Triphenylphosphine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-Dec-05 - 2014-May-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wsserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, D-55116 Mainz

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Triphenylphosphine

EC Number:

210-036-0

EC Name:

Triphenylphosphine

Cas Number:

603-35-0

Molecular formula:

C18H15P

IUPAC Name:

triphenylphosphine

Test material form:

solid: crystalline

Specific details on test material used for the study:

- Purity: 99.71%

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 130034P040.
- Expiration date of the lot/batch: 05 Jul 2015.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle THF over a period of 4 hours was determined analytically. The test substance was stable for 2 hours, after 4 hours a recovery rate of about 88% was obtained. However, all test substance solutions were prepared immediately before administration.
- Solubility and stability of the test substance in the solvent/vehicle: Due to the insolubility of the test substance in several commonly used vehicles, tetrahydrofurane (THF) was selected as vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in tetrahydrofurane (THF).

FORM AS APPLIED IN THE TEST (if different from that of starting material): solution in in tetrahydrofurane (THF).

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media:
Treatment medium (with S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine;
Treatment medium (without S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS)
Culture medium: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS);
Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with hypoxanthine (13.6 x 10E-3 mg/mL), aminopterin (0.18 x 10E-3 mg/mL), thymidine (3.88 x 10E-3 mg/mL), 10% (v/v) fetal calf serum (FCS);
Selection medium ("TG" medium): Hypoxanthine-free Ham's F12 medium supplemented with 6-thioguanine (10 μg/mL), 1% (v/v) stable glutamine (200 mM), 10% (v/v) fetal calf serum (FCS).
All media were supplemented with 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL) and 1% (v/v) amphotericine B (stock solution: 250 μg/mL)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (Each batch used for mutagenicity testing was checked for mycoplasma contamination)
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/β-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

1st Experiment with S9 mix: 6.25; 12.50: 25.00; 50.00; 100.00; 200.00 µg/mL
1st Experiment without S9 mix: 2.5; 5.00; 10.00: 20.00; 40.00; 80.00 µg/mL
2nd Experiment with S9 mix: 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL
2nd Experiment without S9 mix: 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL
3rd Experiment without S9 mix: 0.63; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00 µg/mL

Following the requirements of the current international guidelines and the ICPEMC Task Group (4) a test substance should be tested up to a maximum concentration of 5 mg/mL, 5 μL/mL or 10 mM, whichever is the lowest. In case of toxicity, the top dose should result in approximately 10 - 20% relative survival (relative cloning efficiency), but not less than 10%. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of the exposure period.
In the pretest for toxicity based on the purity and the molecular weight of the test substance 2600 μg/mL (approx. 10 mM) test substance was used as top concentration both with and without S9 mix at 4-hour exposure time.
In the pretests the pH value was not relevantly influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, precipitation of the test substance in the vehicle tetrahydroflurane was not
observed in the stock solution (Test group: 2600 μg/mL). Test substance precipitation (macroscopical detection) occurred by the end of treatment at concentrations of 325.0 μg/mL and above in the absence and presence of S9 mix.
After 4 hours treatment in the absence of S9 mix, cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival at 162.5 μg/mL and above. In addition, in the presence of S9 mix, a clearly reduced relative cloning efficiency was observed after treatment with 40.6 μg/mL and above.

Vehicle / solvent:

- Vehicle/solvent used: tetrahydrofurane (THF)
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in several commonly used vehicles, tetrahydrofurane (THF) was selected as vehicle,which has been demonstrated to be suitable in the CHO/HPRT assay and for which historical contral data are available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.

DURATION
- Exposure duration: 4 h (with and without S9).
- Expression time (cells in growth medium): 7 - 9 days.
- Selection time (if incubation with a selection agent): 6 - 7 days.
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days.

SELECTION AGENT (mutation assays): 6-thioguanidine .
STAIN (for cytogenetic assays): Giemsa.

NUMBER OF REPLICATIONS: Duplicate cultures were used for all experimental groups.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency.

Evaluation criteria:

ACCEPTANCE CRITERIA:
The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0.00 – 16.43 mutants per 10E+06 clonable cells.
- The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data).
- At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

ASSESSMENT CRITERIA:
A finding is assessed as positive if the following criteria are met:
- Increase of the corrected mutation frequencies (MF[corr.]) both above the concurrent negative control values and our historical negative control data range.
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10E+06 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency (MF[corr.]) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Osmolarity and pH values were not influenced by test substance treatment.
In this study, in the absence of S9 mix, test substance precipitation was macroscopically observed in culture medium 3 – 4 hours after start of treatment at 200.00 µg/mL in the 1st Experiment, and at 50.00 µg/mL and above in the 2nd Experiment. Additionally, in the presence of S9 mix, precipitates were found in culture medium at the end of treatment at 50.00 µg/mL and above in the 2nd Experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in all three experiments in the absence and presence of S9 mix, at least at the highest applied concentrations.
In the 1st Experiment in the absence of metabolic activation only two surviving test groups (6.25 to 12.50 µg/mL) were obtained after scoring the survival rate (CE1). Due to missing the recommendations of the current OECD Guideline 476 which demands the evaluation of at least four concentrations, this experimental part was discontinued.
In detail, without S9 mix, there was a strong decrease in the number of colonies from 25.00 µg/mL (CE1 relative: 2.5%) onward in the 2nd Experiment and from 20.00 µg/mL (CE1 relative: 0.0%) in the 3rd Experiment, each. The cell densities were distinctly reduced.
In addition, with S9 mix, there was a clear decrease in the number of colonies at 80.00 µg/mL (CE1 relative: 0.0%) in the 1st Experiment and from 50.00 µg/mL (CE1 relative: 11.6%) onward in the 2nd Experiment, respectively. The cell densities were distinctly reduced.

CELL MORPHOLOGY:
After 4 hours of treatment the morphology and attachment of the cells treated with at least the highest applied concentration were adversely influenced in all experimental parts scored for gene mutations. This occurred in samples regardless of the presence or absence of metabolic activation.

Any other information on results incl. tables

Table 1: Summary of results - experimental parts without S9 mix

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	Cytotoxicity***
						CE1 [%]	CE2 [%]
1	4	Vehicle control^1	-	n.d.	n.c.^3	100.0	n.c.^3
		6.25	-	-	n.c.^3	99.0	n.c.^3
		12.50	-	-	n.c.^3	93.1	n.c.^3
		25.00	-	-	n.c.^3	5.7	n.c.^3
		50.00	-	-	n.c.^3	0.0	n.c.^3
		100.00	-	-	n.c.^3	0.0	n.c.^3
		200.00	-	+	n.c.^3	0.0	n.c.³
		Positive control^2	-	n.d	n.c.^3	110.2	n.c.^3
2	4	Vehicle control^1	-	n.d.	4.45	100.0	100.0
		0.78	-	-	n.c.^1	105.3	n.c.^1
		1.56	-	-	3.51	114.8	97.5
		3.13	-	-	0.32	116.3.	99.5
		6.25	-	-	2.33	90.8	99.2
		12.50	-	-	1.12	91.5	109.3
		25.00	-	-	0.65	80.6	102.1
		20.00	-	-	n.c.^2	2.5	n.c.^2
		50.00	-	+	n.c.^2	0.0	n.c.^2
		100.00	-	+.	n.c.²	0.0	n.c.²
		Positive control²	-	n.d.	63.37	113.8	95.7
3	4	Vehicle control^1	-	n.d.	2.69	100.0	100.0
		0.63	-	-	n.c.^1	104.6.	n.c.^1
		1.25	-	-	0.73	109.4	92.4
		2.5.0	-	-	1.85	104.3	93.8
		5.00	-	-	0.00	111.5	96.4
		10.00	-	-	0.65	80.6	102.1
		20.00	-	-	n.c.^2	0.0	n.c.^2
		40.00	-	-	n.c.^2	0.0	n.c.^2
		Positive control^2	-	n.d.	145.02	97.8	85.5

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MF_corr.: mutant colonies per 10⁶ cells corrected with the CE₂ value

*** Cloning efficiency related to the respective vehicle control

n.c.¹ Culture was not continued since a minimum of only four analysable concentrations are required

n.c.² Culture was not continued due to strong cytotoxicity

n.c.³ Culture was not continued due to missing the OECD recommendations

n.d. not determined

1 THF 0.5% (v/v) ² EMS 300 µg/ml

 

Table 2: Summary of results - experimental parts with S9 mix

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	Cytotoxicity***
						CE1 [%]	CE2 [%]
1	4	Vehicle control^1	+	n.d.	1.43	100.0	100.0
		2.50	+	-	2.56	109.0	111.3
		5.00	+	-	4.77	89.1	93.4
		10.00	+	-	2.07	88.4	102.9
		20.00	+	-	2.49	118.4	96.3
		40.00	+	-	0.71	56.1	96.3
		80.00	+	-	n.c.²	0.0	n.c.²
		Positive control^2	+	n.d.	433.00	104.9	78.6
2	4	Vehicle control^1	+	n.d	0.96	100.0	100.0
		3.13	+	-	1.02	91.7	94.2
		6.25	+	-	0.77	79.1	^92.7
		12.50	+	-	2.02	96.1	94.2
		25.00	+	-	0.72	98.8	85.3
		50.00	+	-	0.00	11.6	108.7
		100.00	+	-	n.c.²	0.0	n.c.²
		Positive control^2	+	n.d.	293.78	66.3	67.2

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MF_corr.: mutant colonies per 10⁶ cellscorrected with the CE₂ value

*** Cloning efficiency related to the respective vehicle control

n.c.¹ Culture was not continued since a minimum of only four analysable concentrations are required

n.c.² Culture was not continued due to strong cytotoxicity

n.c.³ Culture was not continued due to missing the OECD recommendations

n.d. not determined

1 THF 0.5%(v/v) ² MCA 20 µg/mL

Applicant's summary and conclusion

Conclusions:

The test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro.

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations.

In this study, in all experiments at least the highest concentrations tested for gene mutations were clearly cytotoxic in the absence and the presence of metabolic activation.

The 1st Experiment in the absence of S9 mix failed the requirements of the current OECD Guideline 476 due to strong cytotoxicity. A repeat experiment was performed.

On the basis from the results of the present study, the test substance did not cause any relevant and dose-dependent increase in the mutant frequencies both without S9 mix and after adding a metabolizing system in three experiments performed independently of each other.

Conclusion: Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.