Fatty acids, C18-36, esters with ethylene glycol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test the substance was tested in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strainWP2uvrAin presence and absence of metabolic activation. No increase in mutant frequency was reported in any of the strains tested both in presence and absence of metabolic activation. The substance is considered to be non-mutagenic under the conditions of this test.

Duplicate cultures of human lymphocytes, treated with the substance, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The substance did not demonstrate any marked toxicity 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. The substance was considered to be non-clastogenic to human lymphocytes in vitro.

The analogue substance was tested in the L5178Y TK +/- Mouse  Lymphoma Assay according to OECD 490 up to precipitation level. Exposure was either 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). No increase in the mutant frequency at the TK +/- locus was reported (evaluation with the Global Evaluation Factor (GEF) of 126 E-06). Therefore it is concluded that the substance is non-mutagenic in this assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2017 to 12 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

NA

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: human peripheral lymphocytes
- Suitability of cells: standard cells (checked for exposure to high levels of radiation or hazardous chemicals and not knowingly recently suffered from a viral infection)
- Cell cycle length, doubling time or proliferation index: AGT 16 hours
- Sex, age and number of blood donors if applicable: Preliminary Toxicity Test: male, aged 31 years; Main Experiment: female, aged 30 years
- Whether whole blood or separated lymphocytes were used if applicable: fresh heparinized whole blood
- Methods for maintenance in cell culture: grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air
- Modal number of chromosomes: 44-48
- Normal (negative control) cell cycle time: 16 hours

MEDIA USED
- Type and identity of media including CO2 concentration: MEM at approximately 37 ºC with 5 % CO2
- Properly maintained: yes

Cytokinesis block (if used):

demecolcine (Colcemid 0.1 µg/mL) 2.5 hours before the required harvest time.

Metabolic activation:

with and without

Metabolic activation system:

S9 from rat livers induced with phenobarbital/beta-naphthoflavone

Test concentrations with justification for top dose:

Exposure Group Final concentration of

4(20)-hour without S9 0*, 1.25, 2.5, 5, 8*, 10*, 20*, 40, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 5, 8, 10, 15*, 20*, 40*, 80, CP 1*
24-hour without S9 0*, 1.25, 2.5, 5, 8*, 10*, 20*, 40, MMC 0.1*


* Dose levels selected for metaphase analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)(0.25% in 25 µl aliquots; maximum achieved dosing concentration 625 µg/mL)
- Justification for choice of solvent/vehicle: insoluble in dimethyl sulphoxide at 125, 250 and 500 mg/mL and acetone at 250 and 500 mg/mL. However, the substance was soluble in THF at 500 mg/mL

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DURATION
- pre-incubation: 48 hours at approximately 37 ºC, 5% CO2 in humidified air
- Exposure duration: 4 hour with and without metabolic activation, 24 hours without metaboliv activation at approximately 37 ºC, 5% CO2 in humidified air
- Expression time (cells in growth medium): 4 hour tests: 20 hours at approximately 37 ºC, 5% CO2 in humidified air

SPINDLE INHIBITOR: Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) 2.5 hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2/concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: 2000/concentration for mitotic index

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE : 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: cells with 69 chromosomes or more were scored as polyploid cells
- Determination of endoreplication: recorded separately and included in the polyplod cell total

Rationale for test conditions:

standard according to the guidance

Evaluation criteria:

•The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range
•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
•The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
•The required number of cells and concentrations were analyzed

Statistics:

Fisher's Exact test (p<0.05)

Key result

Species / strain:

lymphocytes: human lymphocytes

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Water solubility: see vehicle selection
- Precipitation: yes at 20 ug/mL in tests without metabolic activation and at 40 ug/mL in test with metabolic activation

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: available (all findings were within historical control values)
- Negative (solvent/vehicle) historical control data: available (all findings were within historical control values)

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity observed

details see attached document

Conclusions:

The substance is considered not clastogenic under the conditions of the test

Executive summary:

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated;4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour 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. The dose levels selected for the Main Test were as follows:

Group	Final concentration of substance evaluated
4(20)-hour without S9	0, 8, 10, 20
4(20)-hour with S9 (2%)	0. 15, 20, 40
24-hour without S9	0, 8, 10, 20

All vehicle (Tetrahydrofuran) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items 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 item did not demonstrate any marked toxicity 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. 

The test item,was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September 2016 to 27 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells (Salmonella/E.coli): University of California, Berkeley, on culture discs, on 04 August 1995/British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
- Methods for maintenance in cell culture if applicable: stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

Test 1 (plate incorporation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Test 2 (pre-incubation): 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: not soluble in distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and in acetone at 100 mg/mL but fully soluble in tetrahydrofuran at 200 mg/mL.

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: exp 1: plate incorporation; exp 2: preincubation

DURATION
- Preincubation period: 20 min at 37 ± 3 °C
- Exposure duration: 48 h at 37 ± 3 °C

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY: no cytotoxicity observed up to 5000 ug/plate (There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix))

Evaluation criteria:

A substance is considered positive when:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

Statistics:

Dunnetts Regression Analysis

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100 and WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

precipitate at and above 500 ug/plate (no interference with scoring)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

A test item precipitate (particulate and white in appearance) was noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.
A small, statistically significant increase in WP2uvrA revertant colony frequency was observed in the first mutation test at 50 µg/plate in the absence of S9-mix. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose level was within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.7 times the concurrent vehicle control.

Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 27 September 2016					To: 30 September 2016
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (THF)	96 92 103	(97) 5.6#	26 14 10	(17) 8.3	18 24 18		(20) 3.5	32 28 28	(29) 2.3	27 10 8	(15) 10.4
	1.5 µg	104 101 100	(102) 2.1	12 12 9	(11) 1.7	17 24 29		(23) 6.0	24 22 28	(25) 3.1	8 7 9	(8) 1.0
	5 µg	92 70 88	(83) 11.7	11 22 10	(14) 6.7	35 18 32		(28) 9.1	25 35 27	(29) 5.3	12 12 19	(14) 4.0
	15 µg	73 84 76	(78) 5.7	15 28 20	(21) 6.6	26 18 38		(27) 10.1	25 19 34	(26) 7.5	18 17 28	(21) 6.1
	50 µg	99 93 104	(99) 5.5	27 24 17	(23) 5.1	28 38 33		* (33) 5.0	36 33 33	(34) 1.7	17 18 18	(18) 0.6
	150 µg	113 96 91	(100) 11.5	19 38 20	(26) 10.7	18 31 32		(27) 7.8	38 28 39	(35) 6.1	19 15 9	(14) 5.0
	500 µg	120 P 95 P 110 P	(108) 12.6	25 P 20 P 21 P	(22) 2.6	30 P 25 P 26 P		(27) 2.6	28 P 24 P 28 P	(27) 2.3	24 P 21 P 20 P	(22) 2.1
	1500 µg	107 P 85 P 76 P	(89) 15.9	19 P 22 P 31 P	(24) 6.2	21 P 23 P 22 P		(22) 1.0	30 P 21 P 23 P	(25) 4.7	15 P 14 P 18 P	(16) 2.1
	5000 µg	105 P 94 P 104 P	(101) 6.1	18 P 22 P 17 P	(19) 2.6	26 P 27 P 28 P		(27) 1.0	22 P 23 P 26 P	(24) 2.1	16 P 13 P 16 P	(15) 1.7
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		422 337 408	(389) 45.6	374 240 288	(301) 67.9	613 661 578		(617) 41.7	101 116 117	(111) 9.0	444 442 347	(411) 55.4

      Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 27 September 2016					To: 30 September 2016
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (THF)	103 114 88	(102) 13.1#	14 11 13	(13) 1.5	22 38 30		(30) 8.0	37 29 32	(33) 4.0	12 16 11	(13) 2.6
	1.5 µg	92 98 94	(95) 3.1	11 8 14	(11) 3.0	28 32 25		(28) 3.5	36 30 43	(36) 6.5	13 14 17	(15) 2.1
	5 µg	92 94 99	(95) 3.6	16 15 15	(15) 0.6	35 32 36		(34) 2.1	34 39 35	(36) 2.6	19 16 16	(17) 1.7
	15 µg	92 96 94	(94) 2.0	17 9 18	(15) 4.9	31 32 43		(35) 6.7	31 34 38	(34) 3.5	17 17 14	(16) 1.7
	50 µg	92 79 101	(91) 11.1	15 17 16	(16) 1.0	32 31 26		(30) 3.2	26 26 23	(25) 1.7	18 12 18	(16) 3.5
	150 µg	87 103 112	(101) 12.7	12 13 21	(15) 4.9	32 37 38		(36) 3.2	23 31 30	(28) 4.4	16 23 13	(17) 5.1
	500 µg	117 P 113 P 111 P	(114) 3.1	10 P 10 P 22 P	(14) 6.9	28 P 29 P 31 P		(29) 1.5	34 P 31 P 32 P	(32) 1.5	14 P 20 P 15 P	(16) 3.2
	1500 µg	75 P 73 P 81 P	(76) 4.2	17 P 14 P 18 P	(16) 2.1	31 P 27 P 34 P		(31) 3.5	28 P 23 P 27 P	(26) 2.6	9 P 13 P 13 P	(12) 2.3
	5000 µg	103 P 94 P 91 P	(96) 6.2	13 P 13 P 20 P	(15) 4.0	36 P 29 P 24 P		(30) 6.0	19 P 16 P 22 P	(19) 3.0	11 P 13 P 10 P	(11) 1.5
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		2720 1734 1794	(2083) 552.8	228 212 219	(220) 8.0	338 315 321		(325) 11.9	166 172 167	(168) 3.2	264 210 176	(217) 44.4

 

        Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 04 October 2016					To: 07 October 2016
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (THF)	102 109 90	(100) 9.6#	12 11 9	(11) 1.5	18 21 36		(25) 9.6	32 15 18	(22) 9.1	8 14 8	(10) 3.5
	15 µg	103 106 122	(110) 10.2	15 11 13	(13) 2.0	24 32 22		(26) 5.3	19 16 20	(18) 2.1	16 12 13	(14) 2.1
	50 µg	90 122 103	(105) 16.1	7 10 9	(9) 1.5	32 33 21		(29) 6.7	18 33 19	(23) 8.4	11 19 11	(14) 4.6
	150 µg	114 111 116	(114) 2.5	9 11 11	(10) 1.2	33 31 22		(29) 5.9	21 19 21	(20) 1.2	18 16 14	(16) 2.0
	500 µg	109 P 104 P 121 P	(111) 8.7	9 P 19 P 9 P	(12) 5.8	25 P 20 P 31 P		(25) 5.5	28 P 20 P 16 P	(21) 6.1	17 P 10 P 10 P	(12) 4.0
	1500 µg	105 P 87 P 90 P	(94) 9.6	11 P 12 P 10 P	(11) 1.0	23 P 34 P 23 P		(27) 6.4	18 P 23 P 21 P	(21) 2.5	16 P 6 P 12 P	(11) 5.0
	5000 µg	113 P 86 P 93 P	(97) 14.0	11 P 7 P 9 P	(9) 2.0	20 P 17 P 14 P		(17) 3.0	16 P 19 P 18 P	(18) 1.5	16 P 11 P 10 P	(12) 3.2
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		774 876 909	(853) 70.4	1931 1537 1800	(1756) 200.7	714 734 848		(765) 72.3	104 112 147	(121) 22.9	258 187 218	(221) 35.6

 

        Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 04 October 2016					To: 07 October 2016
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (THF)	91 111 122	(108) 15.7#	19 9 11	(13) 5.3	37 30 26		(31) 5.6	32 24 27	(28) 4.0	10 12 12	(11) 1.2
	15 µg	129 123 98	(117) 16.4	14 15 12	(14) 1.5	34 17 25		(25) 8.5	37 20 21	(26) 9.5	7 19 10	(12) 6.2
	50 µg	115 112 113	(113) 1.5	15 12 10	(12) 2.5	29 31 31		(30) 1.2	24 26 25	(25) 1.0	21 8 11	(13) 6.8
	150 µg	107 115 124	(115) 8.5	12 9 16	(12) 3.5	23 34 21		(26) 7.0	21 20 32	(24) 6.7	12 16 13	(14) 2.1
	500 µg	106 P 120 P 130 P	(119) 12.1	17 P 14 P 16 P	(16) 1.5	40 P 34 P 33 P		(36) 3.8	25 P 27 P 34 P	(29) 4.7	18 P 16 P 23 P	(19) 3.6
	1500 µg	86 P 109 P 104 P	(100) 12.1	10 P 11 P 8 P	(10) 1.5	28 P 35 P 31 P		(31) 3.5	15 P 31 P 22 P	(23) 8.0	7 P 12 P 4 P	(8) 4.0
	5000 µg	119 P 106 P 87 P	(104) 16.1	14 P 10 P 10 P	(11) 2.3	22 P 37 P 29 P		(29) 7.5	26 P 20 P 24 P	(23) 3.1	10 P 6 P 14 P	(10) 4.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1150 1228 1095	(1158) 66.8	277 254 243	(258) 17.3	178 223 213		(205) 23.6	100 107 119	(109) 9.6	259 206 223	(229) 27.1

Conclusions:

Based on the findings in this test the substance is considered non-mutagenic in bacteria

Executive summary:

In an Ames test the substance was tested in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strain WP2uvrA in presence and absence of metabolic activation. No increase in mutant frequency was reported in any of the strains tested both in presence and absence of metabolic activation. The substance is considered to be non-mutagenic under the conditions of this test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May 2017 to 25 July 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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

``Kanpoan No. 287 - - Environment Protection Agency``
``Eisei No. 127 - - Ministry of Health and Welfare``
``Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry``

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED: L5178Y TK+/- 3.7.2c mouse lymphoma cell line
- Source of cells: MRC Cell Mutation Unit at the University of Sussex, Brighton, UK
- Cell cycle length:ca 12 hours
- Methods for maintenance in cell culture if applicable: stored in liquid nitrogen at approximately -196°C

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes, before freezing

Additional strain / cell type characteristics:

other: TK+/-

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthaflavone induced rat liver S9

Test concentrations with justification for top dose:

4 h and 24 h exposure: 0.61, 1.22, 2.44, 4.88, 9.77, 19.53, 39.06 and 78.13 µg/mL

Top dose based on presence of precipitate

Vehicle / solvent:

THF Due to formulation issues the maximum achievable concentration was set at 625 µg/mL using THF as a vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

A pretest was conducted on cell cultures at 5 x 10 E5 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E5 cells/mL using a 24-hour exposure period without S9. The dose range was set at 0.31 to 78.13 µg/mL (due to precipitation). After exposure cells were washed twice, resuspended and counted. The cultures were serially diluted to 2 x 10E5 cells/mL and incubated at 37 C with 5% CO2 in air and sub-cultured after 24 hours. After a further 24 hours the cultures were counted to assess Suspension Growth (SG)

In the main study exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) in R0 or R10 medium (total volume to 20 mL). The solutions were incubated at 37°C for 4 or 24 hours with continuous shaking. the initial cell number was 1 x 10E6 cells/mL for 4 hour exposures and 0.3 x 10E6 cells/mL for 24 hour exposure.
At the end of the treatment period, for each experiment, the cells were washed twice, resuspended in R20 medium and incubated at 37°C with 5% CO2 (subcultured and counted every 24 hours -> Relative Suspension Growth (%RSG)) for 2 days.

On Day 2 of the experiment, the cells were counted again, diluted to 10E4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates.On this day additional cells were diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37°C with 5% CO2 in air. These plates were also scored for the presence of large and small colonies.

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 MF above the concurrent background exceeds the 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.

Statistics:

Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989).

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no (7.16-7.33)
- Effects of osmolality: no (326-341 mOs)
- Precipitation:
main study: onset at 39.07 µg/mL in all cultures

RANGE-FINDING/SCREENING STUDIES: no cytotoxicity, onset of precipitate at at 39.07 µg/mL in the 4-hour cultures and at 19.53 µg/mL in the 24-hour culture without metabolic activation

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) are reported in an annex to the report

Remarks on result:

other: 4 hour exposure

Treatment (µg/ml)		4-hours-S-9				Treatment (µg/ml)		4-hours+S-9				Treatment (µg/ml)		24-hours-S-9
		%RSG	RTG	MF§				%RSG	RTG	MF§				%RSG	RTG	MF§
0		100	1.00	115.74		0		100	1.00	147.74		0		100	1.00	174.31
0.61	Ø	90				0.61	Ø	130				0.61	Ø	75
1.22		89	0.88	120.90		1.22		136	1.06	147.56		1.22		93	1.16	146.71
2.44		100	0.68	195.26		2.44		102	0.65	145.28		2.44		87	0.92	204.73
4.88		96	0.85	129.53		4.88		99	0.72	160.81		4.88		138	0.92	184.06
9.77		93	0.67	154.02		9.77		99	0.94	154.35		9.77		86	0.86	161.45
19.53		99	0.79	176.14		19.53		93	0.91	164.89		19.53		76	0.70	179.87
39.06		104	0.69	158.45		39.06		90	0.84	176.44		39.06		135	0.84	193.15
78.13	Ø	79				78.13	Ø	86				78.13	Ø	77
EMS						CP						EMS
400		78	0.60	1267.36		1.5		85	0.72	812.12		150		63	0.41	1128.46

 

Treatment (µg/ml) 4-hours-S-9		Proportion small colony mutants	Treatment (µg/ml) 4-hours+S-9	Proportion small colony mutants	Treatment (µg/ml) 24-hours-S-9	Proportion small colony mutants
0		0.48	0	0.46	0	0.43
1.22		0.48	1.22	0.49	1.22	0.41
2.44		0.34	2.44	0.56	2.44	0.43
4.88		0.41	4.88	0.48	4.88	0.47
9.77		0.46	9.77	0.58	9.77	0.56
19.53		0.47	19.53	0.68	19.53	0.35
39.06		0.45	39.06	0.52	39.06	0.42
400 EMS		0.49	1.5 CP	0.68	150 EMS	0.44

Conclusions:

The substance is considered non-mutagenic in the L5178Y TK +/- Mouse Lymphoma Assay

Executive summary:

The substance was tested in the L5178Y TK +/- Mouse  Lymphoma Assay according to OECD 490 up to precipitation level. Exposure was either 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). No increase in the mutant frequency at the TK +/- locus was reported (evaluation with the Global Evaluation Factor (GEF) of 126 E-06). Therefore it is concluded that the substance is non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test on mutagenicity in mammalian cells is performed on an analogue with a similar structure and similar hydrolysis products. The ethylene glycol that could be formed is not mutagenic.

Justification for classification or non-classification

Based on the information available the substance does not need to be classified as mutagen according to Regulation (EC) No 1272/2008 (CLP).