Diisopropylbiphenyl and triisopropylbiphenyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Mutagenicity (OECD 471, Ames): negative with and without metabolic activation

Gene mutation in mammalian cells (OECD 476): negative with and without metabolic activation

Cytogenicity/chromosome aberration in mammalian cells (OECD 473): negative with and without metabolic activation

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 14, 1991 - Nov 22, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

yes

Remarks:

no strain to detect cross-linking mutagens included

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 (prepared from male Sprague-Dawley rats)

Test concentrations with justification for top dose:

Range finding test (tester strain TA 100, with and without S9 mix): 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, 5000 µg per plate and vehicle control
(vehicle: DMSO)
Main experiment and confirmatory assay: 100, 333, 1000, 3333, 5000 µg per plate and vehicle control (vehicle: ethanol)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (range finding test), ethanol (main experiment)
- Justification for choice of solvent/vehicle: In the range finding test precipitation but no appreciable toxicity was observed.
Therefore ethanol was chosen as an appropriate solvent in the main experiment.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see table below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 3 per tester strain and concentration, with or without S9 mix

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth/colony formation

Evaluation criteria:

A test substance is considered as mutagenic
- if a clear and dose-related increase in the number of revertants occurs in at least one tester with or without metabolic activation
and/or
- if a biologically relevant positive response for at least one of the dose groups occurs in at least one tester with or without metabolic activation.

An increase is considered relevant
- if in TA 98 and TA 100 mutation rate is at least twice as high as the rate of the solvent control;
- if in TA 1535, TA 1537, and TA 1538 the mutation rate is at least 3x higher than that of the solvent control.

Statistics:

According to the OECD guidelines, the biological relevance is the criterion for the interpretation of the results: A statistical evaluation was not
considered necessary under this premise.

Key result

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Yes (Range finding test with DMSO as solvent)

RANGE-FINDING/SCREENING STUDIES: Results indicated that precipitate of test item but no appreciable toxicity was observed.

A 1.8 -fold dose-responsive increase with tester strain TA98 (with S9) was not reproducible. Also a 2.3 -fold dose-responsive increase in tester strain TA1537 (with S9) was not reproducible.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the conditions of the study, alkylation and transalkylation products of biphenyl with propene did not cause a positive response with any of the tester strains with or without metabolic
activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 28, 1996 - Jan 2, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

heterozygous at the thymidine kinase (TK) locus; designated as clone 3.7.2C
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 (prepared from male Sprague-Dawley rats)

Test concentrations with justification for top dose:

Preliminary dose range-finding experiment: 500 µg/ml (highest concentration) followed by nine lower concentrations prepared in two-fold dilution steps with each vehicle (with and without metabolic activation).
Main experiment: see table below

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol, acetone (concentration in medium: 1%)
- Justification for choice of solvent/vehicle:

Untreated negative controls:

yes

Remarks:

without test item, with and without S9

Negative solvent / vehicle controls:

yes

Remarks:

ethanol or acetone, with and without S9

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Methyl methanesulfonate (MMS; 5.0 nl/ml and 10 nl/ml, without S9); methylcholanthrene (MCA; 2.0 µg/ml and 4.0 µg/ml, with S9)

Details on test system and experimental conditions:

Independant repeat trials were performed with duplicate cultures using either ethanol or acetone as vehicle.
Cells from exponentially growing cultures were seeded in a series of tubes (approx. 6 x 10E6 cells per tube); after pelleting by centrifugation
resuspension in 10 ml of treatment medium, incubation at 37°C in an orbital shaker incubator.

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: no data
- Exposure duration: 4 h, 37°C
- Expression time (cells in growth medium): 48 h, 37°C
- Selection time (if incubation with a selection agent): 10 - 14 d

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT, 3 µg/ml) in cloning medium

NUMBER OF REPLICATIONS: 2x per dose level and assay, vehicle control: 3x per assay, positive control: 2x per assay

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:

Minimum criterion considered necessary for mutagenicity of any treatment:
- Mutant frequency equal or higher the 2-fold the concurrent background mutant frequency (= average mutant frequency of vehicle control)

Further criteria must be fullfilled:
- A dose-related or toxicity-related increase in mutant frequency for at least three dose steps
- Signifiant mutagenic activity should be confirmed in replicates of the same dose level
- A mutagenic dose-response in one assay should be confirmed in a second assay

A test article is evaluated as nonmutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 10% to 20% relative growth or, in the case of relative nontoxic materials, a range of applied concentrations extending to the maximum of 500 µg/ml

Assay acceptance criteria:
- Average absolute cloning efficiency of the vehicle controls should be between 60% and 130%.
- Minimum value for suspension growth of the average vehicle controls: 8.0-fold incrase in two days from the original cell number
- Mutation frequency of positive controls must be must be equal or higher than 200 x 10E-6

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Precipitation: White precipitate was observed in culture medium at concentrations between 35.0 µg/ml and 500 µg/ml

RANGE-FINDING/SCREENING STUDIES:
- Vehicle ethanol ( with and without metabolic activation): weakly cytotoxic to noncytotoxic in a dose range from 0.977 µg/ml to 31.3 µg/ml,
cytotoxic at 62.5 µg/ml, and lethal at higher concentrations.
- Vehicle acetone (without metabolic activation): moderately cytotoxic in a dose range from 0.977 µg/ml to 7.81 µg/ml.
Higher concentrations were highly cytotoxic or lethal.
- Vehicle acetone (with metabolic activation): weakly cytotoxic to noncytotoxic in a dose range from 0.977 µg/ml to 15.6 µg/ml, highly cytotoxic
at 31.3 µg/ml, and lethal at higher concentrations.

Trial 1 with acetone as vehicle (without metabolic activation) was terminated, because a good range of cytotoxicity was not obtained for analysis.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative

The test item was negative in the mouse lymphoma forward mutation assay both with and without S9 metabolic activation under the conditions of
testing.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 30, 1991 - Oct 18, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 cells (obtained from Americam Culture collection , Rockville, MD, USA
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 (prepared from male Sprague-Dawley rats)

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.5, 1.5, 5.0, 15, 50, 150, 500, 1500, 5000 µg/ml (with and without S9)
Main study: 3.2, 6.3, 12.5, 25, 50 µg/ml without metabolic activation
6.3, 12.5, 25, 50, 100 µg/ml with metabolic activation (S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: compatible with test system

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Triethylenemelamine (TEM, 0.5 µg/ml, without S9); Cyclophosphamide (CP, 50µg/ml, with S9)

Details on test system and experimental conditions:

Exponentially growing CHO-K1 cells were seeded in complete medium (McCoy's 5A medium); approx. 5 x10E5 cells per 25-ml flask;
incubation at 37°C +/- 1°C in humified atmosphere of 5% CO2 +/- 1% in air

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 16-24 hrs
- Exposure duration: 18 hrs (without S9), 2 hrs (with S9)
- Expression time (cells in growth medium): 16 hrs
- Metaphase cells were harvested approximately 20 hrs after intiation of treatment. Due to observed delays in cell cycle kinetics, the harvest time was set at 20 hrs in order to assure that cells were evaluated in first division metaphase after treatment.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml final concentration), incubation for additional 2 hrs.
STAIN (for cytogenetic assays): Chromosome staining with Giemsa (modified fluorescence-plus-Giemsa technique)

NUMBER OF REPLICATIONS: 2 (treatment, untreated control, vehicle control, positive control)

NUMBER OF CELLS EVALUATED: 100

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; aberrant cells and total number of structural chromosome aberrations; cell cycle delay

Evaluation criteria:

A test item is considered to induce a positive response when the percentage of aberrant cells is increased in a dos-responsive manner with on or
more concentrations being statistically elevated in relation to the solvent control group.

A test is valid
- when the frequency of cells with structural chromosome aberrations in either the untreated or solvent control is not higher than 6%.
- The percentage of cells with structural chromosome aberrations in the positive control is statistically increased (p =< 0.05) in relation to the
untreated control.

Statistics:

Fisher's exact test (% of aberrant cells), Cochrain-Armitage test (determination of dose responsiveness)

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility and precipitation: The test article was soluble in solvent at all concentrations tested. Addition of the test item to medium resulted in the
formation of oil droplets at test concentrations of 1500 µ/ml and 5000 µg/ml. At dose levels of 150 mg/ml and 500 mg/ml the medium became
cloudy.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxicity measured by mitotic inhibition relative to solvent control: - approx. 63% at highest concentration evaluated (50 µg/ml) without S9
- approx. 49% at highest concentration evaluated (50 µg/ml), complete
inhibition at 100 µg/ml with S9

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summery of results (excerpt from original report)

Treatment (µg/ml)	S9	Harvest time (h)	Mitotic index	Cells scored	Aberration per cell (Mean +/- SD)	Cells with aberrations (%)
untreated	No	20	4.7	100	0.020 +/- 0.141	2
Ethanol	No	20	4.1	100	0.030 +/- 0.223	2
6.3	No	20	4.0	100	0.060 +/- 0.278	5
12.5	No	20	4.0	100	0.020 +/-0.141	2
25	No	20	3.2	100	0.000	0
50	No	20	1.5	100	0.000	0
TEM (0.5 µg/ml)	No		2.6	100	0.660 +/- 1.423	31
untreated	Yes	20	7.3	100	0.020 +/- 0.141	2
Ethanol	Yes	20	6.1	100	0.020 +/- 0.141	2
6.3	Yes	20	5.9	100	0.020 +/- 0.200	1
12.5	Yes	20	5.6	100	0.020 +/- 0.141	2
25	Yes	20	6.2	100	0.000	0
50	Yes	20	3.1	100	0.020 +/- 0.141	2
100	Yes	20	0.0	0
CP (50 µg/ml)	Yes	20	2.0	100	0.400 +/- 1.163	22

Conclusions:

Interpretation of results:
negative Percentage of cells with structural chromosome aberrations in the groups treated with the test item was not significantly increased above that of the solvent control (p >= 0.05, Fisher's exact test) in both with and without metabolic activation.

No increase in structural chromosome aberrations was observed in either the non-activated or S9-activated test system. The test item was concluded to be negative in CHO cytogenetics assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The available genotoxicity data obtained for the test substance are conclusive but not sufficient for classification according to Regulation (EC) No. 1272/2008 (CLP).