Reaction product of 6H-dibenz[c,e][1,2]oxaphosphorin 6-oxide, itaconic acid and ethylene glycol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test: Under the test conditions, the test item 9,10-Dihydroxy-9-oxa-10-[2,3-Di-(2-hydroxyethoxy)carbonylpropyl]-10-phosphaphenanthren-10-oxid is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535.

Micronucleus-Test: Under the present test conditions, Ukanol FR 70 tested up to a concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.

HPRT-Test: Under the present test conditions, Ukanol FR 70 tested up to a concentration of 1000 µg/mL medium, that led to test item precipitation (4-hour exposure, without and with metabolic activation) was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-02-26 - 20009-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The GLP study was conducted according to an internationally accepted guideline. All study parameters are given in detail.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

other: hisD6610, uvrB, pKM 101, rfa

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: hisD3052, uvrB, pKM 101, rfa

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: hisG46, uvrB, pKM 101, rfa

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: hisG428, pKM 101, rfa

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: hisG46, uvrB, rfa.

Metabolic activation:

with and without

Metabolic activation system:

S9, produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.

Test concentrations with justification for top dose:

First Experiment:
1501 / 500 / 150 / 50 /15 µg/plate

Second Experiment:
1502 / 751 / 376 / 188 / 94 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene,

Details on test system and experimental conditions:

On the day of the start of the first experiment, a stock solution containing 50 g/L of the test item in DMSO was prepared.
In the second experiment, a stock solution containing 15 g/L of the test item in DMSO was prepared.
DMSO was chosen as solvent, because the test item was completely soluble, and this solvent doesn't have any effects on the viability of the bacteria or the number of spontaneous revertants. The stock solutions were used to prepare the geometric series of the concentrations to be tested. Each solution was membrane filtrated to accomplish sterility.
One day before the start of each experiment, one pellet per strain to be used was put into a culture vessel containing nutrient broth. For the incubation of strains TA97a, TA98, TA100 and TA102, ampicilline was added to the medium (25 mg/L). For the incubation of strain TA102, additionally, tetracycline was added (20 mg/L). TA1535 was incubated without addition of antibiotica, as this strain doesn't carry a resistance plasmid.

After incubation for 10 hours at 37 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Evaluation criteria:

A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor > 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The mean revertant values of the four replicates are presented in the following table.

Strain		TA97a		TA98		TA100		TA102		TA1	535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H20	Mean	n.t.	n.t.	n.t.	n.t.	114	n.t.	n.t.	n.t.	10	n.t.
	sd	n.t.	n.t.	n.t.	n.t.	15.3	n.t.	n.t.	n.t.	3.3	n.t.
DMSO	Mean	103	97	10	8	99	131	209	215	9	11
	sd	5.8	13.0	5.4	3.9	7.4	17.7	65.4	26.7	3.1	3.3
Pos.Contr.	Mean	1001	1001	1001	1001	1001	1001	1001	1001	1001	1001
	sd	0	0	0	0	0	0	0	0	0	0
	m	9.72	10.32	100.1	125.1	8.78	7.64	4.79	4.66	100.1	91.00
1502|jg/pl.	Mean	86	82	7	6	105	111	152	181	11	8
	sd	5	6	2	1	17	18	10	64	4	2
	m	0.83	0.85	0.70	0.75	1.06	0.85	0.73	0.84	1.22	0.73
751Mg/pi.	Mean	109	88	6	6	95	114	132	222	8	14
	sd	5	20	1	1	20	6	20	19	1	6
	m	1.06	0.91	0.60	0.75	0.96	0.87	0.63	1.03	0.89	1.27
376ng/pi.	Mean	83	87	8	6	120	99	236	201	11	11
	sd	8	15	4	1	6	15	72	22	2	1
	m	0.81	0.90	0.80	0.75	1.21	0.76	1.13	0.93	1.22	1.00
188Mg/pl-	Mean	123	111	6	6	104	92	134	175	10	12
	sd	11	6	2	5	22	15	5	45	3	5
	m	1.19	1.14	0.60	0.75	1.05	0.70	0.64	0.81	1.11	1.09
94jig/pi.	Mean	106	100	9	5	150	99	157	213	13	10
	sd	8	16	2	3	11	1	13	9	3	2
	m	1.03	1.03	0.90	0.63	1.52	0.76	0.75	0.99	1.44	0.91

Conclusions:

Interpretation of results:
negative

Under the test conditions, the test item 9,10-Dihydroxy-9-oxa-10-[2,3-Di-(2-hydroxyethoxy)carbonylpropyl]-10-phosphaphenanthren-10-oxid is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains TA 1535, TA 97, TA98,TA100 and TA 102 of S. typhimurium were exposed to 9,10-Dihydroxy-9-oxa-10-[2,3-Di-(2-hydroxyethoxy)carbonylpropyl]-10-phosphaphenanthren-10-oxid.

The test item didn't show mutagenic effects in both experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants (solvent only). Cytotoxicity of the test item was not detected: The background lawn was visible and the number of revertants was not significantly decreased. Therefore it can be stated, that under the test conditions, the test item 9,10-Dihydroxy-9-oxa-10-[2,3-Di-(2-hydroxyethoxy)carbonylpropyl]-10-phosphaphenanthren-10-oxid is not mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-23 - 2017-05-17

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: IN VITRO GENE MUTATION ASSAY (HPRT TEST)

Specific details on test material used for the study:

Designation Ukanol FR 70
CAS no. 63562-34-5
EC no. 264-313-6
Batch no. 7505294
Receipt no. 61985
Date of receipt December 22, 2016
Characteristics Solid
Storage conditions At room temperature (at +10°C to +25°C) in a tightly closed container.
Stability / Expiry date No information on stability was available to LPT
Content ≥ 95.0%

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cells were maintained in growth medium (Dulbecco's modified Eagle-Medium ) supplemented with 10% foetal calf serum and 1% penicillin/streptomycin solution . Cultures were incubated at 37°C in a humidified atmosphere (90%) containing 10% CO2. For subculturing, a trypsin (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used. The cells were periodically checked for the absence of mycoplasma contamination. The spontaneous mutation rate was continuously monitored.

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary cytotoxicity test:
The concentrations to be employed in the main experiment were chosen based on the results of a preliminary cytotoxicity study without and with metabolic activation with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL medium. In this preliminary test no signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium. Test item precipitation was noted macroscopically starting at a concentration of 316 µg/mL medium in the experiments without and with S9 mix (see Table 1). No changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO. For details see the Text table 5-1.
Hence, two concentrations of 500 and 1000 µg Ukanol FR 70/mL medium showing test item precipitation were employed as highest concentrations for the genotoxicity tests without and with metabolic activation in the main experiment.
Main study
Concentrations of 62.5, 125, 250, 500 and 1000 µg Ukanol FR 70/mL medium were selected for the mutagenicity experiment without and with metabolic activation. Experiments with and without metabolic activation were conducted in duplicates.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Approximately 1500000 cells were placed in 15 mL DMEM-FCS per 75 cm2 culture flask. On the following day, the growth medium was removed and the cells were resuspended in treatment medium. The cells were exposed to a wide range of concentrations of Ukanol FR 70 in the absence and presence of S9 mix, for 4 hours. For experiments without metabolic activation 0.15 mL test item solution, negative or positive controls were added to 14.85 mL treatment medium.
For experiments with metabolic activation 3 mL of the S9 mix were added to 11.85 mL treatment medium before treatment.
Concurrent positive and negative (vehicle) controls, both with and without metabolic activation were included in each experiment.
Both experiments (in the absence and presence of S9 mix) were performed in independent replicates.
At the end of the exposure period and removal of the test item the cells were washed with PBS and the cells were trypsinised and then suspended in 9 mL growth medium. The cells were pelleted by centrifugation (250 x g for 5 minutes), the supernatant was removed, and the cells were resuspended in 3 mL growth medium.
Then, one part of the cells was used for the determination of the relative plating efficiency for each dose to obtain an accurate measure of the cytotoxic effect of the chemical. Therefore, three replicate plates (60 mm diameter dishes) were used with 150 cells per plate in 5 mL growth medium. After about 8 days, the cells were fixed and stained with methylene blue in ethanol. The colonies were then counted for plating efficiency (PE1).
Another part of the cells was used for the determination of the mutant frequency. The cells were further incubated for 6 days including one cell passage in between. This period was required for expression of the new genotype, i.e. for sufficient dilution and catabolism of the previously expressed HPRT. After the expression period the cells were harvested by trypsinisation and replated at a density of 500000 cells per 100 mm diameter dish in DMEM-FCS containing 6-thioguanine (10 µg/mL) for selection of mutants (4 replicate plates), and 150 cells per 60 mm diameter dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE2), (3 replicate plates). After about 8 days (PE2) or about 12 days (mutant plates), the cells were fixed and stained with methylene blue in ethanol and the colonies were then counted.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

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

Positive controls validity:

valid

Conclusions:

Under the present test conditions, Ukanol FR 70 tested up to a concentration of 1000 µg/mL medium, that led to test item precipitation (4-hour exposure, without and with metabolic activation) was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Executive summary:

Ukanol FR 70 was tested for its mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours in the experiments without and with S9 mix.

Ukanol FR 70 was completely dissolved in dimethyl sulfoxide (DMSO). The vehicle DMSO served as the negative control.

Preliminary cytotoxicity test

The concentrations to be employed in the main experiment were chosen based on the results of a preliminary cytotoxicity study without and with metabolic activation with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL medium. In this preliminary test no signs of cytotoxicity were noted up to the top concentration of 2000 µg/mL medium. Test item precipitation was noted macroscopically starting at a concentration of 316 µg/mL medium in the experiments without and with S9 mix. No changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO.

Hence, two concentrations of 500 and 1000 µg Ukanol FR 70/mL medium showing test item precipitation were employed as highest concentrations for the genotoxicity tests without and with metabolic activation in the main experiment.

Main study

Concentrations of 62.5, 125, 250, 500 and 1000 µg Ukanol FR 70/mL medium were selected for the mutagenicity experiment without and with metabolic activation. Experiments with and without metabolic activation were conducted in duplicates.

Cytotoxicity

No signs of cytotoxicity were noted up to the top concentration of 1000 µg Ukanol FR 70/mL medium in the absence and presence of metabolic activation (4 hour exposure). Test item precipitation was noted macroscopically at concentrations of 500 and 1000 µg/mL medium in the experiments without and with S9 mix.

 

Mutagenicity

Experiments without metabolic activation

The mutation frequency of the solvent control DMSO was 14.88 and 15.60 mutant colonies per 106 cells, for the 1st and the 2nd experiment, respectively. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 62.5, 125, 250, 500 and 1000 µg Ukanol FR 70/mL culture medium ranged from 6.57 to 16.03 mutant colonies per 106 cells. These results are within the normal range of the solvent controls.

Experiments with metabolic activation

The mutation frequency of the solvent control DMSO was 19.73 and 4.00 mutant colonies per 106 cells, for the 1st and the 2nd experiment, respectively. Hence, the solvent controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 62.5, 125, 250, 500 and 1000 µg Ukanol FR 70/mL culture medium ranged from 3.76 to 19.48 mutant colonies per 106 cells. These results are within the normal range of the solvent controls.

The positive controls in the direct test EMS (ethyl methanesulfonate) and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 466.07 to 818.05 mutant colonies per 106 cells in the case of EMS and ranging from 327.15 to 719.41 mutant colonies per 106 cells in the case of DMBA, indicating the validity of this test system. The slightly higher value of 818.05 mutant colonies per 106 cells for EMS compared to the historical positive control data of 159.4 to 703.4 mutant colonies per 106 cells is considered to be within the normal variation, as the mutation frequency is compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.

 

The background mutation frequency at LPT ranges from 1.6 to 40.7 mutant colonies per 106 cells for the solvent controls. The mutation frequency of the positive controls at LPT ranges from 159.4 to 703.4 mutant colonies per 106 cells for EMS and from 220.3 to 711.8 mutant colonies per 106 cells for DMBA.

Under the present test conditions, Ukanol FR 70 tested up to a concentration of 1000 µg/mL medium, that led to test item precipitation (4-hour exposure, without and with metabolic activation) was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-12-23 - 2017-03-30

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)

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:

Designation Ukanol FR 70
CAS no. 63562-34-5
EC no. 264-313-6
Batch no. 7505294
Receipt no. 61985
Date of receipt December 22, 2016
Characteristics Solid
Storage conditions At room temperature (at +10°C to +25°C) in a tightly closed container.
Stability / Expiry date No information on stability was available to LPT
Content ≥ 95.0%

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Human peripheral blood was obtained by venipuncture from young, healthy, non-smoking male or female individuals with no known recent exposures to genotoxic chemicals or radiation, and collected in heparinised vessels. Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin . The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.

Cytokinesis block (if used):

CytoB (Cytochalasin B)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg Ukanol FR 70/mL medium were employed. Test item precipitation was noted macroscopically starting at a concentration of 316 µg/mL medium in the experiments without and with S9 mix (24- or 4-hour exposure). No signs of cytotoxicity were noted up to the top concentration of 2000 µg Ukanol FR 70/mL medium. Hence, 2000 µg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.
In the main study no signs of cytotoxicity were noted up to the top concentration of 2000 µg Ukanol FR 70/mL medium in the experiments without and with metabolic activation. Test item precipitation was noted macroscopically at all tested concentrations in the experiments without and with S9 mix.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

other: colchicine

Details on test system and experimental conditions:

0.5 mL of freshly prepared blood lymphocytes were seeded with 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin. After initiation appropriate concentration of the test item in the vehicle were added to the cell cultures for each target concentration of the test item in the test medium and each experiment. Precipitation of the test item was checked before and after each experiment. Evaluation of precipitation was done by light microscopy at the beginning and end of treatment. Theoretical considerations, together with published data, indicate that most aneugens and clastogens are detected by a short term treatment period of 4 hours in the presence and absence of S9, followed by removal of the test item and a growth period of 1.5 cell cycles. Cells were sampled at a time equivalent to about 1.5 times the normal (i.e. untreated) cell cycle length either after the beginning or at the end of treatment. Sampling or recovery times would have been extended if it is known or suspected that the test item affects the cell cycling time (e.g. when testing nucleoside analogues). Because of the potential cytotoxicity of S9 preparations for cultured mammalian cells, an extended exposure treatment was used only in the absence of S9.
All treatments were conducted while the cells were growing exponentially
Each culture was harvested and processed separately. After the test item incubation, mitotic activity was arrested by the addition of CytoB to each culture at a final concentration of 5 µg/mL. After an additional incubation of 20 hours the cultures were centrifuged for 10 minutes at 800 rpm, the supernatant was discarded and the cells resuspended in KCl (0.56%). After incubation for 17 minutes at 37°C, the cell suspensions were centrifuged for 10 minutes at 800 rpm. The supernatant was discarded and 5 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added. The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm. The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette. Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide. The slides were then stained using 10% Giemsa and left to air-dry at room temperature.
At least 500 cells per replicate cell culture (two cultures per concentration in the main study, one culture per concentration in the preliminary test) were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity. The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
• the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
• any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).
The test chemical is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the present test conditions, Ukanol FR 70 tested up to a concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.
The results for the vehicle controls were within historical control range.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Executive summary:

Test samples of Ukanol FR 70 were assayed in anin vitromicronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a ratliver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied.

The test item wascompletely dissolved in dimethylsulfoxide (DMSO).The vehicle DMSO served as the negative control.

The concentrations employed were chosen based on the results of a cytotoxicity study.In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg Ukanol FR 70/mL medium were employed. Test item precipitation was noted macroscopically starting at a concentration of 316 µg/mL medium in the experiments without and with S9 mix (24- or 4-hour exposure). Nosigns ofcytotoxicitywere noted up to the top concentration of 2000 µg Ukanol FR 70/mL medium. Hence, 2000 µg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation.

In the main study nosigns ofcytotoxicitywere noted up to the top concentration of 2000 µg Ukanol FR 70/mL medium in the experiments without and with metabolic activation. Test item precipitation was notedmacroscopicallyat all tested concentrations in the experiments without and with S9 mix.

Mitomycin C (at 0.2 µg/mL) and colchicine (at 0.02 µg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 µg/mL) in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The micronucleus frequencies of cultures treated with the concentrations of 250, 500, 1000 and 2000 µg Ukanol FR 70/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 1.0 to 7.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 2000 µg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies.In this test frequencies of 1.5 or 4.0 micronucleated cells per 1000 binucleated cells for the 4-hour and 24-hour exposure, respectively, were observed. The vehicle result was within the historical control ranges.

In the positive control cultures the micronucleus frequencies were increased to 18.5 or 15.0 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively.This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the concentrations of250, 500, 1000 and 2000 µg Ukanol FR 70/mL medium(4-h exposure) in the presence of metabolic activation ranged from 2.5 to 3.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei upto the top concentration of 2000 µg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls.

Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test a mean frequency of 4.0 micronucleated cells per 1000 binucleated cells was observed. The vehicle result was within the historical control ranges.

In the positive control culture the micronucleus frequency was increased to 17.0 micronucleated cells per 1000 binucleate cells for the 4-hour exposure.This demonstrated that cyclophosphamide induced significant chromosomal damage.

Under the present test conditions, Ukanol FR 70 tested up to a concentration of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance does not meet the criteria for classification and labelling for this endpoint, as set out in Regulation (EC) NO. 1272/2008.