1,4-Benzenediol, 2-(6-oxido-6H-dibenz[c,e][1,2]oxaphosphorin-6-yl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

3 Genotoxicity studies were performed on DOPO HQ.

 1.   Ames test (OECD 471) was conducted with DOPO HQ - This study was performed to investigate the potential of DOPO-HQ to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:       3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate as well. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No relevant toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains with and without metabolic activation. Only in a few strains a reduction below the indication factor of 0.5 was observed.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with DOPO-HQ at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies

 2.   An in vitro mammalian gene mutation test using the Hprt genes (OECD 476) was performed to investigate the potential of10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone)to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79). The assay comprised a pre-test and three independent experiments (experiment I, I_2 and I_3) whereby experiment I_2 was declared as invalid. This experiment is not reported, all documentation is kept with the raw data and will be archived at the GLP test facility. Experiment I_3 was only performed because one acceptability criterium (the highest test item concentration of 2 mg/mL was not tested) was not fulfilled in experiment I. Nevertheless, experiment I is declared as valid since all further acceptability criteria were fulfilled. The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.

Experiment I and I_3 were performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. Since the results of experiment I and experiment I_3 was clearly negative no experiment II was performed with a treatment period of 24 hours without metabolic activation.

The highest nominal concentration (experiment I (+S9 and -S9): 1 mg/mL; experiment I_3 (+S9 and -S9): 2 mg/mL) applied, was chosen with regard to the solubility of the test item in organic solvents and aqueous media as well as the results of the pre-test.

Precipitation or turbidity of the test item was not visible in experiment I at the end of treatment. In experiment I_3 experimental precipitates were visible at the two highest test item concentrations (2 mg/mL and 1 mg/mL).

Ethylmethane sulfonate (EMS) and 7,12-Dimethylbenzanthracene (DMBA) as appropriate reference mutagens were used as positive controls. Both induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

The evaluated experimental points and the results are summarized in chapter8, page24.

In experiment I statistically significantly increased mutant frequency in both replicates as well as the mean value was only detected at one test item concentration (0.5 mg/mL) and only in the approach without metabolic activation. This value was also outside the historical control range. At the next higher test item concentration, only in replicate B a statistically significant value was observed but this one is within the historical control range of the solvent control and is therefore considered as not biologically relevant. In both approaches, no dose dependent increase in mutant colony numbers was observed in experiment I. Since the significant increase in mutant frequency at concentration 0.5 mg/mL was not reproducible in experiment I_3, experiment I was declared as negative.

In experiment I_3 +S9 only at the lowest test item concentration replicate B induced a statistically significant increase in mutant frequency. However, since this effect appeared only in one replicate and only at the lowest test item concentration it was declared as not biologically relevant. In the approach without metabolic activation (-S9) no relevant statistically significantly increased mutant frequency was detected at any of the tested concentrations. All values remained within the historical control data. No dose dependent increase in mutant colony numbers was observed in experiment I_3 in both approaches. For that reason, this experiment is declared as clearly negative.

Taken all data together, no biological relevant increase in mutant colony numbers was observed in both experiments I up to the maximal concentration of the test item.

 

3. In Vitro Mammalian Cell Micronucleus Test (OECD 487 and EU B.49). This study was performed to assess the potential of DOPO HQ to induce formation of micronuclei in human lymphocytes cultured in vitro in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

Human peripheral blood lymphocytes in whole blood culture were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item and positive control. All cell cultures were set up in duplicates. After exposure and harvesting, slides were prepared and stained. In order to assess the toxicity of the test item to the cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all evaluable cultures. On the basis of these data, the appropriate concentrations were selected to determine the proportion of binucleated cells containing micronuclei.

In the experimental part without metabolic activation, a slight cytotoxic effect could be observed at the highest test item concentration (0.95 mg/mL). Apart from that, no cytotoxicity could be found.

In both experimental conditions tested, the 2 highest test item concentrations showed statistically significantly increased values of binucleated cells with micronuclei (MBNC), lying above the range of the historical laboratory control data for the concurrent solvent control DMSO. In addition, a clear increase of MBNC at higher doses could be observed. Regression analysis revealed a (not statistically significant) dose-response relationship in the experimental approach with metabolic activation. In summary, all criteria for a positive result are fulfilled. All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported,10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone)is able to induce the formation of micronuclei in human lymphocytesin vitro.

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:

18 June 2019 - 12 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Required by REACH

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: DOPO-HQ
Batch: 108376001
Purity: 99.7%
Appearance: White powder
Expiry Date: 20 March 2020
Storage Conditions: At 2-8°C

Target gene:

S. typhimurium: histidine
E. coli: tryptophan

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: his C 3076; rfa-; uvrB- his D 3052; rfa-; uvrB-; R-factor his G 46; rfa-; uvrB- his G 46; rfa-; uvrB-; R-factor

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: trp-; uvrA-

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 were used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the SOP for rat liver S9 preparation.

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: strains: TA 1537, TA 98 4-nitro-o-phenylene-diamine (10 µg/plate in strain TA 98, 50 µg/plate in strain TA 1537) With S9: strains: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA 2-aminoanthracene ; 2.5 µg/plate ; 10.0 µg/plate in WP2 uvrA

Details on test system and experimental conditions:

For each strain and dose level, including the controls, three plates were used.
Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level (solvent or reference mutagen solution
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution
buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. ‎3.4.3 Precultures),
2000 µL Overlay agar
Experiment II (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37°C for 60 minutes.
100 µL Test solution at each dose level (solvent or reference mutagen solution
(positive control)),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution
buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. ‎3.4.3 Precultures),
After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube.
The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 µL of the stock solution, 500 µL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

Rationale for test conditions:

The study was performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium and Escherichia coli reverse mutation assays. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.
The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform and give reliable data on the ability of an agent to interact with DNA and produce mutations.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

plate incorporation test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

NaN3

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

plate incorporation test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

4-NOPD

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

plate incorporation test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

4-NOPD

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

plate incorporation test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

NaN3

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

plate incorporation test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

MMS

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

plate incorporation test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-AA

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

plate incorporation test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

a minor reduction in number of revertants below the indication factor of 0.5

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-AA

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

plate incorporation test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-AA

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

plate incorporation test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-AA

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

plate incorporation test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-AA

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

pre incubation test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

NaN3

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

pre incubation test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

4-NOPD

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

pre incubation test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

4-NOPD

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

pre incubation test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

NaN3

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

pre incubation test

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

a minor reduction in number of revertants below the indication factor of 0.5

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

MMS

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

pre incubation test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-AA

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

pre incubation test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-AA

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

pre incubation test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

a minor reduction in number of revertants below the indication factor of 0.5

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-AA

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

pre incubation test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-AA

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

pre incubation test

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

2-AA

Conclusions:

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, DOPO-HQ is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of DOPO-HQ to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:       3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate as well. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No relevant toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains with and without metabolic activation. Only in a few strains a reduction below the indication factor of 0.5 was observed.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with DOPO-HQ at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

202-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29. Jul. 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EU-Method B.17 of the Commission Regulation (EC) No. 440/2008, adopted 30. May 2008: “Mutagenicity – In vitro Mammalian Cell Gene Mutation Test”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Test Item:
Condition at Receipt: Ambient temperature, in proper conditions
Specification
The following information concerning identity and composition of the test item was pro-vided by the sponsor.
Name 10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone)
Batch no. 108376001
CAS no. 99208-50-1
Composition 10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide > 99 %
Storage room temperature (20 ± 5 °C)
Expiry date 20. Aug. 2020
Stability stable under storage conditions
Appearance White powder
Purity 99.7%
Homogeneity homogeneous

Molecular formula C18H13O4P
Molecular weight 324 g/mol
Vapour pressure not stated
Solubility in solvents H2O: < 0.1 g/L; EtOH: > 1 g/L; acetone: unknown; CH3CN: unknown; DMSO: unknown;
Stability in solvents H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated

See also a copy of the certificate of analysis, attached

Target gene:

The endogenous hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on chromosome X in Chinese Hamster cells (V79)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The Chinese hamster cells V79 (V79-4 clone) are lung fibroblasts, growing adherent. They have been used successfully in in vitro experiments for many years because of its sensitivity to chemical mutagens. Especially the high proliferation rate (doubling time 12 – 14 h in stock cultures) and a high cloning efficiency (80 %) of untreated cells, both nec-essary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 (Bradley et al., 1981). The cells were purchased by ATCC and were sold under the name ATCC® CCL-93™.

Metabolic activation:

with and without

Metabolic activation system:

In vitro methods are valuable when it is desired to accurately control the concentration and exposure time of cells to the test item under study. However, due to the limited ca-pacity for metabolic activation of potential mutagens an exogenous metabolic activation system is necessary. For this purpose, the cells were exposed to the test item with (+S9) and without (-S9) exogenous metabolic activation for 4 h+rs (experiment I, experiment I_3). Following an expression time, the descendants of the treated cell population were monitored for the loss of functional HPRT enzyme by culturing in selective medium.

Test concentrations with justification for top dose:

Dose Selection
According to OECD 476, the highest concentration should be 0.01 M or 2 mg/mL or 2 µL/mL (whichever is the lowest), unless limited by the solubility or toxicity of the test item. Relative survival values below 20 % are considered toxic. If the pre-test reveals cytotoxici-ty, a minimum of one concentration of the test item should reduce the relative survival (RS) below 20 % in the main experiments (optimally the RS of the highest test item con-centration is between 10 % - 20 %). When the test chemical is not of defined composition, e.g., substance of unknown or variable composition, complex reaction products or biolog-ical materials (i.e., Chemical Substances of Unknown or Variable Composition (UVCBs)), testing may be started at a higher concentration to increase the amount of each of the test item components. In case of precipitates or turbidity in culture medium, the highest evalu-ated concentration in the experiments should produce turbidity or precipitates.
According to the results of the pre-test, 6 concentrations were chosen for the main exper-iment and tested with and without metabolic activation:

Experiment I (+S9 and -S9)
Nominal concentrations of test solutions (mg/mL) 100 50 25 13 6 3 -
Resulting nominal concen-trations in experiment (mg/mL) 1.0 0.5 0.25 0.13 0.06 0.03 -

Experiment I_3 (+S9 and -S9)
Nominal concentrations of test solutions (mg/mL) 200 133 89 59 40 26 18
Resulting nominal concen-trations in experiment (mg/mL) 2.0 1.33 0.89 0.59 0.40 0.26 0.18

Vehicle / solvent:

The test item was insoluble in DMEM, ethanol and acetone but solved in DMSO after son-ication.

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

The study was performed to investigate the potential of the test item 10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone) to induce mutations at the HPRT locus on chromosome X Chinese Ham-ster V79 cells.

The assay was performed in two independent experiments, using two parallel cultures each (duplicates). In experiment I, 6 concentrations of the test item were tested with and without metabolic activation. The exposure time was 4 hours. The following nominal con-centrations of the test item were investigated in experiment I (+S9 and -S9):
1.0 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.13 mg/mL, 0.06 mg/mL, 0.03 mg/mL
In experiment I_3, 7 concentrations of the test item were tested with and without metabolic activation. The exposure time was 4 hours. The following nominal concentrations of the test item were investigated in experiment I_3 (+S9 and -S9):
2.0 mg/mL, 1.33 mg/mL, 0.89 mg/mL, 0.59 mg/mL, 0.40 mg/mL, 0.26 mg/mL, 0.18 mg/mL
Since the results of both experiments I (experiment I and experiment I_3) were declared as negative, no experiment II was performed.

Precipitation or cytotoxicity of the test item was not observed up to the highest tested con-centration in experiment I. For that reason, not the maximal required test item concentra-tion (2 mg/mL) was tested in experiments which is a deviation of the acceptability criteria. However, since all further acceptability criteria were fulfilled, experiment I was declared as valid but a second experiment I (experiment I_3) was performed using 2 mg/mL as highest test item concentration. In this experiment precipitation of the test item, visible to the na-ked eye, was noted the concentrations 2 mg/mL and 1.33 mg/mL in both approaches (+S9 and -S9). In addition, cytotoxicity (RS < 20 %) was observed at the test item concentration 1.33 mg/mL in the approach without metabolic activation. For that reason, the test item concentrations 2 mg/mL as well as 1.33 mg/mL are not analysable or mutagenicity accord-ing to OECD 476. Therefore, the results of those two concentrations were not included in the final evaluation.

EMS (750 µg/mL) and DMBA (5 µg/mL) were used as positive controls and showed a dis-tinct increase in induced total mutant colonies, without substantial reduction of the viabil-ity.
The study is valid. It can be used to assess the mutagenic potential of the test item 10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone).

Rationale for test conditions:

Test System
Reasons for the Choice of the Cell Line V79
The Chinese hamster cells V79 (V79-4 clone) are lung fibroblasts, growing adherent. They have been used successfully in in vitro experiments for many years because of its sensitivity to chemical mutagens. Especially the high proliferation rate (doubling time 12 – 14 h in stock cultures) and a high cloning efficiency (80 %) of untreated cells, both nec-essary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 (Bradley et al., 1981). The cells were purchased by ATCC and were sold under the name ATCC® CCL-93™.
6.4.2 Cell Cultures
Prior to use in the experiments, a Master Cell Bank (MCB) and a Working Cell Bank (WCB) were prepared. Stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH. The cells of the MCB were screened for mycoplasma contamination, the doubling time was determined (14h) and the cells were cleansed of pre-existing mutant cells by culturing in HAT medium (medium containing Hypoxanthine, Aminopterin and Thymidine). Cleansed cells build the WCB. Directly after Cleansing the modal chromo-some number was analysed and the cells were expanded (max. 2 passages), stocks were frozen in liquid nitrogen.
MCB and WCB are routinely checked regarding modal chromosome number, doubling time and mycoplasma contamination to allow a continuous working stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells. The data are stored in the archive of LAUS GmbH.
The cells were thawed 9 d prior treatment and cultivated in DMEM complete culture medi-um with 10 % FBS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Evaluation criteria:

Acceptability
The gene mutation assay is considered acceptable if it meets the following criteria:

1. Concurrent negative controls should ideally be within the 95% control limits of the dis-tribution of the laboratories historical negative control database. Where concurrent negative control data fall outside the 95% control limit, they may be acceptable for in-clusion in the historical control distribution as long as these data are not extreme out-liers and there is evidence that the test system is under control and there is evidence of no technical or human failure.
2. the positive control substances must produce a significant increase (p < 0.05) in mu-tant frequency compared with the concurrent solvent controls and should induce re-sponses that are compatible with those, generated in the historical data base.
3. two experimental conditions (+S9 and -S9) are tested unless one resulted in positive results.
4. adequate number of cells (treatment: 20*106) and never less than 2*106 cells must be cultured during the expression period and plated for mutant selection. At least 4 con-centrations, that meet the acceptance criteria are analysable.
5. If the maximum concentration is based on cytotoxicity, the RS of the highest concen-tration is between 20 % and 10 %. If no precipitate or limiting cytotoxicity is observed, the highest test item concentration should be 10mM, 2 mg/mL or 2µL/mL whichever is the lowest. For poorly soluble test items, the highest evaluated concentration in the experiment should produce turbidity or precipitates at the end of treatment. The pre-cipitate should not interfere with the conduct of the test.

If these criteria are not completely fulfilled, the acceptability of the study should be evaluated by the study director. In case of an invalid experiment, the experiment will be repeated under the same conditions.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation or cytotoxicity of the test item was not observed up to the highest tested con-centration in experiment I. For that reason, not the maximal required test item concentra-tion (2 mg/mL) was tested in experiments which is a deviation of the acceptability criteria. However, since all further acceptability criteria were fulfilled, experiment I was declared as valid but a second experiment I (experiment I_3) was performed using 2 mg/mL as highest test item concentration. In this experiment precipitation of the test item, visible to the na-ked eye, was noted the concentrations 2 mg/mL and 1.33 mg/mL in both approaches (+S9 and -S9). In addition, cytotoxicity (RS < 20 %) was observed at the test item concentration 1.33 mg/mL in the approach without metabolic activation. For that reason, the test item concentrations 2 mg/mL as well as 1.33 mg/mL are not analysable or mutagenicity accord-ing to OECD 476. Therefore, the results of those two concentrations were not included in the final evaluation.

EMS (750 µg/mL) and DMBA (5 µg/mL) were used as positive controls and showed a dis-tinct increase in induced total mutant colonies, without substantial reduction of the viabil-ity.
The study is valid. It can be used to assess the mutagenic potential of the test item 10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone).

In experiment I statistically significantly increased mutant frequency in both replicates as well as the mean value was only detected at one test item concentration (0.5 mg/mL) and only in the approach without metabolic activation. This value was also outside the histori-cal control range. At the next higher test item concentration, only in replicate B a statisti-cally significant value was observed but this one is within the historical control range of the solvent control and an is therefore considered as not biologically relevant. In both ap-proaches, no dose dependent increase in mutant colony numbers was observed in exper-iment I. Since the significant increase in mutant frequency at concentration 0.5 mg/mL was not reproducible in experiment I_3, experiment I was declared as negative.
In experiment I_3 +S9 only at the lowest test item concentration replicate B induced a sta-tistically significant increase in mutant frequency. However, since this effect appeared only in one replicate and only at the lowest test item concentration it was declared as not biologically relevant. In the approach without metabolic activation (-S9) no relevant statis-tically significantly increased mutant frequency was detected at any of the tested concen-trations. All values remained within the historical control data. No dose dependent in-crease in mutant colony numbers was observed in experiment I_3 in both approaches. For that reason, this experiment is declared as clearly negative.
Taken all data together, no biological relevant increase in mutant colony numbers was observed in both experiments I up to the maximal concentration of the test item.

Conclusions:

Conclusion:
It can be stated that under the experimental conditions of this study10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone)did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.
Therefore, the test item10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone)is considered to be “non-mutagenic under the conditions of the HPRT assay”.

Executive summary:

This study was performed to investigate the potential of10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone)to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79).

The assay comprised a pre-test and three independent experiments (experiment I, I_2 and I_3) whereby experiment I_2 was declared as invalid. This experiment is not reported, all documentation is kept with the raw data and will be archived at the GLP test facility. Experiment I_3 was only performed because one acceptability criterium (the highest test item concentration of 2 mg/mL was not tested) was not fulfilled in experiment I. Nevertheless, experiment I is declared as valid since all further acceptability criteria were fulfilled.

The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.

Experiment I and I_3 were performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. Since the results of experiment I and experiment I_3 was clearly negative no experiment II was performed with a treatment period of 24 hours without metabolic activation.

The highest nominal concentration (experiment I (+S9 and -S9): 1 mg/mL; experiment I_3 (+S9 and -S9): 2 mg/mL) applied, was chosen with regard to the solubility of the test item in organic solvents and aqueous media as well as the results of the pre-test.

Precipitation or turbidity of the test item was not visible in experiment I at the end of treatment. In experiment I_3 experimental precipitates were visible at the two highest test item concentrations (2 mg/mL and 1 mg/mL).

Ethylmethane sulfonate (EMS) and 7,12-Dimethylbenzanthracene (DMBA) as appropriate reference mutagens were used as positive controls. Both induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

The evaluated experimental points and the results are summarized in chapter8, page24.

In experiment I statistically significantly increased mutant frequency in both replicates as well as the mean value was only detected at one test item concentration (0.5 mg/mL) and only in the approach without metabolic activation. This value was also outside the historical control range. At the next higher test item concentration, only in replicate B a statistically significant value was observed but this one is within the historical control range of the solvent control and is therefore considered as not biologically relevant. In both approaches, no dose dependent increase in mutant colony numbers was observed in experiment I. Since the significant increase in mutant frequency at concentration 0.5 mg/mL was not reproducible in experiment I_3, experiment I was declared as negative.

In experiment I_3 +S9 only at the lowest test item concentration replicate B induced a statistically significant increase in mutant frequency. However, since this effect appeared only in one replicate and only at the lowest test item concentration it was declared as not biologically relevant. In the approach without metabolic activation (-S9) no relevant statistically significantly increased mutant frequency was detected at any of the tested concentrations. All values remained within the historical control data. No dose dependent increase in mutant colony numbers was observed in experiment I_3 in both approaches. For that reason, this experiment is declared as clearly negative.

Taken all data together, no biological relevant increase in mutant colony numbers was observed in both experiments I up to the maximal concentration of the test item.

 

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

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

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29. Jul. 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Test Items
Name 10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone)
Batch no. 108376001
CAS no. 99208-50-1
Composition 10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide > 99 %
Storage room temperature (20 ± 5 °C)
Expiry date 20. Aug. 2020
Stability stable under storage conditions
Appearance White powder
Purity 99.7%
Homogeneity homogeneous
Source of test item Bromine Compounds Ltd (ICL-IP)
Production date not stated
EC no. not stated
Molecular formula C18H13O4P
Molecular weight 324 g/mol
Vapour pressure not stated
Solubility in solvents H2O: < 0.1 g/L; EtOH: > 1 g/L; acetone: unknown; CH3CN: unknown; DMSO: unknown;
Stability in solvents H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated

See also Copy of Certificate of Analysis

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human peripheral blood lymphocytes

Cytokinesis block (if used):

In order to assess the toxicity of the test item to the cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all evaluable cultures. On the basis of these data, the appropriate concentrations were selected to determine the proportion of binucleated cells containing micronuclei.

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male rats, treated with Aroclor 1254

Test concentrations with justification for top dose:

Dose Selection
According to OECD 487 and EU B.49, the maximum concentration of the test item should be 2 µl/mL, 2 mg/mL or 10 mM, whichever is the lowest. When cytotoxicity occurs, the highest concentration should aim to produce 55 ± 5% cytotoxicity. When the test item is a substance of unknown or variable composition, a complex reaction product or of biologi-cal origin (UVCB), testing may be started at a higher concentration to increase the amount of each of the test item components. Generally, concentration intervals of approximately 2 to 3-fold are used. When solubility is a limiting factor, the maximum concentration, if not limited by cytotoxicity, should be the lowest concentration at which turbidity or minimal precipitate is visible in the cultures.
For the test item 10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone), the maximum concentration according to these criteria and regarding the results of the pre-tests for solubility (see chapter 6.1.3, page 11) was 0.95 mg/mL.

Vehicle / solvent:

DMSO
DMSO was used as solvent control for the test item.
0.9% NaCl was used as solvent control for the positive controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Experiment I without and with metabolic activation
After centrifugation (10 min, 500 * g), the cells were resuspended in minimal culture me-dium and solvent control, positive control or the single test item concentrations were add-ed.
In the case of metabolic activation, 50 µL S9 mix per mL medium were used. The cell cul-tures were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 4 h (exposure period).
After the exposure time of 4 h, the cells were spun down by gentle centrifugation for 5 min (500 * g). The supernatant was discarded, the cells were re-suspended in 5 mL Sa-line G and centrifuged again. The washing procedure was repeated once as described.
After washing, the cells were re-suspended in complete culture medium RPMI 1640, cyto-chalasin B (final concentration 5 µg/mL) was added and the cells we

Harvesting Procedure
Each cell culture was harvested and processed separately. The cells were spun down by gentle centrifugation (10 min, 500 * g). The supernatant was discarded and the cells were re-suspended in 11 mL hypotonic KCl solution. The cell suspension was allowed to stand for 5 min at room temperature (20 ± 5°C). After stopping the hypotonic treatment by addi-tion of approximately 1 ml fixative, the tubes were centrifuged (500 * g, 10 min), the su-pernatant was discarded and the cell pellet was treated with fixative (a mixture of metha-nol and glacial acetic acid (3:1)) at 2 – 8 °C, minimum 30 min. Centrifugation and fixation steps were repeated until the cell pellet was white.

Preparation of Slides
The slides were prepared by dropping the cell suspension onto a clean microscope slide. The cells were then stained with a 10 % solution of Giemsa. All slides were independent-ly coded before microscopic analysis.
Determination of the Cytokinesis-Block Proliferation Index
In all replicates, the cytokinesis-block proliferation index (using at least 500 cells per cul-ture) was determined in order to assess the cytotoxicity of the test item. From these deter-minations, the test item concentrations which were evaluated for scoring of micronuclei were defined.

Determination of Binucleated Cells with Micronuclei
At least 1000 binucleated cells per culture were scored for micronuclei. Only cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included in the analysis.

Evaluation Procedure
Evaluation of the slides was performed using Zeiss microscopes with appropriate magni-fication. The generated data were recorded on raw data sheets.
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.

Evaluation criteria:

Acceptability
The genotoxicity assay is considered acceptable if it meets the following criteria:
• All experimental conditions are tested (short exposure with and without metabolic activation, extended exposure without metabolic activation) unless a positive result is achieved in any experiment.
• In each experiment, an adequate number of cells is analysable both in the controls and in at least 3 test item concentrations.
• The micronucleus induction of the solvent and positive controls is compatible with the historical laboratory control data or the literature data.
• The positive control shows a statistically significant increase of binucleated cells with micronuclei compared with the concurrent solvent control.
• The criteria for cell proliferation and for the selection of concentrations are fulfilled.

Classification
The test item is considered unable to induce chromosome breaks and/or loss in this test system (negative result) if, in all experimental conditions, the following criteria are met:
• Neither a statistically significant nor a concentration-related increase of the num-ber of micronucleate cells in the evaluated test concentrations is observed.
• The obtained results lie within the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5 % control limits where appropriate.
The test item is considered able to induce chromosome breaks and/or loss in this test sys-tem (positive result) if, in any of the experimental conditions, all of the following criteria are met:
• At least one test concentration shows a statistically significant increase of micro-nucleate cells compared to the concurrent solvent control.
• In at least one experimental condition a dose-related increase of micronucleate cells can be observed using trend analysis.
• Any of the results lies outside the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5 % control limits where appropriate.

Statistics:

Statistical Evaluation
The number of binucleated cells with micronuclei in each treatment group was compared with the solvent control. Statistical significance was tested using Fisher’s exact test at the five per cent level (p 0.05) with the following equation:

a number of binucleated cells with micronuclei of the solvent control
b number of binucleated cells with micronuclei of the test item of the respective concentration
c number of binucleated cells without micronuclei of the solvent control
d number of binucleated cells without micronuclei of the test item of the respective concentration
n total number of cells
 

Key result

Species / strain:

other: Primary cultures of human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

n conclusion, under the experimental conditions reported,10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone)is able to induce the formation of micronuclei in human lymphocytesin vitro. The result of the micronucleus test with the test item 10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone) is considered as “positive” under the conditions of the test.

Executive summary:

 Summary

This study was performed to assess the potential of10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone)to induce formation of micronuclei in human lymphocytes culturedin vitro in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

The test item was dissolved in DMSO to prepare a stock solution with a concentration of 190 mg/mL, corresponding to the highest concentration (0.95 mg/mL) in the test. In addition, a geometric series of dilutions was prepared from the stock solution to get the single concentration to be tested.

One valid experiment (4 h exposure, without and with metabolic activation) was performed.

Human peripheral blood lymphocytes in whole blood culture were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item and positive control. All cell cultures were set up in duplicates. After exposure and harvesting, slides were prepared and stained. In order to assess the toxicity of the test item to the cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all evaluable cultures. On the basis of these data, the appropriate concentrations were selected to determine the proportion of binucleated cells containing micronuclei.

In the experimental part without metabolic activation, a slight cytotoxic effect could be observed at the highest test item concentration (0.95 mg/mL). Apart from that, no cytotoxicity could be found.

In both experimental conditions tested, the 2 highest test item concentrations showed statistically significantly increased values of binucleated cells with micronuclei (MBNC), lying above the range of the historical laboratory control data for the concurrent solvent control DMSO. In addition, a clear increase of MBNC at higher doses could be observed. Regression analysis revealed a (not statistically significant) dose-response relationship in the experimental approach with metabolic activation. In summary, all criteria for a positive result (see chapter7.5, page20) are fulfilled.

All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported,10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone)is able to induce the formation of micronuclei in human lymphocytesin vitro. The result of the micronucleus test with the test item10-(2,5-Dihydroxyphenyl)-10H-9-oxa-10-phospha-phenantbrene-10-oxide (DOPO-Hydroquinone)is considered as “positive” under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

based on the 3 studies performed, DOPO HQ is not subject to classification under CLP.