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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In 2 Ames tests diphenyl cresyl phosphate was negative in Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98 and Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA.

The HPRT assay in V79 cells of the Chinese hamster was negative.

In a chromosome aberration test with Chinese hamster CHL/IU cells in the absence of metabolic activation no indications of a chromosome-damaging effect was seen. With metabolic activation in the highest concentration an increase in chromosomal damage and a significant increase of polyploidies were observed.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
Diphenyl cresyl phosphate was investigated in the Salmonella / microsome test for point mutagenic effects in doses up to 12500 µg per plate on four Salmonella typhimurium LT2 mutants. These were the histidine auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Ames assay: detection of base pair substitutions and frameshift mutations
Species / strain / cell type:
other: S. typhimurium TA 98, 100, 1535, 1537
Metabolic activation:
with and without
Test concentrations with justification for top dose:
12500; 2500; 500; 100; 20 µg DPK per plate
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Sodium azide; Nitrofurantoin; 4-nitro-1,2-phenylenediamine; 2-aminoanthracene
Details on test system and experimental conditions:
Ames test
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:
cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid

Summary of the results with diphenylcresylphosphate in the Salmonella/ microsome test

 S-9 mix  TA 1535  TA 100  TA 1537  TA98
 without  negative    negative    negative    negative
 with    negative    negative    negative    negative

The positive controls ( Sodium azide; Nitrofurantoin; 4-nitro-1,2-phenylenediamine; 2-aminoanthracene) increased the mutant counts to well over double those of the negative controls, and so demonstrated the system´s sensivity and the activity of the S-9 mix.

Conclusions:
Dipehnyl cresyl phosphate was negative in Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98 with and without metabolic activation.
Executive summary:

DPK was investigated in the Salmonella/ microsome test for point-mutagenic effects in doses up to 12500 µg per plate on four Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98.

In all doses, the substance had a strain-specific bacteriotoxic effect, so that the tested range could only be used to a limited extent up to 12500 µg per plate for evaluation purposes. Substance precipitation occured from the dose 2500 µg per plate.

No evidence for mutagenic activity for DPK was found.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Principles of method if other than guideline:
Diphenyl cresyl phosphate was tested in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA in doses up to 5000 µg/plate.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Ames test
Species / strain / cell type:
other: TA100, TA1535, TA98, TA1537, E.coli WP2 uvrA
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0, 312.5, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S): AF-2 (TA100, WP2, TA98), sodium azide (TA1535) and 9-aminoacridine (TA1537); +S9: 2-aminoantracene (all strains).
Species / strain:
other: TA100, TA1535, TA98, TA1537, E.coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid

     Genetic effects:     E.coli WP2 uvrA
   +  ?  -

 with metabolic activation

 /  /  X
 without metabolic activation  /  /  X

 +  ?  -

 with metabolic activation

 /  /  X
 without metabolic activation  /  /  X
Conclusions:
The test substance was negative in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA with and without metabolic activation.
Executive summary:

The test substance was not mutagenic with or without metabolic activation in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA.

The doses tested were: 0, 312.5, 625, 1250, 2500, 5000 µg/plate.

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)
Principles of method if other than guideline:
The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT gene-locus
Species / strain / cell type:
other: Chinese Hamster V79 cells
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment I:
without S9 mix: 2.5, 5.0, 10.0, 20.0 and 30.0 µg/mL
with S9 mix: 10.0, 20.0, 30.0, 40.0 and 60.0µg/mL
Experiment II:
without S9 mix: 2.5, 5.0, 10.0, 20.0 and 30.0 µg/mL
with S9 mix: 5.0, 10.0, 20.0, 40.0 and 60.0 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: ethylmethane sulfonate (without metabolic activation), 7,12-dimethylbenz(a)anthracene (with metabolic activation)
Key result
Species / strain:
other: Chinese Hamster V79 cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first and second experiment the treatment period was 4 hours with and without metabolic activation. The experimental part of the first experiment without metabolic activation was terminated prior to the generation of any data on mutagenicity due to exceedingly severe cytotoxic effects. This experimental part was repeated as experiment IA. The data of experiment IA are reported as experiment I without metabolic activation.

The cell cultures were evaluated at the following concentrations:

Experiment I:

without S9 mix: 2.5, 5.0, 10.0, 20.0 and 30.0 µg/mL

with S9 mix: 10.0, 20.0, 30.0, 40.0 and 60.0µg/mL

Experiment II:

without S9 mix: 2.5, 5.0, 10.0, 20.0 and 30.0 µg/mL

with S9 mix: 5.0, 10.0, 20.0, 40.0 and 60.0 µg/mL

The maximum concentration of the test item in the main experiments was limited by cytotoxic effects. Cytotoxicity defined as a relative cloning efficiency below 50% in both parallel cultures occurred in the second experiment without metabolic activation at 20 µg/mL and above. In the presence of metabolic activation cytotoxic effects as described above were noted at 20 µg/mL and above in the first and at 10 µg/mL and above in the second experiment except at 40 µg/mL. Surprisingly, only moderate or virtually no cytotoxicity reproducibly occurred in both cultures of both experiments at 40 µg/mL. Such a biphasic trend of cytotoxicity in the presence of metabolic activation is often based on substrate inhibition of liver microsomal enzymes involved in the metabolism of a test item.

Precipitation was observed in the second experiment at 80 µg/mL with and 20-40 µg/mL without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutation frequency generally remained within the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the first experiment at 5.0 µg/mL without metabolic activation and at 30-60 µg/mL in the second culture with metabolic activation. However, statistical evaluation of the data revealed that there was no dose dependent increase of the mutation frequency. The results discussed above were consequently judged as biologically irrelevant fluctuations.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies.

No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.6 up to 17.3 mutants per 106 cells; the range of the groups treated with the test item was from 9.1 up to 38.8 mutants per 106 cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Conclusions:
The test item was negative in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first and second experiment with and without metabolic activation. The maximum dose of the pre-experiments (5000.0 µg/mL) was chosen with regard to the current OECD Guideline 476. The dose range of the main experiments was limited by cytotoxicity of the test item. The tested concentrations were following:

Experiment I:

without S9 mix: 2.5, 5.0, 10.0, 20.0 and 30.0 µg/mL

with S9 mix: 10.0, 20.0, 30.0, 40.0 and 60.0µg/mL

Experiment II:

without S9 mix: 2.5, 5.0, 10.0, 20.0 and 30.0 µg/mL

with S9 mix: 5.0, 10.0, 20.0, 40.0 and 60.0 µg/mL

Precipitation was noted at the three highest concentrations of the second experiment without metabolic activation and at the maximum concentration of the second experiment with metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Disflamoll DPK is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
No positive control was used without S9 mix in the 6 hour experiment.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Micronuclei
Species / strain / cell type:
other: Chinese hamster CHL/IU cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
-S9 (continuous treatment): 0, 0.004, 0.008, 0.016 mg/ml;
-S9 (short-term treatment): 0, 0.011, 0.022, 0.043 mg/ml;
+S9 (short-term treatment): 0, 0.011, 0.022, 0.043 mg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S9: Mitomycin C; +S9, Cyclophosphamide
Key result
Species / strain:
other: Chinese hamster CHL/IU cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: Chinese hamster CHL/IU cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No positive control was used without S9 mix in the 6 hour experiment. All other positive and negative controls gave the expected results.

Genotoxic effects:

         clastogenicity        polyploidy
   +  ?  -  ?  -
 without metabolic activation  /  /  X  /  /  X
 with metabolic activation  X  /  /  /  /  X
Conclusions:
In a chromosome aberration test with Chinese hamster CHL/IU cells the test substance was clastogen with metabolic activation..
Executive summary:

In a chromosome aberration test with Chinese hamster CHL/IU cells the test substance was tested in following dosages:

-S9 (continuous treatment; 24 hours): 0, 0.004, 0.008, 0.016 mg/ml;

-S9 (continuous treatment; 48 hours): 0, 0.004, 0.008 mg/ml;

-S9 (short-term treatment; 6 hours): 0, 0.011, 0.022, 0.043 mg/ml;

+S9 (short-term treatment; 6hours): 0, 0.011, 0.022, 0.043 mg/ml;

second experiment: +S9 (short-term treatment; 6 hours): 0, 0.022, 0.032, 0.043 mg/ml.

Short-term treatment with the highest concentration (0.043 mg/ml) of the test substance, incorporated with S9 mix, resulted in 8.6% of the cells with structural aberrations (including gaps). Without gaps 7.5% chromosomal aberrations were observed. However, there were insufficient metaphase cells for analysis because of cytotoxicity. Therefore, a confirmation test was performed and the reproducibility was confirmed (11% chromosomal aberrations including gaps; 10% chromosomal aberrations without gaps). Cytotoxicity was not checked in the main study, but it can nevertheless be deduced from the small numbers of cells evaluated at this concentration in the first test series, that cytotoxicity was high. The main study with 24 and 48 -hour exposures was conducted with test substance concentrations of 0.016, 0.008 and 0.004 mg/ml only in the absence of metabolic activation and gave no indications of a chromosome-damaging effect. With metabolical activation in the highest concentration (0.043 mg/ml) an increase in chromosomal damage and a signifficant increase of polyploidies could be observed. Only 93 (instead of 200) cells could be evaluated concearning clastogenicity and 124 (instead of 800) concerning poliploidies. This indicates a high cytotoxicity. In the second experiment the no. of expected cells was reached (JETOC, 1997b); Tanaka, 1995).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A Micronucleus Test according OECD 474 in male and female Crj: BDF mice was negative. The frequency of micronucleated polychromatic erythrocytes was not significantly increased in male or female mice up to the dose of 1250 mg/kg after 24h after the application by oral gavage.

In another micronucleus test the o-cresyl-free test substance was tested for its clastogenic potential in male and female NMRI mice. No increase in micronucleated polychromatic erythrocytes was observed in any dose group or time point. The test substance did not produce a chromosome-damaging effect in this study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study; Purity of test substance uncertified.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: CrJ:BDF1
Sex:
male/female
Route of administration:
oral: gavage
Duration of treatment / exposure:
24 h
Frequency of treatment:
no data
Post exposure period:
no data
Remarks:
Doses / Concentrations:
0, 312.5, 625 and 1250 mg/kg
Basis:

No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
Cyclophosphamide 50 mg/kg
Tissues and cell types examined:
micronucleated polychromatic erythrocytes
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid

The frequency of micronucleated polychromatic erythrocytes was not significantly increased in male or female mice up to the dose of 1250 mg/kg 24 h after the application by oral gavage. Inhibition of bone marrow cell proliferation was not observed under the test conditions used. Maximum tolerated dose: greater than 1250 mg/kg in males and females.

Conclusions:
The in-vivo micronuleus test in male and female mice was negative.
Executive summary:

In an Micronucleus Test according OECD 474 male and female Crj:BDF mice received following doses by gavage: 0, 312.5, 625 and 1250 mg/kg.

The frequency of micronucleated polychromatic erythrocytes was not signifficantly increased in male or female mice up to the dose of 1250 mg/kg after 24h after the application by oral gavage. Inhibition of bone marrow cell proliferation was not observed under the test conditions used. The maximum tolerated dose was found to be greater than 1250 mg/kg in males and females. No genotoxicity could be observed.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No OECD guideline defined.
Qualifier:
according to
Guideline:
other: EEC Directive 92/69, L 383 A, Annex V, B 12 dated December 29, 1992
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Route of administration:
intraperitoneal
Vehicle:
The test article was formulated in corn oil. The vehicle was chosen to its reletive non-toxicity for the animals.
Duration of treatment / exposure:
single administration
Frequency of treatment:
All animals received a single standard volume of 10 ml/kg body weight intraperitoneally.
Post exposure period:
Sampling of the bone marrow from animals treated with the highest dose was done 16,24, 48 hours after treatment. Bone marrow samples from animals treated with the low and medium dose were taken only at preparation interval 24 hours.
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg
Basis:
other: intraperitoneally
No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
CPA; Cyclophosphamide
Tissues and cell types examined:
The bone marrow cells were collected for micronuclei analysis; 1000 polychromatic erythrocytes per animal were scored for micronuclei
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Summary of results:

 test group dose (mg/kg bw)  sampling time (h)  PCEs with micronuclei (%)  range  PCE/NCE 
 test article  1000 16  0.17  0 -5  1000/ 801 
 vehicle  0  24  0.11  0 -5  1000/ 721
 test article  100  24  0.09  0 -4  1000/ 746
 test article  300  24  0.14  0 -3  1000/ 905
 test article  1000  24  0.22  1 -5  1000/1033
 cyclophosphamide  30  24  1.70  6 -31  1000/ 829
 test article  1000  48  0.08  0 -2  1000/ 824

The animals treated with 1000 mg/kg bw Diphenyl cresylphosphate expressed toxic reactions. Reduction of spontaneous activity, eyelid closure, and convulsion (females only within the first hour after administration of the test article) followed by apathy were observed. One out of 18 femeles treated with 1000 mg/kg bw Diphenyl cresylphosphate died.

Conclusions:
The in-vivo micronuleus test in male and female mice was negative. In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the micronuclei at any preparation interval after application of the test article and with any dose level used.
Executive summary:

A micronucleus assay was performed to investigate the potential of Diphenyl cresyl phosphate to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in corn oil. This vehicle was used as negative control. The volume administered intraperitoneally was 10 ml/kg bw. 16h, 24h, and 48h after a single application of the bone marrow cells were collected for micronuclei analysis. The occurrence of micronuclei in ten animals (5males, 5 females) per test group was evaluated. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose levels of the test article were investigated:

16 h preparation interval: 1000 mg/kg bw;

24 h preparation interval: 100, 300, and 1000 mg/kg bw.;

48 h preparation interval: 1000 mg/kg bw.

In a pre-experiment the highest dose administered was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. Reduction of spontaneous activity, eyelid closure, and convulsions (females only within the first hour after administration of the test article) followed by apathy were observed. In the micronucleus assay the same toxic symptoms occurred and 1 out of 18 females treated with 1000 mg/kg bw Diphenyl cresylphosphate died. After treatment with the highest test article dose at preparation interval 24 hours the number of NCEs was slightly increased as compared to the corresponding negative control thus indicating that Diphenyl cresylphosphate had a cytotoxic effect. In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the micronuclei at any preparation interval after application of the test article and with any dose level used. 30 mg/kg bw cyclophosphamide administered intraperitoneally was used as positive control which induced a distinct increase of the micronucleus frequency. In conclusion it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, Diphenyl cresylphosphate is considered to be nonmutagenic in this micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro tests:

The test substance was investigated in the Salmonella/ microsome test for point-mutagenic effects in doses up to 12500 µg per plate on four Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98. In all doses, the substance had a strain-specific bacteriotoxic effect, so that the tested range could only be used to a limited extent up to 12500 µg per plate for evaluation purposes. Substance precipitation occurred from the dose 2500 µg per plate. No evidence for mutagenic activity for the test substance was found (Bayer AG/ Herbold, 1988).

In a second Ames assay the test substance was not mutagenic with or without metabolic activation in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA. The doses tested were: 0, 312.5, 625, 1250, 2500, 5000 µg/plate. No data on cytotoxicity mentioned (JETOC, 1997; Shibuya, 1995).

In addition a study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first and second experiment with and without metabolic activation. The maximum dose of the pre-experiments (5000.0 µg/mL) was chosen with regard to the current OECD Guideline 476. The dose range of the main experiments was limited by cytotoxicity of the test item. The tested concentrations were following:

Experiment I:

without S9 mix: 2.5, 5.0, 10.0, 20.0 and 30.0 µg/mL

with S9 mix: 10.0, 20.0, 30.0, 40.0 and 60.0µg/mL

Experiment II:

without S9 mix: 2.5, 5.0, 10.0, 20.0 and 30.0 µg/mL

with S9 mix: 5.0, 10.0, 20.0, 40.0 and 60.0 µg/mL

Precipitation was noted at the three highest concentrations of the second experiment without metabolic activation and at the maximum concentration of the second experiment with metabolic activation. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Disflamoll DPK is considered to be non-mutagenic in this HPRT assay (Wollny, 2010).

In a chromosome aberration test with Chinese hamster CHL/IU cells the test substance was tested in following dosages:

-S9 (continuous treatment; 24 hours): 0, 0.004, 0.008, 0.016 mg/ml;

-S9 (continuous treatment; 48 hours): 0, 0.004, 0.008 mg/ml;

-S9 (short-term treatment; 6 hours): 0, 0.011, 0.022, 0.043 mg/ml;

+S9 (short-term treatment; 6hours): 0, 0.011, 0.022, 0.043 mg/ml;

second experiment: +S9 (short-term treatment; 6 hours): 0, 0.022, 0.032, 0.043 mg/ml.

Short-term treatment with the highest concentration (0.043 mg/ml) of the test substance, incorporated with S9 mix, resulted in 8.6% of the cells with structural aberrations (including gaps). Without gaps 7.5% chromosomal aberrations were observed. However, there were insufficient metaphase cells for analysis because of cytotoxicity. Therefore, a confirmation test was performed and the reproducibility was confirmed (11% chromosomal aberrations including gaps; 10% chromosomal aberrations without gaps). Cytotoxicity was not checked in the main study, but it can nevertheless be deduced from the small numbers of cells evaluated at this concentration in the first test series, that cytotoxicity was high. The main study with 24 and 48 -hour exposures was conducted with test substance concentrations of 0.016, 0.008 and 0.004 mg/ml only in the absence of metabolic activation and gave no indications of a chromosome-damaging effect. With metabolic activation in the highest concentration (0.043 mg/ml) an increase in chromosomal damage and a significant increase of polyploidies could be observed. Only 93 (instead of 200) cells could be evaluated concerning clastogenicity and 124 (instead of 800) concerning polyploidies. This indicates a high cytotoxicity. In the second experiment the no. of expected cells was reached (JETOC, 1997b); Tanaka, 1995).

In vivo tests:

In a Micronucleus Test according OECD 474 male and female Crj: BDF mice received following doses by gavage: 0, 312.5, 625 and 1250 mg/kg. The frequency of micronucleated polychromatic erythrocytes was not significantly increased in male or female mice up to the dose of 1250 mg/kg after 24h after the application by oral gavage. Inhibition of bone marrow cell proliferation was not observed under the test conditions used. The maximum tolerated dose was found to be greater than 1250 mg/kg in males and females. No genotoxicity could be observed (CIPC Japan, 1996).

In another micronucleus test the o-cresyl-free test substance was tested for its clastogenic potential in male and female NMRI mice. The doses tested were: 0 (controls), 100, 300 and 1000 mg/kg bw. A single intraperitoneal dose was administered. The femoral bone marrow was prepared from 5 animals per dose and sex as followed: 16 hours (1000 mg/kg bw), 24 hours (100, 300 and 1000 mg/kg bw) and 48 hours (1000 mg/kg bw). The ratios of polychromatic and normochromatic erythrocytes were determined. Additionally the number of micronucleated polychromatic erythrocytes was determined. 1000 cells were scored in each case. Signs of toxicity were observed in the animals of the highest dose group (depressed spontaneously activity, closed eyelids, convulsion). 1 of 18 mice died. The number of normochromatic erythrocytes was slightly increased 24 hours after administration of 1000 mg/kg bw. This finding was interpreted as a cytotoxic effect. No increase in micronucleated polychromatic erythrocytes was observed in any dose group or time point. The test substance did not produce a chromosome-damaging effect in this study (BG Chemie, 1993).


Short description of key information:
Overall the test substance was found to be clastogenic in an in vitro chromosome aberration assay, but two reliable in vivo chromosome aberration tests performed similar to actual guidelines revealed that the test compound is not clastogenic in vivo. The substance is not mutagenic in different bacterial reverse mutation assays similar to actual guidelines.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In 2 Ames tests and the available HPRT assay diphenyl cresyl phosphate was negative. An in-vitro chromosome aberration test with Chinese hamster CHL/IU cells was negative without metabolic activation and positive with metabolic activation. Two in-vivo Micronucleus tests were negative and revealed no clastogenic potential.

Overall, on a weight-of-evidence consideration diphenyl cresyl phosphate is regarded as negative for genotoxicity.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.