Butylidenebis[2-tert-butyl-5-methyl-p-phenylene]-P,P,P',P'-tetratridecylbis(phosphine)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

• Negative Ames Assay (OECD 471)
• Negative in vitro Chromosome Abberation Test (OECD 473)

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:

2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TEST MATERIAL NAME (as stated in study report): Doverphos 479

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Dover Chemical Corp., Batch No. 162T022807
- Expiration date of the lot/batch: March 30, 2009
- Purity: 100 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, moisture protected, under nitrogen
- Stability under test conditions: Stable for 3 - 4 days in acetone at room temperature

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:

Phenobarbital / beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiments I and II: 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Test concentrations were selected based on results of a pre-experimental to determine toxicity.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I in agar (plate incorporation); Experiment II by preincubation.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: three

DETERMINATION OF CYTOTOXICITY
- Method: a reduction in the number of spontaneous revertants or a
clearing of the bacterial background lawn

Rationale for test conditions:

Guidelines and preliminary toxicity results

Evaluation criteria:

per guidelines

Statistics:

Statistical analyses were not performed, and are not required by the guidelines.

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium 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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The laboratory´s historical control range was slightly exceeded in the solvent control of strains TA 100 without S9 mix and WP2 uvrA with S9 mix in experiment I. This minor deviation is judged to be based on biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.

Precipitation of the test item in the test tubes was observed at 2500 µg/plate and 5000 µg/plate in both experiments. Precipitation of the test item on the incubated agar plates was observed in both experiments at 5000 µg/plate in the absence of metabolic activation and from 333 µg/plate up to 5000 µg/plate in the presence of metabolic activation. The undissolved particles had no influence on the data recording.

Conclusions:

Doverphos 479 is considered to be non-mutagenic in this guideline Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

Doverphos 479 is considered to be non-mutagenic in this guideline Salmonella typhimurium and Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

TEST MATERIAL NAME (as stated in study report): Doverphos 479

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Dover Chemical Corp / Batch No. 162T022807
- Expiration date of the lot/batch: March 30, 2009
- Purity: 100 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, moisture protected, under nitrogen
- Stability under test conditions: Stable for 3 - 4 days in acetone at room temperature

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing, LMP,
Technical University Darmstadt, 64287 Darmstadt, Germany
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimal Essential Medium (SEROMED; 12247 Berlin, Germany), supplemented with
10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany). Incubated at 37° C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I (4 hrs with and without S9): 31.3 - 4000 µg/mL
Experiment II (4 hrs with S9): 15.6 - 500 µg/mL
Experiment II (18 hrs without S9): 31.3 - 1000 µg/mL
Experiment II (28 hrs without S9): 62.5 - 500 µg/mL
Dose selection was performed considering pre-test toxicity data and the occurrence of precipitation.

Vehicle / solvent:

Acetone. The solvent was chosen based on its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (MEM with 10 % FCS)
- Cell density at seeding (if applicable): 1 x 104 - 6 x 104 cells

DURATION
- Exposure duration: 4, 18 or 28 hrs

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 well spread metaphase plates per
culture

DETERMINATION OF CYTOTOXICITY
- Method: reduced cell numbers

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Rationale for test conditions:

Guidelines and pre-test results

Evaluation criteria:

per guidelines

Statistics:

Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In a range finding pre-test on toxicity, clear toxic effects were observed after treatment with 1000 µg/mL in the absence of S9 mix. In contrast, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. 24 hrs continuous treatment with 500 and 1000 Lg/mL in the absence of S9 mix induced strong toxic effects.

In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 62.5 µg/mL and above in the absence and presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control
373 mOsm, pH 7.4 versus 332 mOsm and pH 7.4 at 4000 µg/mL).

In the main experiments, in the absence and presence of S9 mix, precipitation of the test item in culture medium was observed after treatment with 125 µg/mL and above.

Conclusions:

Under these guideline test conditions, Doverphos 479 did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to cytotoxic or precipitating concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were 7 to 8 weeks old on day one of dosing. The females were nulliparous and not pregnant. Male weights: 32-37 g; Female weights: 23-34 g; at the start of the study.
TEST ANIMALS

- Source:
- Age at study initiation: 7 to 8 weeks old on day one of dosing
- Weight at study initiation: Males: 32-37 g; Female weights: 23-34 g
- Assigned to test groups randomly: yes
- Housing: Group (5 animals of same sex per cage)
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23°C
- Humidity (%): 65 - 67 %
- Air changes (per hr): Minimum 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 h artificial light (06:00 - 18:00 h) and 12 h darkness

Route of administration:

oral: gavage

Vehicle:

vegetable oil

Details on exposure:

2000 mg/kg bw of TiTDP was given in vegetable oil via oral gavage for two consecutive days.

Duration of treatment / exposure:

2 days

Frequency of treatment:

once daily

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Basis: actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin-C, 1 mg/kg bw was used as a positive, concurrent, control

Tissues and cell types examined:

Femoral bone marrow

Details of tissue and slide preparation:

SAMPLING TIMES: Vehicle control and treated groups (18 - 24 h after last treatment); Positive control (24 h after last treatment)

Evaluation criteria:

per Test Guideline

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No
- Ratio of PCE/NCE (for Micronucleus assay): P/E ratio was 12.40% lower in treated male animals and 1.18% higher in treated female animals than in their respective vehicle control animals. These differences were not considered to be biologically significant.

Conclusions:

Interpretation of results (migrated information): negative
Based on the results of the study, it is concluded that TiTDP does not have micronucleus induction potential in male and female mice.

Executive summary:

In a reliable OECD 474 Guideline GLP study, TiTDP did not cause micronucleus induction in male and female mice when administered at a limit dose of 2000 mg/kg bw by gavage for two consecutive days.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The results of a guideline gene mutation study of DP-479 in bacteria, and a guideline in vitro chromosome aberration study in mammalian cells were both negative. Therefore, it is concluded that DP-479 is not genotoxic and does not warrant classification for mutagenicity according to CLP Regulation EC 1272/2008.