Reaction mass of 2-ethylhexyl (6-isocyanatohexyl)-carbamate and bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in 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:

July-Aug 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Stability under test conditions: A stability test in the formulation at 0.1 and 100 mg/mL revealed no significant degradation of the test item up to at least 4 hours (A 01/0207/02 LEV).

Target gene:

Histidine-deficient mutant  strains of Salmonella typhimurium LT2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from the liver of Aroclor 1254 induced rats

Test concentrations with justification for top dose:

Pre-test for cytotoxicity: doses up to 5000 µg/plate (recommended maximum test concentration)
Initial test (plate incorporation): due to bacteriotoxic effects doses up to 128 µg/plate were used (4, 8, 16, 32, 64, and 128 µg/mL)
Independent repeat (pre-incubation method): doses up to 64 µg/tube were used (1, 2, 4, 8, 16, 32, and 64 µg/mL)

Vehicle / solvent:

Ethylene glycol dimethylether (EGDE; dried with molecular sieve)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

EGDE

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other:

Remarks:

Na-azide, NF, 4-NPDA, MMC, and Cumene are only used without S9 mix, 2-AA is only used with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A pre-test for cytotoxicity and the initial mutagenicity test were performed as plate incorporation test, the independent repeat as pre-incubation modification (Pre-incubation time 20 minutes at 37 °C). Each strain and concentration was tested in triplicate.

DETERMINATION OF CYTOTOXICITY:
1. gross appraisal of background growth, 2. mutant count per plate

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100 and TA98 this increase should be about twice that of negative controls, whereas for TA1537 at least a threefold increase should be reached. For TA102 an increase of about 100 mutants should be reached.

Species / strain:

S. typhimurium, other: TA1535, TA100, TA1537, TA98, and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Evidence of mutagenic activity of the test substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Doses up to and including 4 µg per plate did not cause any bacteriotoxic  effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 128 µg per plate for assessment purposes.

Executive summary:

A bacterial reverse mutation test (Ames) equivalent to OECD TG 471 was conducted for the evaluation of point mutagenic effects. In this assay five histidine-deficient mutant strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537, TA 98, and TA 102) were used, with and without a metabolic activating system. The study was performed as initial plate incorporation with the pre-incubation modification as independent repeat. Doses ranged from 1 to 5000 μg/plate.

Doses up to and including 4 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 128 µg/plate for assessment purposes.

Evidence of mutagenic activity of the test substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls, and by thus showed the sensitivity of the test system and the activity of the metabolic activating system.

Therefore, the test substance was considered to be non-mutagenic without or with a metabolic activating system in bacteria.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June-Aug 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)

Version / remarks:

(2015)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Stability under test conditions: A stability test in the formulation at 0.1 and 100 mg/mL revealed no significant degradation of the test item up to at least 4 hours (A 01/0207/02 LEV).

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM containing Hank's salts supplemented with 10 % FBS, neomycin (5 µg/mL) and amphotericin B (1 %); for the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
- 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:

S9-Mix from the liver of phenobarbital/beta-naphthoflavone induced rats.

Test concentrations with justification for top dose:

Pre-test for cytotoxicity: doses up to 2 mg/mL were used (recommended maximum test concentration)
Main experiment (4 hour-exposure time, two cultures): without S9-mix 0.28, 0.56, 1.1, 2.2, 4.4, 6.6, 8.8, and 17.5 µg/mL, with S9-mix 8.8, 17.5, 35, 70, 140, and 280 µg/mL

The following concentrations were selected for reading: without S9-mix 0.56, 1.1, 2.2, 4.4, and 6.6 µg/mL, with S9-mix 8.8, 17.5, 35, 70, and 140 µg/mL

Vehicle / solvent:

EGDE (Ethylene glycol dimethylether)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

EGDE

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE). The pre-experiment was performed in the presence and absence (4 h treatment) of metabolic activation. Test item concentrations between 0.1 μg/mL and 2000 μg/mL (adjusted to the substance's purity) were used.

Treatment Protocol for main test:
Approximately 0.7 to 1.2×10exp7 were seeded in plastic flasks. The cells were grown for 24 hours prior to 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 and sub-cultivated. At least 2.0×10exp6 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. Three or four days after first sub-cultivation approximately 2.0×10exp6 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 5×10exp5 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.

DETERMINATION OF CYTOTOXICITY
Toxicity of the test item is indicated by a reduction of the cloning efficiency.

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 analysis (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 using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95 % confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation/phase separation: 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. Phase separation occurred after 4 hours treatment at 138.9 μg/mL and above with metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the pre-experiment relevant cytotoxic effects, indicated by a relative cloning efficiency of 50 % or below, were observed at 4.5 μg/mL and above without metabolic activation and at 1111.0 μg/mL and above with metabolic activation.

Executive summary:

A study according to OECD TG 476 was conducted in order to investigate the potential of the substance to induce gene mutations at the HPRT locus in mammalian cells. The treatment period was 4 hours with and without metabolic activation. The maximum concentration of the pre-test on toxicity was approximately 2000 μg/mL as requested by the guideline. The concentration range of the main experiment was limited by phase separation of the test item, leading to chosen concentrations of 0.28 to 17.5 µg/mL without and 8.8 to 280 µg/mL with metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions the test item did not induce gene mutations in mammalian cells.

 

 

 

 

 

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July-Oct 2016

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:

(2014)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- Stability under test conditions: A stability test in the formulation at 0.1 and 100 mg/mL revealed no significant degradation of the test item up to at least 4 hours (A 01/0207/02 LEV).

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) FBS.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from the liver of phenobarbital/beta-naphthoflavone induced rats.

Test concentrations with justification for top dose:

Pre-test for cytotoxicity: doses up to 2 mg/mL were used (recommended maximum test concentration)
Exp. I (pulse exposure, 4 hour-exposure time, two cultures): with and without S9 mix 0.4, 0.8, 1.5, 3.1, 6.2, 12.3, 24.7, 74.1, 222, 667, and 2000 µg/mL
Exp. II (continuous exposure, 24 hour (continuous)-exposure time, two cultures): without S9 mix 6.0, 10.4, 18.3, 32.0, 56.0, 98.0, 171, and 300 µg/mL

The following concentrations were selected for reading: pulse exposure, without and with S9 mix 24.7, 74.1, and 222 µg/mL; continuous exposure, without S9 mix 56.0, 98.0, and 171 µg/mL.

Vehicle / solvent:

EGDE (Ethylene glycol dimethylether)

Untreated negative controls:

no

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

EGDE

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Griseofulvin

Details on test system and experimental conditions:

A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the Cytokinesis-block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture.

Treatment Protocol for main test:
1) Pulse exposure (4 hours)
The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2). The cells will then be cultured in complete medium containing 10 % (v/v) FBS in the presence of Cytochalasin B (1.5 μg/mL) for the remaining culture time of 20 hours.
2) Continuous exposure (without S9 mix, 24 hours)
The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item. At the same time Cytochalasin B was added to the cell culture (1.5 μg/mL). The medium was not changed until preparation of the cells.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity is characterized by the percentages of reduction in the CBPI (Cytokinesis-block proliferation index) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate.

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95 % confidence interval).
A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition.
− The results are outside the range of the laboratory historical solvent control data (95 % confidence interval).

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no, but tested up to concentrations at which phase-separation occurred.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the absence and presence of S9 mix, no relevant increases in the number of micronucleate cells were observed after treatment with the test item. However, in Exp. I in the absence of S9 mix after treatment with 74.1 μg/mL (conc. based on test material) one statistically significant increase in micronucleate cells was observed (1.10 %). Since the value is in the range of the historical solvent control data (0.0 – 2.62 % micronucleate cells), the finding can be regarded as biologically irrelevant.
Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH or osmolality: No relevant influence on osmolarity or pH was observed.
- Precipitation/phase separation: No precipitation of the test item in the culture medium was observed. Phase separation was observed in Exp. I at 222 μg/mL and above in the absence and presence of S9 mix and in Exp. II at 171 μg/mL in the absence of S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.

Executive summary:

The substance was assessed for its potential to induce micronuclei in human lymphocytes in vitro in accordance to OECD TG 487. In two independent experiments the cells were exposed for either 4 hours (pulse exposure) with or without S9 mix and 20 hours (continuous exposure) without S9 mix. The highest treatment concentration in this study, 2000 μg/mL was chosen with respect to the guideline.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.

In the absence and presence of S9 mix, no relevant increases in the number of micronucleate cells were observed after treatment with the test item. However, in Experiment I in the absence of S9 mix after treatment with 74.1 μg/mL one statistically significant increase in micronucleate cells was observed (1.10 %). Since the value is in the range of the historical solvent control data (0.0 – 2.62 % micronucleate cells), the finding was regarded as biologically irrelevant.

In conclusion, the test item was considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest evaluable concentrations.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A bacterial reverse mutation test (Ames) equivalent to OECD TG 471 was conducted for the evaluation of point mutagenic effects. In this assay five histidine-deficient mutant strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537, TA 98, and TA 102) were used, with and without a metabolic activating system. The study was performed as initial plate incorporation with the pre-incubation modification as independent repeat. Doses ranged from 1 to 5000 μg/plate.

Doses up to and including 4 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 128 µg/plate for assessment purposes.

Evidence of mutagenic activity of the test substance was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Therefore, the test substance was considered to be non-mutagenic without or with a metabolic activating system in bacteria.

A study according to OECD TG 476 was conducted in order to investigate the potential of the substance to induce gene mutations at the HPRT locus in mammalian cells. The treatment period was 4 hours with and without metabolic activation. The maximum concentration of the pre-test on toxicity was approximately 2000 μg/mL as requested by the guideline. The concentration range of the main experiment was limited by phase separation of the test item, leading to concentrations of 0.28 to 17.5 µg/mL without and 8.8 to 280 µg/mL with metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions the test item did not induce gene mutations in mammalian cells.

The substance was also assessed for its potential to induce micronuclei in human lymphocytes in vitro in accordance to OECD TG 487. In two independent experiments the cells were exposed either for 4 hours (pulse exposure) with or without S9 mix, or for 20 hours (continuous exposure) without S9 mix. The highest treatment concentration in this study, 2000 μg/mL was chosen with respect to the guideline.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.

In the absence and presence of S9 mix, no relevant increases in the number of micronucleate cells were observed after treatment with the test item. However, in Experiment I in the absence of S9 mix after treatment with 74.1 μg/mL one statistically significant increase in micronucleate cells was observed (1.10 %). Since the value is in the range of the historical solvent control data (0.0 – 2.62 % micronucleate cells), the finding can be regarded as biologically irrelevant.

In conclusion, the test item was considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest evaluable concentrations.

 

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008, Annex I, classification for genetic toxicity is not justified.