1-bromohexadecane

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:

22nd May to 20th June 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no deviations from standard test guidelines and no methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-bromohexadecane

EC Number:

204-008-7

EC Name:

1-bromohexadecane

Cas Number:

112-82-3

Molecular formula:

C16H33Br

IUPAC Name:

1-bromohexadecane

Details on test material:

- Substance type: White semi-solid block on arrival which melts into a clear colourless viscous liquid.
- Date received: 12th April 2002
- Storage condition of test material: Room temperature in a dark room

Method

Target gene:

S. typhimurium - histidine
E. coli - tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% Liver S9 in standard co-factors

Test concentrations with justification for top dose:

S. typhimurium - 15, 50, 150, 500, 1500 and 5000 µg/plate (15 µg/plate concentration was omitted in experiement 2).
E. coli - 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Positive controls without S9-mix: N-ethyl-N'-nitro-N-nitrosoguanidine (TA100, TA1535, WP2uvrA); 9-Aminoacridine (TA1537) and 4-Nitroquinoline-1-oxide (TA98). Positive controls with S9-mix: 2-Aminoanthracene (all other strains) and Benzo(a)pyrene (TA98)

Details on test system and experimental conditions:

BACTERIA SOURCE AND MAINTENANCE
- Salmonella strains were obtained from the University of California at Berkeley on 4th August 1995; E. coli strain was obtained from the British Industrial Biological Research Association on 17th August 1987.
- Stored at -196ºC in a Statebourne liquid nitrogen freezer, model SXR 34
- Characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

BACTERIAL PREPERATION
- Cultures were prepared overnight in nutrient broth (Oxoid Limited; lot no. 250177 05/06 and 257111 10/06) and incubated for approximately 10 hours at 37ºC.

TEST MATERIAL PREPERATION
Concentrations were weighed and mixed with acetone in a vortex mixer and sonication for 15 minutes at 40ºC on the day of the experiment. Prior to use the preparation was dried using molecular sieves.

METHOD OF APPLICATION
Direct plate incorporation method.

AGAR PREPERATION
Prepared with 0.6% Difco Bacto agar and 0.5% sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top agar.

NUMBER OF REPLICATIONS
For each experiment the plates were replicated in triplicate for each bacterial strain and each concentration.

DURATION
All of the plates were incubated at 37ºC for approximately 48 hours.

COLONIES
Colonies counted using a Domino colony counter.

Evaluation criteria:

The test material may be considered positive if the following criteria are met:

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

Statistics:

Dunnett's method of linear regression.

Results and discussion

Test results

Test results 1

Species / strain:

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

Test results 2

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:

PRELIMINARY TOXICITY STUDY
Weakened lawns were recorded in TA100 without metabolic activation (S9) at > 1500 µg/plate. This observation was considered to not be valid, as no other evidence of toxicity was noted for any other strain.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2. Mean Colony Counts for experiments 1 and 2 including control results

Experiment 1
		Mean number of revertant colonies per plate
		Base-pair substitution type			Frameshift type
With or without S9 mix	Test material concentration (µg/plate)	TA100	TA1535	WP2uvrA	TA98	TA1537
-	0	75	20	23	17	16
-	15	69	17	Not tested	20	9
-	50	75	17	19	16	11
-	150	69	17	19	15	7
-	500	76	17	22	16	10
-	1500	79	18	20	15	9
-	5000	83	18	19	14	11
Positive controls		497	339	745	139	1319
+	0	103	16	27	33	19
+	15	91	12	Not tested	28	18
+	50	70	14	22	30	18
+	150	74	12	18	27	15
+	500	91	14	21	26	11
+	1500	77	14	24	27	13
+	5000	93	11	18	23	8
Positive controls		1847	273	1350	221	347
Experiment 2
-	0	93	21	20	23	18
-	50	85	20	20	19	13
-	150	100	19	19	21	17
-	500	93	22	22	17	18
-	1500	93	23	20	18	14
-	5000	89	17	18	22	16
Positive controls		677	698	1136	123	2632
+	0	99	16	23	30	19
+	50	97	17	21	28	18
+	150	87	14	19	25	19
+	500	99	16	19	29	17
+	1500	104	15	18	31	18
+	5000	101	16	18	30	19
Positive controls		1750	107	880	305	1365

Applicant's summary and conclusion

Conclusions:

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

Under the conditions of this assay, the test material gave a negative i.e. a non-mutagenic response in all strains, with or without metabolic activation. The test material is therefore considered to be non-mutagenic.

Executive summary:

The potential of the test material to cause gene mutation in bacteria was assessed in accordance with the following guidelines: OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100. Salmonella typhimurium (strains; TA100, TA1537, TA 1535 and TA98) and Escherichia coli (strain: WP2uvrA) were exposed to concentrations of the test material ranging from 15 – 5000µg/plate with and without metabolic activation (S9-mix derived of rats liver). The test material gave a negative response where none of the assays showed a significant change in revertant colony numbers.

Under the conditions of the test the test material is therefore considered not to cause genetic mutation in bacteria.