4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro: Gene mutation (Bacterial reverse mutation, Ames test), OECD 471, GLP: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102: negative with and without metabolic activation

Genetic toxicity in vitro: Gene mutation (mouse lymphoma assay), OECD 476, GLP: mouse lymphoma L5178Y cells: negative with and without metabolic activation

Genetic toxicity in vitro: Chromosome aberration (Micronucleus test), OECD 487, GLP: human lymphocytes: negative with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-17 - 2015-07-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well-documented GLP OECD 487 guideline study without deviations on the registered substance itself.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 487 “In vitro Mammalian Cell Micronucleus Test”, adopted 26 September 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Human lymphocytes
Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (31 years old) for Experiment I and from a male donor (25 years old) for Experiment II. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Test: 1.2 to 1200.0 μg/mL (with regard to the solubility of the test item)
Experiment I, ±S9, 4h exposure: 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, 600.0, 1200.0 µg/ml
Experiment II, -S9, 20h exposure: 29.1, 34.9, 41.9, 50.2, 60.3, 72.3, 86.8, 104.2, 125.0, 150.0 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

0.5% DMSO (99.99% purity)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h (pulse exposure) or 20h (continuous exposure)
- Expression time (cells in growth medium): 16h or 0h in medium + 20h with CytB
- Fixation time (start of exposure up to fixation or harvest of cells): 40h

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 μg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 parallel cultures

NUMBER OF CELLS EVALUATED: at least 1000 binucleate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index as indication of cytostasis

Evaluation criteria:

Evaluation of cytotoxicity and cytogenetic damage:
Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI (Cytokinesis-block proliferation index) was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

Statistics:

Statistical analysis
Statistical significance will be confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control. Other statistical methods may be used if appropriate.

Key result

Species / strain:

lymphocytes: human

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH / osmolality: No relevant influence on osmolarity or pH was observed.
- Precipitation: Precipitation of the test item in the culture medium was observed in Experiment I at 37.5 μg/mL and above in the absence of S9 mix and at 75.0 μg/mL and above in the presence of S9 mix and in Experiment II in the absence of S9 mix at 86.8 μg/mL and above at the end of treatment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In both cytogenetic experiments, in the absence and presence of S9 mix, no cytotoxicity was observed.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: negative

The study was conducted under GLP according to OECD guideline 487 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin to induce micronuclei in mammalian cells.
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating or the highest evaluable concentrations.

Executive summary:

In a mammalian cell cytogenetics assay, Micronucleus test (OECD 487), primary human lymphocyte cultures were exposed to 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin in DMSO at concentrations of 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0, 300.0, 600.0, 1200.0 µg/ml for 4h with and without metabolic activation and 29.1, 34.9, 41.9, 50.2, 60.3, 72.3, 86.8, 104.2, 125.0, 150.0 µg/ml for 20h without metabolic activation (Phenobarbital/β-naphthoflavone induced rat liver S9).

4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin was tested up to precipitating concentrations. Positive controls induced the appropriate response.  There was no evidence or a concentration related positive response of Micronuclei induced over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 487 for in vitro cytogenetic mutagenicity data.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-08-22 - 1998-05-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted under GLP according to EU method B.14, OECD guideline 471, and US EPA OPPTS 870.5265 on the registered substance itself without deviations. Although the study was nominally performed according to OECD 471 as adopted in 1983, five strains including S. typhimurium TA 102 were used, which was first foreseen in the revised version of 1997. Consequently, the study does not show any deviations from the most recent guideline.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

as set out in EEC Directive 92/69/EEC, method B.14

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

as of 1983-05-26

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

"public draft" as of June 1996

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: deep rough

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 mix

Test concentrations with justification for top dose:

0, 50, 158, 500, 1581, 5000 µg/plate (first experiment, plate incorporation test)
0, 16, 50, 158, 500, 1581 µg/tube (second experiment, preincubation method)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

other: nitrofurantoin; 4-nitro-1,2-phenylene diamine; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, first experiment); preincubation (second experiment)

DURATION
- Preincubation period: 20 min (preincubation method)
- Exposure duration: 48h
- Expression time (cells in growth medium): corresponds to exposure duration

SELECTION AGENT (mutation assays): histidine-free medium

NUMBER OF REPLICATIONS: each three plates perdose, strain, ±S9

DETERMINATION OF CYTOTOXICITY
- Method: other: gross appraisal of background growth and a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls.

Evaluation criteria:

Assessment Criteria
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 150 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 1581 µg per plate, the substance started to precipitate, so that at 5000 µg per plate no further evaluation was possible.

ADDITIONAL INFORMATION ON CYTOTOXICITY: There was no indication of a bacteriotoxic effect of the test item at doses of up to and including 1581 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: negative

The study was conducted under GLP according to EU method B.14, OECD guideline 471, and US EPA OPPTS 870.5265 on the registered substance itself without deviations. The method is to be considered scientifically reasonable, the validity criteria are fulfilled. Hence, the results can be considered as sufficiently reliable to assess the ability of 12H-Dibenzo(d,g)(1,3,2)dioxaphosphocin, 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl- to induce gene mutations in bacteria.
No indications of mutagenic effects of the test item could be found at assessable doses of up to 1581 µg per plate in any of the Salmonella typhimurium strains used. Hence, 12H-Dibenzo(d,g)(1,3,2)dioxaphosphocin, 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl- has to be regarded as non-mutagenic.

Executive summary:

In a reverse gene mutation assay in bacteria (EU method B.14, OECD guideline 471, US EPA OPPTS 870.5265), histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102 of S. typhimurium LT2 were exposed to 12H-Dibenzo(d,g)(1,3,2)dioxaphosphocin, 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl- in DMSO at concentrations of 0, 50, 158, 500, 1581, 5000 µg/plate (first experiment, plate incorporation test) and 0, 16, 50, 158, 500, 1581 µg/tube (second experiment, preincubation method as independent repeat) in the presence and absence of mammalian metabolic activation.

12H-Dibenzo(d,g)(1,3,2)dioxaphosphocin, 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl- was tested up to insoluble concentrations resp. limit concentration (5000 µg/plate).

Doses up to and including 1581 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 1581 µg per plate and above. Therefore, the test was no longer interpretable at 5000 µg per plate.

Evidence of mutagenic activity of the test item was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-amino-anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Therefore, 12H-Dibenzo(d,g)(1,3,2)dioxaphosphocin, 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl- was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

This study is classified as acceptable. This study satisfies the requirement for EU method B.14, OECD guideline 471, and US EPA OPPTS 870.5265 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-08 - 2015-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well-documented GLP OECD 476 guideline study without deviations on the registered substance itself.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: “Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Ministry of Agriculture, Forestry and Fisheries of Japan. MAFF Notification No. 12 Nousan-8147, 24 November 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with: Hypoxanthine 5.0 × 10-3 M, Aminopterin 2.0 × 10-5 M, Thymidine 1.6 × 10-3 M, Glycin 5.0 × 10-3 M.
The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in RPMI 1640 medium containing: Hypoxanthine 1.0 × 10-4 M, Thymidine 1.6 × 10-3 M.
After this incubation the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

2.3, 4.7, 9.4, 18.8, 37.5, 56.3, 75.0 µg/ml (Experiment 1, 4h, -S9)
2.3, 4.7, 9.4, 18.8, 37.5, 56.3, 75.0 µg/ml (Experiment 1, 4h, +S9)
7.0, 14.0, 28.0, 56.0, 70.0, 84.0, 98.0 µg/ml (Experiment 2, 24h, -S9)
7.0, 14.0, 28.0, 35.0, 42.0, 49.0, 56.0 µg/ml (Experiment 2, 4h, +S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO, 99.9%
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 99.9%, 0.5% (v/v)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

See "Any other information on materials and methods"

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 or 24 h
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): 10 - 15 days

SELECTION AGENT (mutation assays): Trifluorothymidine

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Evaluation of Results
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10exp6 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10% of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10exp6 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using R Script LM.Rnw statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

experimental group p-value
experiment I, culture I without S9 mix 0.144
experiment I, culture II without S9 mix 0.351
experiment I, culture I with S9 mix 0.188
experiment I, culture II with S9 mix 0.314
experiment II, culture I without S9 mix 0.185
experiment II, culture II without S9 mix 0.756
experiment II, culture I with S9 mix 0.031S
experiment II, culture II with S9 mix 0.522S

S = significant trend

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: vehicle control valid

Positive controls validity:

valid

Additional information on results:

See "Any other information on results"

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: negative

The study was conducted under GLP according to OECD guideline 476 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin to induce mutations in mammalian cells. No substantial and reproducible increase of the mutation frequency was noted in both experiments with and without metabolic activation.
In conclusion it can be stated that under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

In a mammalian cell mutation assay (Mouse lymphoma assay, OECD 476), L5178Y cell cultures were exposed to 4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin in DMSO at concentrations of 0 to 98.0 µg/ml with and without metabolic activation (Phenobarbital/β-naphthoflavone induced rat liver S9).

4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin was tested up to cytotoxic concentrations. Relevant cytotoxic effects indicated by a relative total growth of less than 50% in both parallel cultures were observed in experiment I at 37.5 µg/mL with metabolic activation. In experiment II cytotoxicity as described above was noted at 70.0 µg/ml and above in the absence of metabolic activation and at 35.0 µg/mL in the presence of metabolic activation. The recommended cytotoxic range of approximately 10-20% relative total growth was covered with and without metabolic activation. Positive controls induced the appropriate response. There was no evidence of mutations induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 487, EU method B.17, EPA OPPTS 870.5300, and Japanese Guidelines for in vitro mutagenicity data. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

As all available and required in vitro genotoxicity tests, i.e. testing for gene mutations in both bacteria and mammalian cells as well as chromosome mutations in mammalian cells, revealed negative results, no conclusion on a mode of action for genotoxic events can be drawn. No indication is given that the obtained results are not relevant for humans, as in vivo metabolism of the test item is sufficiently mimicked by addition of S9 mix, human lymphocytes were also tested and direct genotoxins act commonly species-independent.

Additional information

Additional information from genetic toxicity in vitro:

There are several data available sufficiently covering all required endpoints according to REACH Annex VIII and above, i.e. gene mutation in bacteria and mammalian cells and chromosome mutations (micronucleus test) in vitro, and which are all assessed as Klimisch 1. All available data is consistently negative for genotoxic effects.

No indication is given that the obtained results are not relevant for humans, as in vivo metabolism of the test item is sufficiently mimicked by addition of S9 mix, human lymphocytes were also tested and direct genotoxins act commonly species-independent.

The data base is so of good quality, sufficient to exclude that any risk with regard to genotoxic effects may arise for humans from CAS 73912-21-7, no data gaps could be identified and no additional testing is required.

Justification for selection of genetic toxicity endpoint
One of three equally reliable in vitro genotoxicity tests, which gave consistently negative results. Hence, endpoint selection has no influence on the endpoint conclusion.

Justification for classification or non-classification

All available test results for gene mutation and chromosome aberrations (micronucleus test) in vitro are consistently negative, and no need for classification as mutagen or directly genotoxic carcinogen was identified.