Bottom residues resulting from the synthesis of propylene oxide and styrene from epoxidation of propylene with ethylbenzene hydroperoxide

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:

From 19.January.2011 to 15.March.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: AMES STudy according to OECD 471 under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

Bottom product of propylene oxide and styrene production

IUPAC Name:

Bottom product of propylene oxide and styrene production

Details on test material:

- Name of test material (as cited in study report): KORE grade V
- Substance type: UVCB
- Physical state: Brown viscous liquid
- Stability under test conditions: months
- Storage condition of test material: room temperature

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I. Based on the toxic effects observed, eight concentrations were tested and 5000 µg/plate were chosen as maximal concentration for experiment II.
The concentration range included two logarithmic decades. The following concentrations were tested:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S9 mix: sodium azide (TA 1535, TA 100), methyl methane sulfonate (TA 102), 4-nitro-o-phenylene-diamine (TA 1537, TA 98) // Without S9 mix : 2-aminoanthracene (all strains)

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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 

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			14 ± 4	10 ± 3	23 ± 3	155 ± 9	409 ± 18
	Untreated			10 ± 0	11 ± 2	23 ± 2	164 ± 17	346 ± 46
	bottom product	3 µg		15 ± 1	10 ± 3	22 ± 1	156 ± 14	336 ± 15
	of propylene	10 µg		16 ± 4	9 ± 1	23 ± 3	153 ± 10	386 ± 22
	oxide and	33 µg		15 ± 0	11 ± 4	21 ± 7	145 ± 7	413 ± 27
	styrene	100 µg		12 ± 5	10 ± 1	21 ± 1	128 ± 13	388 ± 30
	production	333 µg		17 ± 7	9 ± 3	24 ± 6	73 ± 4	331 ± 18
		1000 µg		7 ± 1^P M R	5 ± 2^P M	19 ± 5^P	68 ± 5^P R	219 ± 12^P
		2500 µg		5 ± 1^P M R	1 ± 1^P M R	7 ± 2^P M R	26 ± 5^P M R	117 ± 14^P
		5000 µg		2 ± 1^P M R	0 ± 0^P M R	1 ± 1^P M R	7 ± 2^P M R	8 ± 3^P M R
	NaN3	10 µg		1777 ± 75			1925 ± 111
	4-NOPD	10 µg				296 ± 22
	4-NOPD	50 µg			73 ± 1
	MMS	3.0 µL						4890 ± 347
With Activation	DMSO			21 ± 4	14 ± 1	39 ± 8	142 ± 13	485 ± 40
	Untreated			12 ± 5	17 ± 5	40 ± 2	144 ± 7	461 ± 28
	bottom product	3 µg		21 ± 1	16 ± 3	38 ± 3	145 ± 11	434 ± 36
	of propylene	10 µg		20 ± 6	14 ± 6	42 ± 3	146 ± 9	499 ± 50
	oxide and	33 µg		17 ± 3	14 ± 5	35 ± 5	122 ± 7	509 ± 17
	styrene	100 µg		20 ± 5	15 ± 7	38 ± 10	136 ± 32	465 ± 9
	production	333 µg		17 ± 5	17 ± 3	38 ± 8	120 ± 12	353 ± 23
		1000 µg		16 ± 4^P	16 ± 4^P	23 ± 5^P	73 ± 3^P	294 ± 23^P
		2500 µg		8 ± 3^P M	11 ± 3^P	15 ± 2^P M	42 ± 4^P M R	127 ± 8^P M R
		5000 µg		1 ± 1^P M R	2 ± 1^P M R	11 ± 2^P M R	12 ± 2^P M R	56 ± 7^P M R
	2-AA	2.5 µg		337 ± 43	373 ± 18	2370 ± 191	2428 ± 153
	2-AA	10.0 µg						2013 ± 321

 

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	P R M	Precipitate Reduced background growth Manual count

 

Summary of Results Experiment II

 

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			19 ± 3	8 ± 1	29 ± 8	134 ± 11	350 ± 31
	Untreated			17 ± 5	6 ± 1	31 ± 3	168 ± 10	393 ± 29
	bottom product	3 µg		17 ± 3	7 ± 1	30 ± 7	140 ± 19	349 ± 14
	of propylene	10 µg		14 ± 1	8 ± 0	25 ± 5	126 ± 14	338 ± 20
	oxide and	33 µg		18 ± 4	8 ± 2	21 ± 1	130 ± 16	330 ± 15
	styrene	100 µg		11 ± 3	8 ± 1	19 ± 5	84 ± 11	340 ± 47
	production	333 µg		6 ± 2^M R	3 ± 1^M R	23 ± 1	58 ± 9^M R	249 ± 22
		1000 µg		6 ± 2^M R	2 ± 1^M R P	16 ± 1	54 ± 9^M R	142 ± 19
		2500 µg		2 ± 1^M R P	1 ± 1^P M R	5 ± 2^P M R	31 ± 9^P M R	66 ± 8^P M R
		5000 µg		0 ± 1^P M R	0 ± 0^P M R	2 ± 2^P M R	15 ± 2^P M R	28 ± 8^P M R
	NaN3	10 µg		2124 ± 6			2261 ± 53
	4-NOPD	10 µg				547 ± 39
	4-NOPD	50 µg			62 ± 4
	MMS	3.0 µL						3542 ± 526
With Activation	DMSO			16 ± 3	11 ± 2	38 ± 7	140 ± 12	425 ± 5
	Untreated			22 ± 6	16 ± 1	44 ± 9	173 ± 13	507 ± 2
	bottom product	3 µg		18 ± 4	14 ± 2	39 ± 5	144 ± 12	391 ± 26
	of propylene	10 µg		17 ± 4	10 ± 3	43 ± 5	132 ± 9	486 ± 26
	oxide and	33 µg		15 ± 2	8 ± 1	44 ± 6	131 ± 9	429 ± 44
	styrene	100 µg		17 ± 2	11 ± 2	44 ± 4	147 ± 18	439 ± 15
	production	333 µg		17 ± 3	9 ± 1	33 ± 3	109 ± 11	436 ± 21
		1000 µg		11 ± 5^R M	4 ± 1^M R	22 ± 1^R	53 ± 9^M R	367 ± 90
		2500 µg		1 ± 1^M R P	2 ± 1^P M R	3 ± 2^P M R	10 ± 3^P M R	235 ± 23^P R
		5000 µg		0 ± 0^P M R	0 ± 0^P M R	0 ± 0^P M R	3 ± 1^P M R	46 ± 5^P M R
	2-AA	2.5 µg		325 ± 0	268 ± 1	2196 ± 107	2277 ± 6
	2-AA	10.0 µg						2687 ± 262

 

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	M R P	Manual count Reduced background growth 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 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, bottom product of propylene oxide and styrene production is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of bottom product of propylene oxide and styrene production 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:

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

Reduced background growth was observed at higher concentrations with and without S9 mix in all strains used in both experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without metabolic activation in all strains used in both experiments.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with bottom product of propylene oxide and styrene production 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, bottom product of propylene oxide and styrene production is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.