2,4,6,8,10-pentamethylcyclopentasiloxane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010 -05-26 to 2010-06-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6,8,10-pentamethylcyclopentasiloxane

EC Number:

228-204-7

EC Name:

2,4,6,8,10-pentamethylcyclopentasiloxane

Cas Number:

6166-86-5

Molecular formula:

C5H20O5Si5

IUPAC Name:

2,4,6,8,10-pentamethyl-1,3,5,7,9,2,4,6,8,10-pentaoxapentasilecane

Constituent 2

Reference substance name:

2,4,6,8 ,10-pentamethylcyclopentasiloxane

IUPAC Name:

2,4,6,8 ,10-pentamethylcyclopentasiloxane

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E. coli

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: Better than other

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

TA 1535, TA 100

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium azide, NaN3

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

TA 1537, TA 98

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

WP2 uvrA

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours
NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation:

Precipitation of the test item in the overlay agar was observed in the test tubes at 2,500 µg/plate and 5,000 µg/plate in experiment I. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 2,500 µg/plate and at 5,000 µg/plate without S9 mix and from 1000 µg/plate up to 5000 µg/plate with S9 mix and in experiment II at 5000 µg/plate with S9 mix. The undissolved particles had no influence on the data recording.

- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No reduced background growth was observed in all strains in both experiments with and without metabolic activation.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed in all strains with and without S9 mix in both experiments.

Any other information on results incl. tables

    Summary of Results Experiment I

Study Name: 1323707	Study Code: Harlan CCR 1323707
Experiment: 1323707 VV Plate	Date Plated: 26/05/2010
Assay Conditions:	Date Counted: 01/06/2010

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Ethanol			15 ± 3	13 ± 3	27 ± 4	133 ± 3	46 ± 14
	Untreated			15 ± 4	7 ± 2	31 ± 2	127 ± 12	49 ± 6
	2,4,6,8,10 -Pentamethyl	3 µg		15 ± 2	11 ± 3	31 ± 4	131 ± 9	43 ± 7
	cyclopentasiloxane	10 µg		15 ± 6	11 ± 2	25 ± 4	124 ± 4	43 ± 8
		33 µg		16 ± 4	12 ± 3	28 ± 8	116 ± 2	47 ± 8
		100 µg		17 ± 1	10 ± 3	29 ± 7	126± 8	44 ± 9
		333 µg		13 ± 4	10 ± 3	29 ± 6	119 ± 20	43 ± 1
		1000 µg		15 ± 1	10 ± 4	27 ± 5	127 ± 21	50 ± 8
		2500 µg		15 ± 7^P	13 ± 4^P	26 ± 6^P	127 ± 1^P	55 ± 4^P
		5000 µg		16 ± 4^P	8 ± 2^P	28 ± 7^P	132 ± 12^P	53 ± 3^P
	NaN3	10 µg		1607 ± 44			1981 ± 75
	4-NOPD	10 µg				339 ± 48
	4-NOPD	50 µg			68 ± 6
	MMS	3.0 µL						894 ± 49
With Activation	Ethanol			19 ± 4^B M	15 ± 5	49 ± 11	153 ± 12	67 ± 11
	Untreated			20 ± 3^B M	23 ± 6	45 ± 9	149 ± 6	60 ± 5
	2,4,6,8,10 -Pentamethyl	3 µg		21 ± 2^B M	15 ± 5	46 ± 5	136 ± 5	59 ± 10
	cyclopentasiloxane	10 µg		20 ± 4^B M	13 ± 3	49 ± 7	143 ± 10	57 ± 5
		33 µg		19 ± 4^B M	14 ± 2	48 ± 8	152 ± 8	55 ± 2
		100 µg		20 ± 3^B M	14 ± 2	48 ± 14	146 ± 19	55 ± 7
		333 µg		18 ± 5^B M	14 ± 1	53 ± 2	143 ± 16	53 ± 4
		1000 µg		16 ± 2^B M P	15 ± 1^P	45 ± 9^P	140 ± 13^P	63 ± 8^P
		2500 µg		15 ± 4^B M P	11 ± 1^P M	40 ± 3^P M	144 ± 18^P	72 ± 2^P
		5000 µg		13 ± 3^B M P	11 ± 1^P M	37 ± 3^P M	158 ± 30^P	52 ± 6^P M
	2-AA	2.5 µg		360 ± 16	559 ± 67	1863 ± 139	2919 ± 130
	2-AA	10.0 µg						262 ± 11

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P B M	Precipitate Extensive bacterial growth Manual count

 Summary of Results Experiment II

Study Name: 1323707	Study Code: Harlan CCR 1323707
Experiment: 1323707 HV2 Pre	Date Plated: 15/06/2010
Assay Conditions:	Date Counted: 18/06/2010

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Ethanol			13 ± 1	10 ± 4	38 ± 3	157 ± 22	63 ± 12
	Untreated			16 ± 1	8 ± 1	26 ± 4	169 ± 7	49 ± 6
	2,4,6,8,10 -Pentamethyl	33 µg		11 ± 3	7 ± 2	33 ± 4	146 ± 11	50 ± 6
	cyclopentasiloxane	100 µg		11 ± 4	9 ± 2	34 ± 7	147 ± 20	58 ± 4
		333 µg		15 ± 1	12 ± 2	30 ± 9	153 ± 7	52 ± 12
		1000 µg		17 ± 3	9 ± 2	30 ± 4	158 ± 19	66 ± 3
		2500 µg		16 ± 3	14 ± 0	33 ± 2	165 ± 27	63 ± 12
		5000 µg		14 ± 2	13 ± 2	37 ± 11	171 ± 12	62 ± 7
	NaN3	10 µg		1833 ± 83			1860 ± 166
	4-NOPD	10 µg				516 ± 37
	4-NOPD	50 µg			63 ± 6
	MMS	3.0 µL						412 ± 42
With Activation	Ethanol			22 ± 6	13 ± 3	37 ± 8	179 ± 4	76 ± 8
	Untreated			19 ± 7	18 ± 5	35 ± 6	173 ± 16	72 ± 11
	2,4,6,8,10 -Pentamethyl	33 µg		18 ± 4	12 ± 6	46 ± 4	169 ± 7	64 ± 11
	cyclopentasiloxane	100 µg		20 ± 3	13 ± 3	51 ± 7	167 ± 11	67 ± 7
		333 µg		25 ± 5	7 ± 1	44 ± 17	162 ± 8	70 ± 14
		1000 µg		15 ± 4	12 ± 3	48 ± 6	162 ± 17	74 ± 17
		2500 µg		19 ± 4	9 ± 1	45 ± 9	164 ± 20	68 ± 10
		5000 µg		15 ± 5^P	8 ± 1^P M	41 ± 2^P	168 ± 12^P	77 ± 12^P
	2-AA	2.5 µg		468 ± 26	544 ± 49	2941 ± 199	3505 ± 286
	2-AA	10.0 µg						428 ± 19

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

Applicant's summary and conclusion

Conclusions:

2,4,6,8,10-Pentamethylcyclotetrasiloxane has been tested according to OECD 471 and in compliance with GLP. No increase in the number of revertants per plate was observed in either the initial plate incorporation assay or the independent repeat preincubation assay. Vehicle and positive controls gave expected results. It is concluded that the test substance was negative for mutagenicity to bacteria under the conditions of the test.

Executive summary:

This study was performed to investigate the potential of 2,4,6,8,10- pentamethylcyclopentasiloxane to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1,000; 2,500; and 5,000 Ng/plate

Experiment II: 33; 100; 333; 1,000; 2,500; and 5,000 Ng/plate

No reduced background growth was observed in all strains in both experiments with and without metabolic activation.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed in all strains with and without S9 mix in both experiments.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 2,4,6,8,10-pentamethylcyclopentasiloxane at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, 2,4,6,8,10-pentamethylcyclopentasiloxane is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.