2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Metalink U shows neither mutagenic effects in a Salmonella/microsome test (strains TA 98, TA 100, TA 102, TA 1535 and TA 1537; +/- S9 mix; Jarzombek, 2015) or in a HPRT test (V79 cells; +/- S9 mix; Wollny, 2015) nor clastogenic effects in a micronucleus test (V79 cells; +/- S9 mix; Sutter, 2015).

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:

July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 221305
- Purity: > 98.0 %
- Expiration date of the lot/batch: 2014-07-27

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

0,10, 25, 50, 160, 500, 1600, 5000 µg/plate (without and with S9 mix)

Vehicle / solvent:

Solvents used: ethylene glycol dimethylether (EGDE) dried with a molecular sieve 0.3nm (test substance), phosphate buffer (sodium azide, mitomycin C, cumene hydroperoxide ), DMSO (2-nitrofluorene, 4-nitro-1,2-phenylene diamine, 2-aminoanthracene)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 100, TA 1535), 2-nitrofluorene (TA 98), 4-nitro-o-phenylenediamine (TA 1537), mitomycin C (TA 102, plate incorp.), cumene hyd (TA 102, preincub. trials), 2-aminoanthracene (all strains)

Remarks:

The positive controls sodium azide, 2-nitrofluorene, 4-nitro-o-phenylenediamine, mitomycin C and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.

Details on test system and experimental conditions:

METHOD: Standard plate test and preincubation test

Evaluation 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 1537, TA 100 and TA 98 this increase should be about twice that of negative controls. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

not specified

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Table 1: Summary of results from the Salmonella mutagenicity assay (plate incorporation test) with Metalink U (mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	15	140	9	19	178
10	16	142	8	20	196
25	15	142	10	20	172
50	16	129	9	24	189
160	14	139	11	16	169
500	12	134	9	18	204
1600	15	128	8	18	180
5000	11	128	7	18	177
Positive control	210	489	71	771	538
Dose (µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	17	143	16	42	257
10	15	142	15	43	254
25	16	148	15	45	251
50	17	140	15	49	259
160	17	146	15	42	261
500	15	155	15	37	251
1600	13	153	16	36	230
5000	13	140	12	43	232
Positive control	140	1087	183	1348	379

Table 2: Summary of results from the Salmonella mutagenicity assay (independent preincubation test) with Metalink U (mean values of revertants per plate)

 

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	16	95	9	20	190
10	15	85	9	19	218
25	15	96	8	18	180
50	14	93	9	17	188
160	13	97	8	19	170
500	13	95	9	20	184
1600	12	114	6	18	181
5000	11	92	7	17	137
Positive control	703	805	87	919	537
Dose (µg per plate)	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
Vehicle control (EGDE)	18	98	18	37	258
10	15	95	16	38	261
25	16	111	18	35	257
50	15	97	17	43	243
160	15	116	17	36	266
500	16	137	17	37	268
1600	14	126	18	36	255
5000	14	113	17	38	240
Positive control	90	988	209	679	540

 

Metalink U was initially investigated for point mutagenic effects using the Salmonella/microsome plate incorporation test. The test item, dissolved in EGDE dried with a molecular sieve, was administered in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Substance precipitation occurred at the dose of 5000 µg per plate.

Metalink U was investigated in an independent repeat using the preincubation modification of the Salmonella/microsome test. Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Substance precipitation occurred at the dose of 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 to the solvent controls, was observed in any of the strains tested, without and with S9 mix, in the plate incorporation as well in the preincubation modification, under the experimental conditions applied.

In both experiments, the positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding solvent controls.

Conclusions:

negative

Executive summary:

The mutagenic potential of Metalink U was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was not seen. No biologically relevant increase in the mutant count, in comparison with the solvent controls, was observed. Based on this test, the test substance 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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar to Apr 2016

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 221305
- Purity: > 98.0 %
- Expiration date of the lot/batch: 2016-06-09

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarital/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Pre-experiment: 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2000 µg/mL (-/+ S9 mix)
Main experiment: 0.56, 1.7, 5, 15, 45, 135 µg/mL (-/+ S9 mix)
The dose range of the main experiment was limited by precipitation of the test item.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: ethylene glycol dimethylether (EGDE), dried with a molecular sieve 0.3nm (test substance); nutrient medium (ethylmethane sulfonate); DMSO (7,12-dimethylbenzanthracene)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethylmethane sulfonate (EMS; -S9 mix); 7,12-dimethylbenzanthracene (DMBA; +S9 mix)

Details on test system and experimental conditions:

- DOSE SELECTION: According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye and microscopically. The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 15.6 μg/mL and 2000 μg/mL were used. The highest concentration was chosen with respect to the current OECD Guideline 476. In the pre-experiment no relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed up to the highest concentration with and without metabolic activation. The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Precipitation occurred at the lowest to the highest concentration after 4 hours treatment with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 3.0 to cover soluble as well as insoluble concentrations. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations. The cultures at the highest concentration with and without metabolic activation were not continued to avoid analysis of too many precipitating concentrations.

- CULTURE MEDIUM: For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

- SEEDING: Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×107 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

- TREATMENT: After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with "saline G". Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×106 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium. Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2. The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below. Three or four days after first sub-cultivation approximately 2.0×106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II). The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range. A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits). The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were 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 and statistical significance was considered together. A t-test was performed to evaluate an increase of the mutation frequency at 1.7 μg/mL in the first culture of the main experiment with metabolic activation. The mutation frequency exceeded the 95% confidence interval at this test point. However, the t-test with a p-value of 0.059 was not significant.

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:

not examined

Positive controls validity:

valid

The main experiment was evaluated at the following concentrations: 0.56, 1.7, 5, 10 and 45 µg/mL (-/+ S9 mix).

Precipitation was observed at 15.0 μg/mL and above at the end of treatment. No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiment up to the maximum concentration. The 95% confidence interval was slightly exceeded at 1.7 μg/mL in the first culture with metabolic activation (30.1 versus an upper limit of 28.7 mutant colonies/10⁶ cells). This isolated increase was judged as irrelevant as it was not reproduced in the parallel culture and there was no dose dependent increase and no significant increase compared to the mutation frequency of the corresponding solvent control as indicated by a lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups. Additionally, a t-test was performed to check the mutation frequency generated at 1.7 μg/mL of the first culture of the main experiment with metabolic activation against the corresponding solvent control. Again, a probability value above 0.05 showed no statistical significance.

In the main experiment with and without S9 mix the range of the solvent controls was from 12.4 up to 21.2 mutants per 10⁶ cells; the range of the groups treated with the test item was from 7.5 up to 30.1 mutants per 10⁶ cells.

EMS (300 μg/mL) and DMBA (2.3 μg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

negative

Executive summary:

Metalink U was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476. The cells were exposed to the test item for 4 hours in concentrations up to and including 135 µg/mL with and without metabolic activation. The test item induced no decreases in survival to treatment or in relative population growth. However, precipitation of the test substance in the culture medium was observed at 15 µg/mL and above. No substantial and reproducible dose dependent increase of the mutation frequency above that of the solvent controls was observed for the test item in the cultures with and without S9 mix.

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

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:

2010

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:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 221305
- Purity: > 98.0 %
- Expiration date of the lot/batch: 2014-07-27

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimal essential medium (MEM) with supplements
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

Experiment 1 (4 h treatment): 1.25, 2.5, 5, 10, 12.5, 15 µg/mL (+/- S9 mix)
Experiment 2 (24 h treatment): 1.25, 2.5, 5, 7.5, 10 µg/mL (- S9 mix)

Vehicle / solvent:

Vehicle(s)/solvent(s) used: ethylene glycol dimethylether (EGDE), dried with a molecular sieve 0.3nm

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

EGDE

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C and vinblastine sulfate (- S9 mix); cyclophosphamide (+ S9 mix)

Details on test system and experimental conditions:

- TREATMENT PROTOCOL: About 5 x 10exp4 cells per concentration were seeded in duplicate in 5 mL medium per Quadriperm-well (contains one slide). Cells were allowed to adhere for ca. 4 hours at 37 °C in a humidified atmosphere with ca. 5 % CO2. In the initial assay the medium was then replaced by medium containing the test substance (and 50 µL/mL S9 mix for the experiment with metabolic activation). After a 4 hour treatment period the cells were washed twice and incubated for further 20 hours in culture medium without the test substance. In the second assay, an experiment without metabolic activation was performed, with an extended treatment period of 24 hours. Thereafter, the adherently growing cells were exposed in situ to 0.4 % KCl hypotonic solution and fixed in glacial acetic acid/ethanol (1+3). The air-dried slides were stained with May-Grünwald and Giemsa solutions.

- NUMBER OF CELLS EVALUATED: For the evaluation of the frequency of micronucleus containing cells which represents the genetic endpoint in this study, 2000 cells (1000 cells per slide) per concentration were scored.

- DETERMINATION OF CYTOTOXICITY: Cytotoxic effects of the test item were assessed using the relative increase in cell count (RICC) as well as the proliferation index (PI) in the presence and absence of S9 mix. The results of the solvent controls were set 100% and compared to the cultures treated with the test item. For determination of the RICC, about 5 x 10exp4 cells per concentration were treated in duplicate in 24-well plates with the test item in the absence (pulse or continuous treatment) or presence (pulse treatment) of S9 mix. Thereafter cells were trypsinized and resuspended in medium in a final volume of 1.5 mL. The cells were counted at the time of harvest using a CASY cell counter. RICC values were calculated from these numbers. For determination of the PI, 2000 cell colonies/cells per concentration (1000 per slide) were scored and the number of cells per colony calculated.

Evaluation criteria:

The evaluation of results was performed as follows:
- The test item is classified as mutagenic if one of the test substance concentrations induces a micronucleus frequency that is three times higher than the micronucleus frequency of the concurrent solvent control.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase in the micronucleus frequency. Such an evaluation may be considered independently of the enhancement factor for induced micronucleus frequencies.
- In the evaluation of the test results historical control data obtained in the laboratory and scientific plausibility is taken into consideration.
- Any positive test result should be evaluated for its biological relevance.

Statistics:

So far no satisfactory mathematical methods are available for statistical analysis of mammalian cell mutagenicity experiments such as the in vitro micronucleus assay. Assessment was performed according to the evaluation criteria.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: only in the independent repeat experiment without S9 mix (24 hours treatment) cytotoxicity was observed in the highest test concentration of 10 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Without S9 mix no relevant cytotoxic effects were observed up to the highest concentration used after 4 hours treatment. In contrast, relevant cytotoxic effects were observed at 10 µg/mL after 24 hours treatment. With S9 mix no relevant cytotoxic effects were observed up to the highest tested concentration. Precipitation in the medium could be observed starting at 10 µg/mL.

Therefore, concentrations of 2.5, 5 and 10 µg/mL (without and with S9 mix, 4 hours treatment) were chosen for reading. In addition, 2.5, 5 and 10 µg/mL were chosen for reading (without S9 mix, 24 hours treatment). Higher concentrations were excluded from evaluation for micronuclei based on precipitation at 10 µg/mL.

Solvent controls and appropriate positive controls with known mutagens (mitomycin C, vinblastine sulfate salt, cyclophosphamide) demonstrated the suitability and sensitivity of the test system.

The micronucleus test showed no biologically relevant increase in the frequencies of micronucleus containing V79 cells treated with the test item in the absence (4 hours or 24 hours treatment) or in the presence (4 hours treatment) of S9 mix.

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

Metalink U was examined for chromosome breakage (clastogenic effects) and misdistribution of chromosomes (aneugenic effects) in the in vitro micronucleus test using Chinese hamster V79 cells according to OECD TG 487. Solvent controls and appropriate positive controls with known mutagens demonstrated the suitability and sensitivity of the test system. The micronucleus test showed no biologically relevant increase in the frequency of micronucleus containing V79 cells in the absence (4 hours or 24 hours treatment) or in the presence of S9 mix (4 hours treatment) when tested up to cytotoxic or precipitating concentrations. Thus, Metalink U did not induce clastogenic or aneugenic effects 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

Additional information

The mutagenic potential of Metalink U was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471 (Jarzombek, 2015). Evidence of mutagenic activity was not seen. No biologically relevant increase in the mutant count, in comparison with the solvent controls, was observed. Based on this test, the test substance 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.

Metalink U was tested in an in vitro gene mutation assay in V79 cells (HPRT) according to OECD TG 476 (Wollny, 2016). The cells were exposed to the test item for 4 hours in concentrations up to and including 135 µg/mL with and without metabolic activation. The test item induced no decreases in survival to treatment or in relative population growth. However, precipitation of the test substance in the culture medium was observed at 15 µg/mL and above. No substantial and reproducible dose dependent increase of the mutation frequency above that of the solvent controls was observed for the test item in the cultures with and without S9 mix. In conclusion it can be stated that under the experimental conditions described, the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Metalink U is considered to be non-mutagenic in the HPRT assay.

Metalink U was examined for chromosome breakage (clastogenic effects) and misdistribution of chromosomes (aneugenic effects) in the in vitro micronucleus test using Chinese hamster V79 cells according to OECD TG 487 (Sutter, 2015). Solvent controls and appropriate positive controls with known mutagens demonstrated the suitability and sensitivity of the test system. The micronucleus test showed no biologically relevant increase in the frequency of micronucleus containing V79 cells in the absence (4 hours or 24 hours treatment) or in the presence of S9 mix (4 hours treatment) when tested up to cytotoxic or precipitating concentrations. Thus, Metalink U did not induce clastogenic or aneugenic effects in the in vitro micronucleus test.

Justification for classification or non-classification

Based on the study results a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.