Bottom residues resulting from the synthesis of dimethyldioxane and the synthesis of isoprene from dimethyldioxane and trimethylcarbinol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June to August 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD guideline and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Absorbent A2

IUPAC Name:

Absorbent A2

Details on test material:

- Name of test material (as cited in study report): Absorbent A2
- Substance type: UVCB
- Physical state: Brown liquid
- Stability under test conditions: months
- Storage condition of test material: room temperature, in the dark

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9 is used as the metabolic activation system.

Test concentrations with justification for top dose:

- Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
- Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: WITH S9-MIX: sodium azide (TA 1535, TA 100, 10 µg/pl), 4-nitro-o-phenylene-diamine (10 µg/pl in TA 98, 50 µg/pl in TA 1537), methyl methane sulfonate (TA 102, 3.0 µL/pl) - WITHOUT S9-MIX: 2-aminoanthracene (2.5 µg/pl (10.0 µg/pl in TA 102))

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and pre-incubation test (experiment II)

DURATION
- Preincubation period: 60 minutes in the pre-incubation test
- Exposure duration: at least 48 hours (at 37 °C in the dark)

NUMBER OF REPLICATES: three at each concentration

OTHER: the colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager.

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 TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (2).
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

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

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:

The test item Absorbent A2 was assessed for its potential to induce gene mutations ac¬cording to the plate incorporation test (experi¬ment I) and the pre-incubation test (experiment II) using Salmo¬nella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

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

Pre-Experiment/Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 1000 - 5000 2500 - 5000 333 - 5000 1000 - 5000
TA 1537 1000 - 5000 1000 - 5000 333 - 5000 1000 - 5000
TA 98 5000 / 333 - 5000 1000 - 5000
TA 100 333 - 5000 2500 - 5000 333 - 5000 1000 - 5000
TA 102 / / 333 - 5000 1000 - 5000
/ = no reduced background growth

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 1000 - 5000 / 1000 - 5000 2500 - 5000
TA 1537 1000 - 5000 2500 - 5000 333 - 5000 1000 - 5000
TA 98 / / 1000 5000
TA 100 5000 / 1000 - 5000 2500 - 5000
TA 102 / / 2500 - 5000 2500 - 5000
/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Absorbent A2 at any concentration 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 acknowled¬ged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in¬crease in induced revertant colonies.

In conclusion, it can be stated that during the described mutage¬nicity 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results Pre-Experiment and Experiment I

Study Name: 1417105	Study Code: Harlan CCR 1417105
Experiment: 1417105 VV Plate	Date Plated: 10/06/2011
Assay Conditions:	Date Counted: 16/06/2011

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	DMSO			15 ± 2	13 ± 1	30 ± 6	132 ± 11	465 ± 12
	Untreated			13 ± 1	10 ± 2	32 ± 8	145 ± 4	484 ± 23
	Absorbent A2	3 µg		13 ± 3	13 ± 2	30 ± 5	135 ± 13	462 ± 17
		10 µg		15 ± 4	10 ± 2	23 ± 5	142 ± 12	467 ± 10
		33 µg		14 ± 4	12 ± 3	26 ± 2	126 ± 17	467 ± 33
		100 µg		17 ± 3	8 ± 2	31 ± 6	117 ± 5	471 ± 42
		333 µg		15 ± 2	6 ± 0	23 ± 1	91 ± 10^R	436 ± 5
		1000 µg		6 ± 0^M R P	4 ± 2^P M R	17 ± 2^P	92 ± 10^P R	392 ± 17^P
		2500 µg		4 ± 1^M R P	4 ± 1^P M R	17 ± 2^P	79 ± 6^P M R	323 ± 9^P
		5000 µg		2 ± 1^P M R	3 ± 1^P M R	17 ± 4^P M R	48 ± 14^P M R	275 ± 16^P
	NaN3	10 µg		2091 ± 79			2409 ± 25
	4-NOPD	10 µg				382 ± 11
	4-NOPD	50 µg			76 ± 8
	MMS	3.0 µL						5056 ± 43
With Activation	DMSO			16 ± 5	13 ± 3	38 ± 8	139 ± 11	628 ± 10
	Untreated			20 ± 5	15 ± 5	41 ± 9	170 ± 3	622 ± 9
	Absorbent A2	3 µg		18 ± 5	13 ± 2	41 ± 5	141 ± 18	651 ± 20
		10 µg		16 ± 3	14 ± 2	35 ± 8	153 ± 13	645 ± 48
		33 µg		17 ± 3	11 ± 3	35 ± 2	141 ± 8	652 ± 21
		100 µg		17 ± 2	12 ± 0	33 ± 3	171 ± 21	628 ± 4
		333 µg		17 ± 6	12 ± 5	44 ± 5	149 ± 25	636 ± 4
		1000 µg		15 ± 2	7 ± 0^R	39 ± 3	117 ± 6	642 ± 34
		2500 µg		14 ± 2^P R	6 ± 1^P R	40 ± 4^P	82 ± 10^P R	513 ± 8^P
		5000 µg		11 ± 3^P M R	3 ± 2^P R M	21 ± 3^P M	63 ± 8^P M R	421 ± 21^P M
	2-AA	2.5 µg		468 ± 32	376 ± 22	2274 ± 19	2593 ± 161
	2-AA	10.0 µg						3136 ± 255

 

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

 

 Summary of Results Experiment II

Study Name: 1417105	Study Code: Harlan CCR 1417105
Experiment: 1417105 HV2 Pre	Date Plated: 07/07/2011
Assay Conditions:	Date Counted: 11/07/2011

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	DMSO			18 ± 3	20 ± 4	34 ± 8	113 ± 10	385 ± 33
	Untreated			14 ± 4	27 ± 4	35 ± 5	155 ± 16	384 ± 28
	Absorbent A2	3 µg		17 ± 6	25 ± 5	31 ± 2	117 ± 3	369 ± 22
		10 µg		18 ± 3	20 ± 4	27 ± 5	104 ± 11	391 ± 19
		33 µg		18 ± 5	20 ± 7	36 ± 3	116 ± 13	359 ± 13
		100 µg		17 ± 8	23 ± 3	25 ± 6	97 ± 4	346 ± 13
		333 µg		12 ± 2^R	8 ± 6^R	25 ± 7^R	57 ± 4^R M	372 ± 50^R
		1000 µg		7 ± 1^M R	3 ± 1^M R	15 ± 3^M R	45 ± 10^M R	219 ± 7^M R
		2500 µg		9 ± 2^M R	3 ± 3^M R	16 ± 3^M R	30 ± 13^M R	154 ± 5^M R
		5000 µg		4 ± 2^P M R	1 ± 2^P M R	17 ± 2^P M R	15 ± 4^P M R	120 ± 12^P M R
	NaN3	10 µg		1802 ± 89			1936 ± 99
	4-NOPD	10 µg				387 ± 42
	4-NOPD	50 µg			110 ± 5
	MMS	3.0 µL						2682 ± 94
With Activation	DMSO			16 ± 4	28 ± 8	40 ± 5	137 ± 12	366 ± 68
	Untreated			15 ± 3	27 ± 3	45 ± 7	150 ± 5	369 ± 26
	Absorbent A2	3 µg		15 ± 6	26 ± 4	52 ± 2	132 ± 14	343 ± 51
		10 µg		21 ± 6	21 ± 6	44 ± 4	146 ± 37	379 ± 73
		33 µg		16 ± 3	25 ± 9	37 ± 5	117 ± 5	332 ± 48
		100 µg		13 ± 4	27 ± 4	43 ± 5	137 ± 27	349 ± 38
		333 µg		17 ± 2	25 ± 6	40 ± 4	110 ± 11	354 ± 60
		1000 µg		12 ± 2^M R	8 ± 6^M R	34 ± 3^R	76 ± 8^M R	244 ± 24^M R
		2500 µg		3 ± 1^M R	2 ± 1^M R	20 ± 3^M R	4 ± 2^M R	154 ± 11^M R
		5000 µg		1 ± 1^P M R	0 ± 0^P M R	3 ± 3^P M R	0 ± 0^P M R	92 ± 23^P M R
	2-AA	2.5 µg		265 ± 42	231 ± 40	1157 ± 186	1548 ± 195
	2-AA	10.0 µg						2077 ± 512

 

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

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

In conclusion, it can be stated that during the described mutage¬nicity 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, Absorbent A2 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Absorbent A2 to induce gene muta­tions according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

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 and II:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed reduced back­ground growth in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains at higher concentrations.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with Absorbent A2 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.