2,2,3,3,4,4,5,5-octafluoropentyl methacrylate

Acute Toxicity: other routes

Administrative data

Endpoint:

acute toxicity: other routes

Remarks:

Test for Basal Cytotoxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

6 May 2008 to 20 August 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Guideline:

other: Neutral Red Uptake Cytotoxicity Bioassay

Principles of method if other than guideline:

Cell survival/viability chemosensitivity assay based on the ability of viable cells to incorporate and bind neutral red, a supravital dye. Neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) is a weak cationic dye that readily penetrates cell membranes by non-ionic diffusion and accumulates intracellularly in lysosomes. Alterations of the cell surface or the sensitive lysosomal membrane lead to lysosomal fragility and other changes that gradually become irreversible.
Healthy mammalian cells, when maintained in culture, continuously divide and multiply over time. A toxic chemical, regardless of site or mechanism of action, will interfere with this process and result in a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed as a concentration-dependent reduction of the uptake of the neutral red after chemical exposure. The concentration of test article causing a reduction in neutral red uptake of 50% relative to controls was determined.

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2,3,3,4,4,5,5-octafluoropentyl methacrylate

EC Number:

206-596-0

EC Name:

2,2,3,3,4,4,5,5-octafluoropentyl methacrylate

Cas Number:

355-93-1

Molecular formula:

C9H8F8O2

IUPAC Name:

2,2,3,3,4,4,5,5-octafluoropentyl 2-methylprop-2-enoate

Test material form:

liquid

Test animals

Species:

mouse

Details on test animals or test system and environmental conditions:

BALB/c 3T3 mouse fibroblasts, clone 31, CC-163, from ATCC

Administration / exposure

Route of administration:

other: in vitro toxicity

Vehicle:

ethanol

Details on exposure:

OFPMA was diluted in ethanol to form the 200X stock solutions. The test article was soluble in the ethanol at the highest esting concentration of 500,000 µg/mL.

Doses:

OFPMA (definitive studies): 40.8, 73.5, 132, 238, 429, 772, 1389, 2500 µg/mL
Positive control: sodium lauryl sulfate: 9.49, 13.3, 18.6, 26.0, 36.4, 51.0, 71.4, 100 µg/mL

No. of animals per sex per dose:

6 wells/concentration

Control animals:

other: untreated vehicle control & vehicle control blanks

Details on study design:

The thawed, decontaminated cells were diluted with pre-wared Routine Culture Medium and transferred into a tissue-culture flask. The flask was incubated at standard culture conditions until the cells attached to the culture substrate. Routine culturing procedures were followed until the cells reached 50% to 80% confluence.

A cell suspension of 3.0 x 10E4 cells/mL in Routine Culture Medium was prepared and subcultured into Falcon 96-well plates. Routine Culture Medium was added to the peripheral wells (Blanks).

Plates were incubated at standard culture conditions for 24 ± 2 hours at conditions so that the cells formed an approximately 20% confluent monolayer. The plate cultures were examined under a phase contrast microscope and evaluated for uniform seeding and confluence prior to treating the cells with test article for positive control.

96-Well Plate contained, untreated vehicle control (mean viability set to 100%), test chemical or positive control at eight concentrations, blanks (contain no cells), and vehicle control blanks. Treated cultures were incubated for 48±0.5 hours at standard culture conditions.

Neutral red at a concentration of 25 µg/mL was added to the wells. Following 3-hours incubation, the neutral red medium was removed and neutral red Desorb solution was added to all the wells. The absorption at 550 nm was determined using a microtiter plate reader within 60 minutes of adding the neutral red Desorb solution.

The definitive assay was performed in duplicate as independent repeats.

Results and discussion

Effect levels

Effect levels 1

Dose descriptor:

other: NRU50

Remarks:

Reduction in neutral red uptake of 50% relative to controls

Effect level:

> 2 500 other: µg/mL

Based on:

test mat.

Remarks on result:

other: highest concentration tested

Effect levels 2

Key result

Sex:

not specified

Dose descriptor:

LC50

Effect level:

> 3 183 mg/kg bw

Based on:

test mat.

Remarks on result:

other: estimated based on NRU50 results

Other findings:

The first and second definitive trials were considered valid since the positive control fell within 2 standard deviations of the historical mean, and the left and right mean vehicle control values did not differ by more than 15% from the mean of all vehicle controls.

Applicant's summary and conclusion

Conclusions:

The mean NRU50 value was presented as greater than the highest concentration of 2,500 µg/mL tested in the assay. Based upon the in vitro test results, an estimate of >3,183 mg/kg for the rodent oral LD50 was made.

Executive summary:

The test article was tested in the Neutral Red Uptake Bioassay in BALB/c 3T3 Mouse Fibroblasts-A Test for Basal Cytotoxicity to determine the concentration of test article causing a reduction in neutral red uptake of 50% relative to controls (NRU50). The results of the in vitro assay were used to estimate a rodent oral LD50 value.

 

BALB/c 3T3 mouse fibroblasts were cultured and plated onto a 96-well assay system. The cells were exposed to 40.8 to 2500 µg/mL test material or 9.49 to 100 µg/mL positive control (sodium lauryl sulfate) during two independent cytotoxicity trials. Following a 48-hour incubation, the wells were washed and neutral red was added. Following a further 3-hour neutral red incubation period, the cells were washed and the excess neutral read as absorbed. The absorption at 550 nm was determined using a microtiter plate reader within 60 minutes of adding the neutral red Desorb solution.

 

Since none of the doses resulted in less than 50% relative viability, the mean NRU50 value was presented as greater than the highest concentration of 2500 μg/mL tested in the assay. Based upon the in vitro test results, an estimate of> 3183 mg/kg for the rodent oral LD50 value was made.