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Toxicological information

Genetic toxicity: in vitro

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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

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.