Oxalonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Mouse lymphoma assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Mouse Lymphoma Assay

Test material

Test material information

Constituent 1

Reference substance name:

Oxalonitrile

EC Number:

207-306-5

EC Name:

Oxalonitrile

Cas Number:

460-19-5

Molecular formula:

C2N2

IUPAC Name:

carbononitridic cyanide

Test material form:

gas

Specific details on test material used for the study:

Lot/Batch number 14/07

Purity in accordance with 5-batch analysis

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

subline 3.7.2c

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rats treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Concentration of test substance
Preliminary toxicity test: 0.01, 0.02, 0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5 and 5% v/v

Mutation tests:
-S9 mix (3 hours) 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4 and 0.5 % v/v
+S9 mix (3 hours) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.75 and 1 % v/v
-S9 mix (24 hours) 0.003, 0.006, 0.0125, 0.025, 0.05, 0.1, 0.15 % v/v

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Way of application
The cultures, containing S9 mix where appropriate, were transferred to 25 cm2 flasks. The vessels were sealed and partially evacuated. Appropriate volumes of the test substance were injected via a valve to produce atmospheres at the required concentrations. The vessels were warmed to 37°C and the contents equilibrated to atmospheric pressure, where necessary, by admitting sterile atmospheric air.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at cytotoxic concentrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results and Discussion
Genotoxicity
Without metabolic activation
Main Mutation Test – 3-hour Treatment in the Absence of S9 Mix
There were increases in the mean mutant frequency of the cells when exposed to Ethanedinitrile at 0.4 and 0.5% v/v that exceeded the sum of the mean concurrent vehicle control mutant frequency plus the Global Evaluation Factor (GEF), within acceptable levels of toxicity.

The increases in mean mutant frequency were predominantly due to increased small colony formation when compared to the concurrent vehicle control, which, according to current opinion, suggests large DNA events such as the loss of whole chromosomes, translocations, transversions and large deletions, but not point mutations.

Main Mutation Test – 24-hour Treatment in the Absence of S9 Mix
Cultures were exposed to Ethanedinitrile at concentrations from 0.003 to 0.1% v/v. No precipitate was observed by eye at the end of treatment. Cultures exposed to Ethanedinitrile at concentrations from 0.0125 to 0.15% v/v were assessed for determination of mutation frequency. RTG values from 116 to 25% were obtained relative to the vehicle control. There were increases in the mean mutant frequency of the cells when exposed to Ethanedinitrile at 0.15% v/v that exceeded the sum of the mean concurrent vehicle control mutant frequency plus the Global Evaluation Factor (GEF), within acceptable levels of toxicity. A concentration response relationship was observed across all concentrations (p<0.001). The increases in mean mutant frequency were predominantly due to increased small colony formation when compared to the concurrent vehicle control, which, according to current opinion, suggests large DNA events such as the loss of whole chromosomes, translocations, transversions and large deletions, but not point mutations.

With metabolic activation
Main Mutation Test – 3-hour Treatment in the Presence of S9 Mix
There were increases in the mean mutant frequency of the cells when exposed to Ethanedinitrile at 1 % v/v that exceeded the sum of the mean concurrent vehicle control mutant frequency plus the Global Evaluation Factor (GEF), within acceptable levels of toxicity.
The increases in mean mutant frequency was predominantly due to increased small colony formation when compared to the concurrent vehicle control, which, according to current opinion, suggests large DNA events such as the loss of whole chromosomes translocations, transversions and large deletions, but not point mutations.
Cytotoxicity
No precipitate (observed by eye at the end of treatment) was observed at any concentrations of Ethanedinitrile tested in the absence and presence of S9 mix, following a 3-hour exposure. Exposure to Ethanedinitrile at concentrations from 0.01 to 5% v/v in the absence and presence of S9 mix resulted in relative suspension growth (RSG) values from 93 to 2% and from 108 to 2% respectively. Following a continuous exposure for 24 hours, no precipitation (assessed by eye at the end of treatment) was observed at any concentrations of Ethanedinitrile tested. Exposure to concentrations from 0.01 to 5% v/v resulted in RSG values from 63 to 0%. Concentrations used in the main test were based upon these data.

Applicant's summary and conclusion

Conclusions:

It was concluded that Ethanedinitrile demonstrated mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described. The maximum concentrations tested were limited by toxicity to 0.5 and 1% v/v, with the RTG being reduced to 22 and 16% for the 3-hour treatments in the absence and presence of S9 mix, respectively.
The increases in mean mutant frequency that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF) were associated with decreases in RTG (measurement of toxicity).

Executive summary:

Materials and Methods                               Test material                                    Ethanedinitrile (Oxalonitrile)   Lot/Batch number                        14/07   Specification                                     Specification in accordance with 5-batch analysis, see certificate of analysis within the study                                       Description                                    Colorless gas           Purity              Purity in accordance with 5 batch analysis, see certificate of analysis within the study   Stability                                          1 year   Study Type                                     Mouse Lymphoma Assay Organism/cell type                        Subline 3.7.2c of mouse lymphoma L5178Y cells               Metabolic activation system     S9 mix from rats treated with phenobarbital and 5,6-benzoflavone   Positive control                             Yes                    Administration/Exposure                 Concentration of test substance                            Preliminary toxicity test: 0.01, 0.02, 0.039, 0.078, 0.156, 0.3125, 0.625, 1.25, 2.5 and 5% v/v Mutation tests: -S9 mix (3 hours) 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4 and 0.5 % v/v  +S9 mix (3 hours) 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.75 and 1 % v/v  -S9 mix (24 hours) 0.003, 0.006, 0.0125, 0.025, 0.05, 0.1, 0.15 % v/v   Way of application                         The cultures, containing S9 mix where appropriate, were transferred to 25 cm^2flasks. The vessels were sealed and partially evacuated. Appropriate volumes of the test substance were injected via a valve to produce atmospheres at the required concentrations. The vessels were warmed to 37°C and the contents equilibrated to atmospheric pressure, where necessary, by admitting sterile atmospheric air.   Results and Discussion   Genotoxicity   Without metabolic activation      Main Mutation Test–3-hour Treatment in the Absence of S9 Mix There were increases in the mean mutant frequency of the cells when exposed to Ethanedinitrile at 0.4 and 0.5% v/v that exceeded  the sum of the mean concurrent vehicle control mutant frequency plus the Global Evaluation Factor (GEF), within acceptable levels of toxicity.   The increases in mean mutant frequency were predominantly due to increased small colony formation when compared to the  concurrent vehicle control, which, according to current opinion, suggests large DNA events such as the loss of whole chromosomes, translocations, transversions and large deletions, but not point mutation.                                                            . Main Mutation Test–24-hour Treatment in the Absence of S9 Mix Cultures were exposed to Ethanedinitrile at concentrations from 0.003 to 0.1% v/v. No precipitate was observed by eye at the end of treatment. Cultures exposed to Ethanedinitrile at concentrations from 0.0125 to 0.15% v/v were assessed for determination of mutation frequency. RTG values from 116 to 25% were obtained relative to the vehicle control. There were increases in the mean mutant frequency of the cells when exposed to Ethanedinitrile at 0.15% v/v that exceeded the sum of the mean concurrent vehiccle control mutant frequency plus the Global Evaluation Factor (GEF), within acceptable levels of toxicity. A concentration response relationship was observed across all concentrations (p<0.001). The increases in mean mutant frequency were predominantly due to increased small colony formation when compared to the concurrent vehicle control, which, according to current opinion, suggests large DNA events such as the loss of whole chromosome translocations, transversions and large deletions, but not point mutations.   With metabolic activation            Main Mutation Test–3-hour Treatment in the Presence of S9 Mix There were increases in the mean mutant frequency of the cells when exposed to Ethanedinitrile at 1 % v/v that exceeded the sum of  the mean concurrent vehicle control mutant frequency plus the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The increases in mean mutant frequency was predominantly due to increased small colony formation when compared to the concurrent vehicle control, which, according to current opinion, suggests large DNA events such as the loss of whole chromosomes  translocations, transversions and large deletions, but not point mutations. Cytotoxicity                                     No precipitate (observed by eye at the end of treatment) was observed at any concentrations of Ethanedinitrile tested in the absence and presence of S9 mix, following a 3-hour exposure. Exposure to Ethanedinitrile at concentrations from 0.01 to 5% v/v in the absence and presence of S9 mix resulted in relative suspension growth (RSG) values from 93 to 2% and from 108 to 2% respectively. Following a continuous exposure for 24 hours, no precipitation (assessed by eye at the end of treatment) was observed at any concentrations of Ethanedinitrile tested. Exposure to concentrations from 0.01 to 5% v/v resulted in RSG values from 63 to 0%. Concentrations used in the main test were based upon these data. Summary and conclusion   Materials and methods Ethanedinitrile was tested for mutagenic potential in an in vitro mammalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-). The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix. Concentrations of Ethanedinitrile up to 5% v/v were tested. Ethanedinitrile was not tested at greater concentrations than this due to a known explosion hazard. Sterile air was used as a vehicle control. Results and discussion Toxicity was observed in the preliminary toxicity test. Following a 3-hour exposure to ethanedinitrile at concentrations from 0.01 to 5% v/v, relative suspension growth (RSG) was reduced from 93 to 2% and from 108 to2% in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 63 to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG).   Following 3-hour treatment in the absence of S9 mix, there were increases in mean mutant frequency that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF) at concentrations of 0.4 and 0.5% v/v, with the RTG being reduced to 45 and 16% respectively. A concentration response relationship was observed across all concentrations (p<0.001). Following 3-hour treatment in the presence of S9 mix, there was an increase in mean mutant frequency that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF) at a concentration of 1% v/v, with the RTG being reduced to 22%. A concentration response relationship was observed across all concentrations (p<0.001). Following 24-hour treatment in the absence of S9 mix, there was an increase in mean mutant frequency that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF) at a concentration of 0.15% v/v, with the RTG being reduced to 25%. A concentration response relationship was observed across all concentrations (p<0.001). Conclusion It was concluded that Ethanedinitrile demonstrated mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described. The maximum concentrations tested were limited by toxicity to 0.5 and 1% v/v, with the RTG being reduced to 22 and 16% for the 3-hour treatments in the absence and presence of S9 mix, respectively. The increases in mean mutant frequency that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF) were associated with decreases in RTG (measurement of toxicity).