Octabenzone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1980 - 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

A Compilation of Two Decades of Mutagenicity Test Results with the Ames Salmonella typhimurium and L5178Y Mouse Lymphoma Cell Mutation Assay

Author:

Seifried HE, Seifried RM, Clarke JJ, Junghans TB and San RHC

Year:

2006

Bibliographic source:

Chem. Res. Toxicol. 19: 627-644

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Oxybenzone

EC Number:

205-031-5

EC Name:

Oxybenzone

Cas Number:

131-57-7

Molecular formula:

C14H12O3

IUPAC Name:

(2-hydroxy-4-methoxyphenyl)(phenyl)methanone

Details on test material:

Aquired from NCI contractors and analysed for purity at Midwest Research Institute (USA)
appropriate storage condiations as indicated from supplier

Method

Target gene:

thymidine kinase gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer's medium for leukemic cells of mice (Gibco, Grand Island, NY (USA) supplemented with 10% Horse serum and 0.02% pluronic F-60 (BASF Wyandotte Crop)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data, new cultures were started from cryopreserved cultures every three months
Cells were origally obtained from Dr. Donald Clive, Buroughs Wellcome Co. (Research Triangle Park, USA)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver S9 prepared from Arochlor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

22, 29, 37, 45 and 52 microgramms/mL (without S9)
18, 24, 31, 37, 44 and 50 microgramms/mL (with S9)

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: 4.7 microM without metabolic activation

Controls 2

Positive control substance:

3-methylcholanthrene

Remarks:

Migrated to IUCLID6: 18.6 microM, with metabolic activation

Details on test system and experimental conditions:

Cells at a concentration of 6 x 10exp5/mL (6 x 10 exp6 cells total) were exposed for 4 h to a range of concentrations
from 0.0005 to 10000 microg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37 ( 1 °C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.
The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity.
The mutagenicity assay was performed according to the procedure described by Clive and Spector in 1975 (Mutat. Res. 59: 61 - 180).
A total of 1.2 x 10exp7 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ( 1 °C, washed twice with growth medium, and maintained at 37 ( 1 °C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 x 10exp5/mL at 24 h intervals. They were then cloned (1 x 10exp6 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0. 23% granulated agar (BBL, Inc., Cockeysville, MD). Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 microg/mL) to the cloning medium for mutant selection. The 100x stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37°C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, DynaTech) or ProtoCol colony counter (Synbiosis, Frederick, MD). Only colonies larger than ca. 0.2 mm in diameter were counted. Mutant frequencies were expressed as
mutants per 10exp6 surviving cells.
The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from ca. 0.2 to 1.1 mm.

Evaluation criteria:

Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

Statistics:

none

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

A less than 3 fold increase occured at highly toxic concentrations.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

VC = Viable counts (cloning efficiency)

TFT = Counts after TFT-selection

RTG = Relative total growth

	Non-Activated Cultures					S9-Activated Cultures
Dose (ug/mL)	Average TFT	Average VC	Mut Freq	RTG	Dose (ug/mL)	Average TFT	Average VC	Mut Freq	RTG
22	33	190	0.4	78	18	36	141	0.5	65
	28	167	0.3	70		30	142	0.4	67
29	29	124	0.4	55	24	55	179	0.6	49
	36	172	0.4	64		39	175	0.4	55
37	35	160	0.4	41	31	63	152	0.8	22
	47	172	0.6	41		66	157	0.8	25
45	51	191	0.6	30	37	73	140	1	15
	40	159	0.5	37		58	173	0.7	18
52	53	151	0.7	17	44	89	145	1.2	8
	64	130	0.9	14		91	139	1.3	9
Solvent	29	179	0.3		50	87	143	1.2	6
Positive	33	79	8.4	23		101	155	1.3	8
					Solvent	36	159	0.5
					Positive	107	130	1.7	78