Ethanol, 2-methoxy-, manufacture of, by-products from, esters with boric acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

1990-1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is well documented and performed according to generally valid and/or internationally accepted GLP and testing guidelines. The brake fluid contains B-TTEGME and B-TEGME.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Brake Fluid DOT 4 (lot 4200) containing B-TTEGME

IUPAC Name:

Brake Fluid DOT 4 (lot 4200) containing B-TTEGME

Details on test material:

- Name of test material (as cited in study report): Brake fluid 500 Dot 4
- Substance type: Borated Glycol Ether
- Physical state: Clear colourless liquid
- Analytical purity: 38% B-TTEGME; Confidential details on test material
- Impurities (identity and concentrations): Confidential details on test material
- Composition of test material, percentage of components: Confidential details on test material
- Isomers composition: Not applicable
- Purity test date: Not provided
- Lot/batch No.:4200 (SNC sample No.); Indent 9450/9486; toxicology Ref. No. ST91/267
- Expiration date of the lot/batch: Not provided
- Stability under test conditions: Room temperature

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: obtained from Dr; B. Phillips, BIBRA, Carshalton, Surrey
No. of passages: between 31 and 41.
- Properly maintained: yes
Cell monolayers were grown in 80 cm2 polystyrene tissue culture flasks (NUNC) containing 20 ml Hams F12 medium, supplemented with 10% foetal calf serum and 2 mM glutamine (incubated at 37°C in 5% CO2). Cultures were passaged at confluence by washing in PBS followed by treatment with trypsin. After the cells had rounded up and had become detached from the substratum, culture medium was added and they were aspirated with a pipette and transferred to a fresh culture vessel.
- Periodically checked for Mycoplasma contamination: yes (Mycoplasma experience cell screen test report B16, 1992)
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1st study (-S9): 0, 00, 10, 100, 250, 500, 1000, 1200, 1500, 1875 µg/ml + positive control (MMS)
1 study (+S9): 0, 00, 10, 100, 200, 400, 937.5, 1875 µg/ml + positive control (BP)
2nd study (-/+S9): 0, 00, 1500, 2000, 3000, 4000, 5000 µg/ml + positive control (MMS/BP)

Vehicle / solvent:

- Vehicle(s)/solvent(s) methanol
- Justification for choice of solvent/vehicle: methanol was the most appropriate test compound solvent; concentrations of brake fluid up to 750 mg/ml could be attained in this carrier.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

methanol

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: and benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1. Flasks were seeded with 250000 cells and incubated at 27°C in 5 % CO2 overnight.
2. Test /control substances were diluted in growth medium (2% FCS) +/- S9 and applied to cultures.
3. Medium was removed from cultures +S after 3h exposure; fresh medium (10% FCS) was added.
4.Colcemid was added to all cultures (+/- S9) to give a final concentration of 1.0 µg/ml
5. Metaphase cells were harvested by scraping 24h after initiation of exposure.
6. Cells were processed for metaphase analysis (swelling in 0.56% KCl, fixation in MeOH/CH3COOH)
7. Slides were coded, 200 metaphases were scored; chromosomal aberrations were scored.
8. Mitotic index of each dose group was assessed and mitotic index was calculated.

DURATION
- Preincubation period: overnight (27°C)
- Exposure duration: 3 hours + S9: 24 h – S9 (37°C)

Evaluation criteria:

Signs of cell toxicity & test material precipitation,
Mittotic index & No. of cells with chromosome aberrations incl. gaps, types of aberrations, dose response, statistical findings
Changes in ploidy, if seen.

Statistics:

Fisher’s exact test for heterogeneity were performed on pairs of replicates.
Cocharn-ARmitage trend test on the dose response (including/excluding solvent control)

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: minor (<0.3)
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: Not applicable; good miscible in methanol
- Precipitation: No
- Other confounding effects: NO
RANGE-FINDING/SCREENING STUDIES: Not applicable
COMPARISON WITH HISTORICAL CONTROL DATA: No
ADDITIONAL INFORMATION ON CYTOTOXICITY: No

RANGE-FINDING/SCREENING STUDIES: No

COMPARISON WITH HISTORICAL CONTROL DATA: No

ADDITIONAL INFORMATION ON CYTOTOXICITY: No

Remarks on result:

other: strain/cell type: BIBRA, 31-41 passages

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Cfr. Table 1 in attachment

Applicant's summary and conclusion

Conclusions:

The data generated from this study show that Brake fluid Dot 4 did not induce structural chromosome aberration in cultured human lymphocytes both in the presence and in the absence of S9 mix, under the experimental conditions described.

Executive summary:

Brake Fluid DOT 4 is considered to have a similar toxicological profile as B-TTEGME. The clastogenic potential of the brake fluid containing 38% B-TTEGME was assessed from assays designed to detect chromosome damage in cultured Chinese Hamster Ovary (CHO) cells. Cultures were grown in tissue culture flasks and incubated in medium containing the test compound or relevant controls for either 3 hours in the presence of S9 mix or for 24 hours in the absence of S9 mix. Metaphase cells were prepared on glass microscope slides for the analysis of chromosome aberrations 24 hours following initiation of compound exposure for the cultures both with and without S9 mix.

The data generated from this study show that brake fluid and therefore also B-TTEGME did not induce structural chromosome aberrations in cultured CHO cells both in the presence and in the absence of S9 mix, under the experimental conditions described.