3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471), the test item 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 March 2018 - 06 April 2018

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:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

In the first experiment, the maximum concentration was 5000 µg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

In the second pre-incubation experiment, the dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 µg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

Dimethyl sulphoxide - The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

not specified

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:

EXPERIMENT 1 (PLATE INCORPORATION METHOD)

METHOD OF APPLICATION: An aliquot (0.1 mL) of the appropriate concentration of test item, solvent vehicle or positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel Bonner agar plate. If metabolic activation was required, then 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of the phosphate buffer.

DURATION
- Preincubation period: Not applicable
- Exposure duration: 48 hours at 37 ± 3 °C

NUMBER OF REPLICATIONS: Triplicate

ANALYSIS: The plates were viewed microscopically for evidence of thinning (toxicity).

EXPERIMENT 2 (WITH PRE-INCUBATION)

METHOD OF APPLICATION:A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. If metabolic activation was required, then 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of the phosphate buffer.

DURATION
- Preincubation period:
37 ± 3 °C for 20 minutes (with shaking)
- Exposure duration:
48 hours at 37 ± 3 °C

NUMBER OF REPLICATIONS: Triplicate

ANALYSIS: All plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

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:

not determined

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:

not determined

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:

not determined

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The data obtained for the negative controls were considered acceptable. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1: 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) even at the maximum dose level of 5000 µg/plate.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Experiment 2: Results from Experiment 2 showed that the test item induced a toxic response employing the pre-incubation modification to Salmonella strains TA100 and TA1535 with weakened bacterial background lawns noted in both the absence and presence of S9 mix at 5000 µg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA or Salmonella strains TA98 and TA1537 at any test item dose level in either the absence or presence of S9 mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Table 1            Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
146		19		45		21		9
144	(142)	13	(16)	30	(34)	21	(22)	10	(11)
136		17		28		25		15

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
165		10		31		22		11
149	(158)	11	(11)	26	(26)	19	(22)	14	(10)
159		12		21		26		6

 

Table 2     Test Results: Experiment 1 – Without Metabolic Activation(Plate Incorporation)

Test Period		From: 23 March 2018					To: 26 March 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	152 132 146	(143) 10.3#	23 10 13	(15) 6.8	33 30 23		(29) 5.1	14 23 19	(19) 4.5	15 16 9	(13) 3.8
	1.5 µg	131 139 139	(136) 4.6	14 15 17	(15) 1.5	32 26 33		(30) 3.8	18 26 26	(23) 4.6	7 10 13	(10) 3.0
	5 µg	137 126 148	(137) 11.0	16 10 12	(13) 3.1	27 25 31		(28) 3.1	19 19 17	(18) 1.2	7 8 11	(9) 2.1
	15 µg	139 126 135	(133) 6.7	14 14 8	(12) 3.5	37 29 37		(34) 4.6	17 13 14	(15) 2.1	8 7 15	(10) 4.4
	50 µg	127 150 120	(132) 15.7	14 7 12	(11) 3.6	29 33 29		(30) 2.3	14 16 16	(15) 1.2	9 11 11	(10) 1.2
	150 µg	153 138 129	(140) 12.1	11 13 13	(12) 1.2	33 34 34		(34) 0.6	21 19 23	(21) 2.0	8 15 7	(10) 4.4
	500 µg	125 127 140	(131) 8.1	18 21 15	(18) 3.0	21 36 24		(27) 7.9	22 23 16	(20) 3.8	10 7 13	(10) 3.0
	1500 µg	134 145 147	(142) 7.0	19 21 15	(18) 3.1	26 32 29		(29) 3.0	18 22 16	(19) 3.1	5 8 13	(9) 4.0
	5000 µg	146 117 147	(137) 17.0	26 23 16	(22) 5.1	30 26 22		(26) 4.0	17 16 18	(17) 1.0	16 11 16	(14) 2.9
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
		681 768 764	(738) 49.1	674 728 740	(714) 35.2	820 794 705		(773) 60.3	287 340 333	(320) 28.8	472 338 566	(459) 114.6

Table 3            Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 23 March 2018					To: 26 March 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	123 133 138	(131) 7.6#	19 8 13	(13) 5.5	41 38 36		(38) 2.5	18 29 24	(24) 5.5	11 10 14	(12) 2.1
	1.5 µg	132 132 142	(135) 5.8	15 7 9	(10) 4.2	42 28 37		(36) 7.1	30 26 18	(25) 6.1	16 5 12	(11) 5.6
	5 µg	120 127 128	(125) 4.4	11 9 7	(9) 2.0	32 40 43		(38) 5.7	31 28 28	(29) 1.7	11 6 4	(7) 3.6
	15 µg	140 133 124	(132) 8.0	16 10 20	(15) 5.0	36 37 20		(31) 9.5	34 14 30	(26) 10.6	6 12 7	(8) 3.2
	50 µg	136 139 157	(144) 11.4	7 7 10	(8) 1.7	31 33 42		(35) 5.9	21 18 21	(20) 1.7	10 8 11	(10) 1.5
	150 µg	125 129 123	(126) 3.1	13 17 8	(13) 4.5	35 31 37		(34) 3.1	30 30 12	(24) 10.4	11 15 12	(13) 2.1
	500 µg	135 154 134	(141) 11.3	11 8 13	(11) 2.5	44 37 49		(43) 6.0	13 25 27	(22) 7.6	5 17 6	(9) 6.7
	1500 µg	140 142 147	(143) 3.6	14 10 12	(12) 2.0	49 53 39		(47) 7.2	32 20 26	(26) 6.0	12 12 13	(12) 0.6
	5000 µg	152 122 156	(143) 18.6	18 8 14	(13) 5.0	33 28 44		(35) 8.2	25 26 24	(25) 1.0	12 8 7	(9) 2.6
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
		3315 2885 2890	(3030) 246.8	440 379 398	(406) 31.2	327 408 357		(364) 41.0	200 232 285	(239) 42.9	485 479 495	(486) 8.1

 

Table 4            Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 03 April 2018					To: 06 April 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	160 135 118	(138) 21.1#	17 10 19	(15) 4.7	26 23 39		(29) 8.5	16 21 21	(19) 2.9	14 10 9	(11) 2.6
	15 µg	149 141 146	(145) 4.0	19 12 3	(11) 8.0	23 16 29		(23) 6.5	19 16 18	(18) 1.5	5 9 12	(9) 3.5
	50 µg	137 144 128	(136) 8.0	13 12 22	(16) 5.5	21 28 13		(21) 7.5	23 16 22	(20) 3.8	11 7 9	(9) 2.0
	150 µg	139 132 145	(139) 6.5	12 9 11	(11) 1.5	27 22 22		(24) 2.9	23 21 21	(22) 1.2	8 13 4	(8) 4.5
	500 µg	156 153 149	(153) 3.5	11 9 16	(12) 3.6	22 20 23		(22) 1.5	14 23 19	(19) 4.5	7 10 7	(8) 1.7
	1500 µg	142 138 125	(135) 8.9	14 17 12	(14) 2.5	27 33 26		(29) 3.8	18 25 16	(20) 4.7	16 7 15	(13) 4.9
	5000 µg	106 S 121 S 116 S	(114) 7.6	8 S 8 S 9 S	(8) 0.6	27 28 27		(27) 0.6	24 26 23	(24) 1.5	13 14 12	(13) 1.0
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
		547 529 1380	(819) 486.2	382 485 883	(583) 264.6	865 641 759		(755) 112.1	389 361 375	(375) 14.0	807 353 315	(492) 273.7

Table 5            Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 03 April 2018					To: 06 April 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	141 122 135	(133) 9.7#	18 12 12	(14) 3.5	41 25 38		(35) 8.5	39 30 31	(33) 4.9	8 16 13	(12) 4.0
	15 µg	146 134 160	(147) 13.0	18 15 10	(14) 4.0	39 31 34		(35) 4.0	25 28 24	(26) 2.1	14 15 10	(13) 2.6
	50 µg	142 153 167	(154) 12.5	7 14 8	(10) 3.8	40 48 39		(42) 4.9	24 28 25	(26) 2.1	11 9 11	(10) 1.2
	150 µg	169 145 149	(154) 12.9	15 13 10	(13) 2.5	32 38 36		(35) 3.1	31 19 28	(26) 6.2	10 12 15	(12) 2.5
	500 µg	178 134 155	(156) 22.0	20 8 3	(10) 8.7	39 39 28		(35) 6.4	17 22 29	(23) 6.0	17 12 12	(14) 2.9
	1500 µg	151 145 147	(148) 3.1	11 17 14	(14) 3.0	27 28 27		(27) 0.6	23 24 19	(22) 2.6	10 6 12	(9) 3.1
	5000 µg	109 S 117 S 106 S	(111) 5.7	13 S 6 S 12 S	(10) 3.8	29 28 32		(30) 2.1	19 18 31	(23) 7.2	9 12 15	(12) 3.0
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
		1080 1800 2105	(1662) 526.3	297 319 350	(322) 26.6	164 155 181		(167) 13.2	120 148 140	(136) 14.4	368 330 372	(357) 23.2

Conclusions:

Under the conditions of this test 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane was concluded as non-mutagenic.

Executive summary:

In a Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471), the test item 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix).  Under the conditions of this test, 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane was concluded as non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test, 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane was concluded as non-mutagenic.