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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
0, 312.5, 625, 1250, 2500 and 5000 µg/plate (in the preliminary toxicity study);
0, 8, 40, 200, 1000, and 5000) µg/plate (first experiment);
0, 312.5, 625, 1250, 2500, and 5000) µg/plate (second experiment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: N-methyl-n-nitro-n-nitrosoguanidine
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37°C

SELECTION AGENT (mutation assays): histidine (or tryptophan in the case of WP2uvrA-)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: 0.1 ml of bacterial suspension (TA100 or WP2uvrA-) and 0.1 ml of test solution were added to 2 ml of molten, trace histidine or tryptophan supplemented media (histidine/biotin or tryptophan & top agar) and overlayed onto sterile plates of Vogel-Bonner agar (minimal agar -25 ml/ plate). Five doses of the test substance and a solvent control were tested in duplicate. After 48-72 hours incubation the plates were scored for revertant colonies and examined for a thinning of the background lawn.
Evaluation criteria:
- The test chemical is considered positive in this assay if it induces a dose-related and statistatistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S-9 microsomal enzymes in both experiments.
- The test substance is generally considered non-mutagenic in this test if the number of induced revertants compared to spontaneous revertants is less than 2-fold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity or solubility or up to the maximum recommended dose of 5000 µg/plate.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: observed at the highest tested dose (5000 µg/plate)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mean maximum revertants/plate and the corresponding concentration with/without S-9 mix are resumed below:

- First experiment:

                   Mean maximum revetants observed/plate [corresponding test concentration (µg/plate)]
      Solvent control   Test substance     Positive control (substance)
 Strains  with  without  with  without  with  without
 TA100  78  87  90 [40]  83 [40]  732 [3.3] (2-AA)   472 [2.0] (MNNG)
 TA1535  18  9  19 [40, 5000]  12 [40, 1000]  49 [3.3] (2-AA)   139 [2.0] (MNNG)
WP2uvrA-  24  30  31 [40]  31 [8]  126 [10.0] (2-AA)   734 [3.3] (4NQO)
 TA98  25  18  27 [200, 1000]  22 [200]  501 [3.3] (2-AA)   803 [10.0] (4NOPD)
 TA1537  6  4  6 [8]  4 [8, 200, 1000]  48 [3.3] (2-AA)   202 [50.0] (9-AA)
             

The spontaneous reversion counts/plate was 17.7, 8.3, 29, 107.7 and 33 respectively for TA1535, TA1537, TA 98, TA100 and WP2uvrA-.

- Second experiment

                   Mean maximum revetants observed/plate [corresponding test concentration (µg/plate)]
      Solvent control   Test substance     Positive control (substance)
 Strains  with  without  with  without  with  without
 TA100  70  75  78 [5000]  79 [312.5]  1060 [3.3] (2-AA)   599 [2.0] (MNNG)
 TA1535  14  11  14 [312.5]  12 [312.5, 2500]  74 [3.3] (2-AA)   289 [2.0] (MNNG)
WP2uvrA-  24  21  27 [312.5]  22 [625]  127 [10.0] (2-AA)   823 [3.3] (4NQO)
 TA98  23  19  24 [2500]  19 [5000]  444 [3.3] (2-AA)   741 [10.0] (4NOPD)
 TA1537  7  6  7 [625]  6 [5000]  59 [3.3] (2-AA)   134 [50.0] (9-AA)
             

The spontaneous reversion counts/plate was 15.5, 5, 23.7, 83.3 and 148.3 respectively for TA1535, TA1537, TA 98, TA100 and WP2uvrA-.

Conclusions:
Under the experimental conditions in this study, the test substance is not a mutagenic agent in a bacterial reverse mutation test in vitro.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
according to Japanese MOL/MHW/MITI
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese Hamster Lung cell line (CHL)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Without S9
6-hour treatment (0, 15, 20, 23.3 and 25 µg/ml),
24-hour treatment (0, 5, 10, 15, 20 µg/ml)
48-hour treatment (0, 2.5, 5, 10, 20 µg/ml)

With S9
6-hour treatment (0, 20, 23.3, 26.6, 30 µg/ml)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (test substance concentration: 3 mg/ml)
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 0.075 µg/ml Mitomycin C (MMC) for 24/48 hours treatment without S-9 mix, 10 µg/ml cyclophosphamide (CP) for for 6 hours treatment both with and without S-9 mix.
Details on test system and experimental conditions:
DURATION
- Preincubation period: 24 hours (0.5 x 10E6 cells and 0.25 x 10E6 cells were seeded per flask)
- Exposure duration: without S-9: 6h, 24h, 48h; with S-9: 6h
- Expression time (cells in growth medium): 18 hours (only after 6h exposure)
- Fixation time (start of exposure up to fixation or harvest of cells): cells harvested after exposure (24/48-hour exposure) or after expression time (6-hour exposure)

SPINDLE INHIBITOR (cytogenetic assays): demecolcine 2 hours before the required harvest time.
STAIN (for cytogenetic assays): in 2% Gurrs Giemsa R66 for 5 minutes

NUMBER OF CELLS EVALUATED:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted. If the cell had 23 to 27 chromosomes, any gaps, breaks or rearrangements were noted.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: sample of the cell suspension from each harvest time was counted to measure growth inhibition at each concentration.
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with abberations (gaps included) was equal to or exceeded 10 %, an equivocal response was recorded for values between 5 % and 10 % and a negative response for values less than 5 %. For polyploid cells, an incidence greater than 10 % is generally recorded at positive.
Statistics:
not applicable
Species / strain:
other: Chines Hamster Lung cell line (CHL)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: Chines Hamster Lung cell line (CHL)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in preliminary test the test substance at concentration level of 80 µg/ml caused precipitation.

ADDITIONAL INFORMATION ON CYTOTOXICITY: in preliminary test the test substance at concentration level up to 30 µg/ml (without S-9) or up to 40 µg/ml (with S-9) was found to be cytotoxic after 6 hours treatment. Concentration level up to 20 µg/ml after 24 hours was not found to be cytotoxic (50% cell survival), whereas concentration level at 20 µg/ml was found to be cytotoxic after 48 hours treatment (28% cell survival).
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

The observed chromosome damages and aberrations (number of cells observed: 200 in treatment groups and positive control group without s-9 mix, 50 in positive control group with S-9 mix) are resumed below:

1) After 6 hours treatment

 Treatment group  Total   Chromatid             Chromosome         Others        Total abs.      Total abs.    Total abs.    Cells with abs.       
+S-9 [-S-9]   Gaps    Breaks     Exchanges      Breaks     Exchanges       X (+Gaps)    (-Gaps)      (+Gaps)      (-Gaps)        
(µg/ml)  +S-9 -S-9 +S-9 -S-9  +S-9 -S-9 +S-9 -S-9  +S-9 -S-9 +S-9 -S-9  +S-9 -S-9 +S-9  -S-9  +S-9 -S-9  +S-9  -S-9 
 DMSO  1  0  0  0  0  0  6  0  0  1  0  0  7  1  6  1  2  1  1  1
 20 [15]  1  1  0  0  0  1  0  5  1  1  0  0  2  8  1  7  2  4  1  3
 23.3 [20]  0  1  0  0  0  0  6  1  0  1  0  0  6  3  6  2  2  2  2
 26.6 [23.3]  0  3  0  1  0  1  2  1  1  3  0  0  3  9  3  6  2  7  2  6
30 [25]  2  0  0  0  0  0  12  0  0  0  0  0  14  0  12  0  7  0  6  0
 Pos ctrl  11  0  10  0  10  0  14  3  2  1  0  0  47  4  36  4  27  3  23  3

2) After 24 or 48 hours treatment (without S-9 mix; number of cells observed: 200 in treatment groups excepting 10 [112 cells observed after 48 hours treatment] and 20 µg/ml [18 cells observed after 48 hours treatment] groups, 50 in positive control group [48 hours treatment]:

 Treatment group  Total   Chromatid             Chromosome         Others        Total abs.      Total abs.    Total abs.    Cells with abs.       
24 [48] hours   Gaps    Breaks     Exchanges      Breaks     Exchanges       X (+Gaps)    (-Gaps)      (+Gaps)      (-Gaps)        
(µg/ml)  24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h  24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h  48 h
 DMSO  1  1  0  2  0  0  0  0  1  0  0  0  2  3  1  2  2  2  1  1
 5 [2.5]  9  2  2  0  2  1  2  3  2  1  0  0  17  7  8  5  12  6  7  4
 10 [5]  2  0  0  1  1  2  4  2  0  0  0  0  7  5  5  5  4  2  5
 15 [10]  1  1  1  0  1  0  3  1  1  0  0  0  7  2  6  1  5  2  4  1
20 [20]  5  0  3  0  0  0  5  0  1  0  0  0  14  0  9  0  8  0  5  0
 Pos ctrl  10 6  16  24  13  42  7  15  1  3  0  1  47  90  37  84  26  39  20  39
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 1997
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted May 2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
adopted Aug. 1998
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM containing Hank's salt supplemented with 10% FBS, 1% amphotericin, and 1% neomycin
- 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:
Phenobarbital/ß-naphthoflavone
Test concentrations with justification for top dose:
4h, without S9: 1.3, 2.5, 5.0, 10.0 (20.0, 30.0, 40.0)µg/mL
4h, with S9 (1st exp.): 3.4, 6.8, 13.5, 27.0, 40.5, (54.0)µg/mL
4h with S9 (2nd exp.): 10.0, 20.0, 30.0, 40.0(precipiation occured) (45.0, 50.0, 55.0, 60.0)µg/mL
24h without S9: 10.0, 15.0, 20.0, 25.0, 30.0 (35.0, 40.0)µg/mL
Concentrations in brackets could not be scored due to cytotoxicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: good solubility of the test substance and relative non-toxicity to cell culture
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
The positive control substances should produce a significant increase in mutant colony frequencies.
The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response three times above the spontaneous mutation frequency at one of the test points.

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
linear regression (least squares)
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4h without S9: >=29µg/mL, 4h with S9: >=45µg/mL, 24h without S9: >=35µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: With S9 mix, precipitation occured at and above 40µg/mL, without S9 at and above 30µg/mL and 40µg/mL after 4 or 24h, respectively.

RANGE-FINDING/SCREENING STUDIES:
In a pretest, cytotoxicity was observed after 4h without S9 at 27.1µg/mL, and at 54.4µg/mL after 24h or after 4h (+S9)

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria

Salmonella tyahimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA' were treated with diphenyl(2,4,6 -trimethylbenzoyl)phosphine oxide by the Ames plate incorporation method for 48h at five dose levels in triplicate,

both with and without the addition of Aroclor induced rat liver S9 (BASF Japan 1989). Up to 5000 ug/plate were tested in two independent experiments. No increase in the number of revertant colonies and no cytotoxicity was observed. The vehicle and positive controls gave results within the ranges expected from historical control data.

This result is supported by a second Ames test using the plate incorporation method to expose five Salmonella strains (TA 1535, 100, 1537, 1538, and 98) to up to 2500µg/plate of diphenyl(2,4,6 - trimethylbenzoyl)phosphine oxide for 48 hours in the presence or absence of Aroclor induced rat liver S9 mix (BASF 1979). Cytotoxicity was observed at 2500µg/plates after the addition of S9 mix in all strains and without S9 in TA1535, but no increase in the number of his+-revertants could be detected under all conditions tested.

Chromosome aberration

Chinese hamster lung (CHL) cells were treated with diphenyl(2,4,6 - trimethylbenzoyl)phosphine oxide for 6h (with and without Arcoclor induced rat liver S9), 24h, and 48h (without S9) (BASF Japan 1989). Cells were left to recover for 18h after the 6h treatment. The dose range was selected based on the results of preliminary toxicity tests:

  • 15 to 25 ug/ml for the 6 -hour treatment without S-9
  • 20 to 30 ug/ml for the 6 -hour treatment with S-9
  • 5 to 20 ug/ml for the 24-hour treatment
  • 2 .5 to 20 ug/ml for the 48-hour treatment

No significant, dose-related increases in the frequency of aberrations was demonstrated in any of the treatment cases. Precipitation was observed at 80µg/ml and thus not relevant at the concentrations chosen.

Gene mutation in mammalian cells

In a study according to OECD 476 (BASF 2012) Chinese hamster V79 cells were treated in two independent experiments for 4h with and for 4 and 24h without phenobarbital/ß-naphthoflavone induced rat liver S9. The maximum test substance concentration was

  • 4h, without S9: 1.3, 2.5, 5.0, 10.0 (20.0, 30.0, 40.0)µg/mL
  • 4h, with S9 (1st exp.): 3.4, 6.8, 13.5, 27.0, 40.5, (54.0)µg/mL
  • 4h with S9 (2nd exp.): 10.0, 20.0, 30.0, 40.0(precipiation occured) (45.0, 50.0, 55.0, 60.0)µg/mL
  • 24h without S9: 10.0, 15.0, 20.0, 25.0, 30.0 (35.0, 40.0)µg/mL

Concentrations in brackets could not be scored due to severe cytotoxicity, which was observed only about 5µg/ml above the maximum concentration. After an expression time of 7days, mutant cells were selected using 6 -thioguanine for 8 days. No increase in mutant frequency was detected in any dose group, vehicle and positive controls led to frequencies as expected based on historical control data.


Short description of key information:
Ames negative (BASF Japan 1989, GLP, Guidelines for Screening Mutagenicity Testing Of Chemicals (Japan), equivalent to OECD 471)
Ames negative (BASF 1979, similar to OECD 471)
Chromosome aberration negative (BASF Japan 1989, GLP, Japanese guideline according to MOC/MHW/MITI, similar to OECD 473)
HPRT negative (BASF 2012, GLP, OECD 476)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Since no gene mutation or chromosome aberration was observed in bacteria or mammalian cells, diphenyl(2,4,6 -trimethylbenzoyl)phosphine oxide does not need to be labelled according to 67/548/EEC and CLP/EU-GHS.