tert-butyl bromoacetate

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:

From June 13th to July 20th, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl bromoacetate

EC Number:

226-133-6

EC Name:

tert-butyl bromoacetate

Cas Number:

5292-43-3

Molecular formula:

C6H11BrO2

IUPAC Name:

tert-butyl bromoacetate

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:

S9 mix fraction

Test concentrations with justification for top dose:

Preliminary study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Experiment 1 and 2: 15, 50, 150, 500, 1500 and 5000 µg/plate (assessed in triplicate).

Vehicle / solvent:

Dimethyl sulphoxide

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Varying for metabolic activation or not

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other: 2AA, Danthron

Details on test system and experimental conditions:

METHOD OF APPLICATION
Plate incorporation.
- Aliquots (0.1 ml) of bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine, top agar, 0.1 ml test material formulation, vehicle or positive control and either 0.5 ml of S9 mix or phosphate buffer.
- Contents were mixed and distributed onto the surface of Vogel-Bonner Minimal agar plates.
- Plates were incubated at 37 °C for 48 hours.

An additional second experiment was performed using fresh bacteria following the same method, test material, control solutions and dose range.

REPLICATIONS
Six concentrations of test material were asseyed in triplicate.

COUNTING
Domino colony counter.

Evaluation criteria:

The test material should have induced a reproducible, dose-related and statistically significant increase in the revertnat count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Species / strain:

other: TA100, TA1535, TA102, TA98, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MAIN STUDY
Test substance caused visible reduction in the growth of the bacterial background with and without S9 initially at 1500 µg/plate.
No biologically significant increase in the no. of revertant colonies were recorded for any bacterial strains, with any dose and either with or without S9.
A very small increase in revertant was noted in tester strain TA100 with S9 at 500 µg/plate and only in the first experiment. This response was considered to be of no biological relevance since it was non-reproducible.

All performed positive controls were determined to be valid.

RANGE-FINDING / SCREENING STUDIES
Test material was toxic at and above 1500 µg/plate on the strain TA100.

Any other information on results incl. tables

Experiment 1 results
S9-mix	Test item concentration	Number of revertants (mean no. of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		TA102		TA98		TA1537
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
No	0	116	12.7	13	2.1	223	5.5	11	1.5	12	2.5
	15	123	3.8	13	2.5	212	5.5	10	0.6	9	3.6
	50	122	7.6	13	2.0	213	15.9	13	5.2	12	5.5
	150	141	10.3	14	1.2	204	19.4	13	1.0	9	3.1
	500	137	4.9	7	3.8	189	14.4	14	1.0	9	3.5
	1500	43	19.7	0	0	130 S	13.7	0	0	0	0
	5000	0	0	0	0	0	0	0	0	0	0
Yes	0	142	7.5	13	1.5	310	15.9	27	7.1	14	4.7
	15	149	16.3	11	1.2	301	17.1	29	10.3	15	2.1
	50	156	12.0	11	1.2	320	15.7	27	4.0	17	8.2
	150	161	11.8	12	1.7	303	19.6	26	3.0	11	1.0
	500	171	7.2	8	1.7	276	2.3	30	0.6	11	6.5
	1500	78	15.0	6	1.2	50	4.4	16	8.0	7	1.7
	5000	0	0	0	0	0	0	0	0	0	0

Experiment 2 results
S9-mix	Test item concentration	Number of revertants (mean no. of colonies per plate)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		TA102		TA98		TA1537
		Mean	SD	Mean	SD	Mean	SD	Mean	SD	Mean	SD
No	0	70	2.5	11	2.6	259	24.8	20	3.0	12	1.5
	15	70	3.0	14	6.0	250	20.2	15	0.0	16	0.6
	50	67	6.5	9	3.5	247	30.4	12	0.0	11	4.0
	150	69	8.3	7	1.5	257	20.7	20	0.6	9	0.6
	500	85	12.9	13	7.8	264	5.3	19	2.6	12	3.2
	1500	74	41.1	11	4.7	250	21.9	0	0	0	0
	5000	0	0	0	0	0	0	0	0	0	0
Yes	0	76	12.4	12	4.5	300	27.8	28	4.4	19	3.2
	15	95	13.0	12	2.1	294	13.1	25	3.5	18	4.0
	50	94	17.0	7	4.0	326	8.5	24	1.2	16	2.9
	150	94	4.7	8	1.0	315	22.1	27	4.4	13	5.0
	500	87	13.8	5	0.6	298	2.6	26	2.0	13	4.2
	1500	45	15.9	6	0.6	248	83.2	12	2.3	7	2.6
	5000	0	0	0	0	43	19.1	0	0	0	0

Applicant's summary and conclusion

Conclusions:

The substance did not increase the number of revertant colonies in each of the five tested strains (TA1535, TA1537, TA98, TA100, TA102).

Executive summary:

The test was performed according to OECD 471 (1983).

The mutation assay was conducted on five different bacterial strains (TA100, TA1535, TA102, TA98, TA1537) in two independent experiments. Six test article concentrations, in triplicate, were tested (15, 50, 150, 500, 1500, 5000 µg/plate).

No substantial increases in revertant colony numbers (at any dose level) of any of the five tested strains were observed, either in the presence or absence of S9.

Conclusion

The test substance can be considered as not mutagenic.