mix, respectively.

In the second experiment, the test item was tested up to concentrations of 130 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 10%. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. 

In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.

In conclusion, Fatty acids, C18-unsatd., phosphates. is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

Based on the results of in vitro bacterial gene mutation study, in vitro mammalian chromosomal aberration and gene mutation studies no classification is proposed for genotoxicity according to the criteria of CLP regulation 1272/2008.