Oxalonitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ethanedinitrile was shown to be negative in the Bacterial Reverse Mutation Assay (AMES test) in all bacterial strains (Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and Escherichia coli, strain WP2 uvrA (pKM101). It was concluded that ethanedinitrile showed no evidence of mutagenic activity in this bacterial system under the test conditions employed (Envigo 2016a). This is strongly indicating that the ethanedinitrile and EDN do not have potential to cause point (gene) mutation and interact directly with the DNA in this way. This conclusion is supported by the Mouse Lymphoma Assay since there was an absence of big colonies which would be a consequence of point mutations in this assay. However, Mouse lymphoma assay has indicated presence of structural events such as chromosome aberrations in concentrations accompanied by cytotoxicity (Envigo 2016b). Further there was a dose dependent increase of such events although this was accompanied also with matching dose dependent increase in cytotoxicity. However, the Mouse Lymphoma Assay was often reported as being prone to false positive results (Caldwell 1993). Chromosome aberration assay indicated a presence of chromosomal breaks in presence of cytotoxicity (Envigo 2016c). Cytotoxicity was measured by mitotic index which is only indirect measurement of cytotoxicity very dependent e.g. on the time of the measurement. Range finding cytotoxicity test revealed much higher toxicity (which would disqualify any genotoxicity findings) in the same concentrations as were scored less toxic during the genotoxicity test itself. The highest positive findings were on the edge of historical controls. Chromosome aberration test was found false positive in the assessment of other metabolic poisons than ethanedinitrile, as it is indicated in the report.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

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

Specific details on test material used for the study:

Lot/Batch number 14/07

Purity in accordance with 5-batch analysis

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

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.

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.

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).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Chromosome aberration test in human lymphocytes in vitro

Specific details on test material used for the study:

Lot/Batch number 14/07
Specification in accordance with 5-batch analysis

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Culture of lymphocytes from human blood collected from the healthy non-smoking adult donors

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
Main toxicity tests:
-S9 mix (3h): 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 and 0.4% v/v
+S9 mix (3h): 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 and 0.4% v/v
-S9 mix (21h)
Test N.1: 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5% v/v
Test N.2: 0.00015, 0.0005, 0.001, 0.002, 0.005, 0.01 and 0.02% v/v
Test N.3: 0.00000005, 0.00000015, 0.0000005, 0.0000015, 0.000005, 0.000015, 0.00005, 0.00015, 0.0005% v/v
Mutation test (metaphase analysis)
-S9 mix (3 hours): 0.2, 0.3, 0.4% v/v
+S9 mix (3 hours): 0.15, 0.25, 0.4% v/v
No mutation tests were performed for 21 hours exposure time.

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.
Pre-incubation time 48h

Key result

Species / strain:

lymphocytes: Human blood collected from two healthy, non-smoking, adult (between 18-35 years of age) donors.

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at cytotoxic concentrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Without metabolic activation

Main CA Test - 3-hour Treatment in the Absence of S9 Mix

Ethanedinitrile caused statistically significant increases at 0.2 and 0.3% v/v (p<0.05 excluding gaps only) and at 0.4% v/v (p<0.001 excluding gaps and p<0.01 including gaps) in the proportion of cells with chromosomal aberrations when compared with the vehicle control. The mean values at 0.4% v/v were outside of the laboratory historical range when taken at the 95% confidence limit.

Polyploidy and endoreduplication analysis 3-hour Treatment in the Absence of S9 Mix
A statistically significant increase in the proportion of polyploid metaphase cells was observed at 0.4% v/v (p<0.001).

With metabolic activation
Main CA Test - 3-hour Treatment in the presence of S9 Mix
Ethanedinitrile caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 0.4% v/v (p<0.001 excluding and including gaps) when compared with the vehicle control. The mean values were outside of the laboratory historical control range when taken at the 95% confidence limit.

Polyploidy and endoreduplication analysis 3-hour Treatment in the presence of S9 Mix
A statistically significant increase in the proportion of polyploid metaphase cells was observed at 0.4% v/v (p<0.001).

Cytotoxicity

Cytotoxicity test – 3-hour Treatment in the absence of S9 Mix
Ethanedinitrile caused a reduction in the mitotic index to 49% (cytotoxicity of 51%) of the vehicle control value at 0.4% v/v. The concentrations selected for metaphase analysis were 0.2, 0.3 and 0.4% v/v.

Cytotoxicity test – 3-hour Treatment in the presence of S9 Mix
Ethanedinitrile caused a reduction in the mitotic index to 49% (cytotoxicity of 51%) of the vehicle control value at 0.4% v/v. The concentrations selected for metaphase analysis were 0.15, 0.25 and 0.4% v/v.

Cytotoxicity test – 21-hour Treatment in the absence of S9 Mix
To investigate a possible no observed effect level following a 21-hour treatment with Oxalonitrile, testing was performed on three occasions. Treatment of cultures for 21 hours at concentrations of 0.00000005% v/v and above resulted in overt toxicity. Therefore it was deemed to be impractical to accurately achieve concentrations which would obtain suitable cytotoxicity response for chromosome aberration analysis

Conclusions:

It is concluded that the test substance Ethanedinitrile has shown evidence of causing an increase in the frequency of structural chromosome aberrations and numerical aberrations after 3-hour treatment in both the absence and presence of S9 mix, in this in vitro cytogenetic test system, under the experimental conditions described. The increases in structural and numerical aberrations was seen where a reduction in mitotic index to 49% (cytotoxicity of 51%) was observed. It should be noted that the chromosome aberration test is not specifically designed to measure numerical aberrations and is not routinely used for this purpose. However, the assay can give some indications of the potential of a test item to induce such aberrations.

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 and  certificate of analysis within the study

 

Stability                                         

1 year

 

Study Type 

Chromosome aberration test in human lymphocytesin vitro

 

Organism/cell type                       

Culture of lymphocytes from human blood collected from the healthy non-smoking adult donors

               

Metabolic activation system    

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

 

Positive control                            

Mitomycin C 0.2µg/ml                 

 

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

Main toxicity tests:

-S9 mix (3h): 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 and 0.4% v/v

+S9 mix (3h): 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 and 0.4% v/v

-S9 mix (21h)

Test N.1: 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5% v/v

Test N.2: 0.00015, 0.0005, 0.001, 0.002, 0.005, 0.01 and 0.02% v/v

Test N.3: 0.00000005, 0.00000015, 0.0000005, 0.0000015, 0.000005, 0.000015, 0.00005, 0.00015, 0.0005% v/v

 

Mutation test (metaphase analysis)

-S9 mix (3 hours): 0.2, 0.3, 0.4% v/v

+S9 mix (3 hours): 0.15, 0.25, 0.4% v/v

No mutation tests were performed for 21 hours exposure time.

 

Way of application       

The cultures, containing S9 mix where appropriate, were transferred to 25 cm²flasks. 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.

Pre-incubation time                    

48h

 

Results and Discussion

 

Genotoxicity

 

Without metabolic activation     

Main CA Test - 3-hour Treatment in the Absence of S9 Mix

Ethanedinitrile caused statistically significant increases at 0.2 and 0.3% v/v (p<0.05 excluding gaps only) and at 0.4% v/v (p<0.001 excluding gaps andp<0.01 including gaps) in the proportion of cells with chromosomal aberrations when compared with the vehicle control. The mean values at 0.4% v/v were outside of the laboratory historical range when taken at the 95% confidence limit.

Polyploidy and endoreduplication analysis 3-hour Treatment in the Absence of S9 Mix

A statistically significant increase in the proportion of polyploid metaphase cells was observed at 0.4% v/v (p<0.001).

 

With metabolic activation            

Main CA Test - 3-hour Treatment in the presence of S9 Mix

Ethanedinitrile caused a statistically significant increase in the proportion of cells with chromosomal aberrations at 0.4% v/v 
(p<0.001 excluding and including gaps) when compared with the vehicle control. The mean values were outside of the laboratory
historical control range when taken at the 95% confidence limit.

 

Polyploidy and endoreduplication analysis

3-hour Treatment in the presence of S9 Mix

A statistically significant increase in the proportion of polyploid metaphase cells was observed at 0.4% v/v (p<0.001).

Cytotoxicity                                    

Cytotoxicity test – 3-hour Treatment in the absence of S9 Mix

Ethanedinitrile caused a reduction in the mitotic index to 49% (cytotoxicity of 51%) of the vehicle control value at 0.4% v/v. The
concentrations selected for metaphase analysis were 0.2, 0.3 and 0.4% v/v.

Cytotoxicity test – 3-hour Treatment in the presence of S9 Mix

Ethanedinitrile caused a reduction in the mitotic index to 49% (cytotoxicity of 51%) of the vehicle control value at 0.4% v/v. The 
concentrations selected for metaphase analysis were 0.15, 0.25 and 0.4% v/v.

Cytotoxicity test – 21-hour Treatment in the absence of S9 Mix

To investigate a possible no observed effect level following a 21-hour treatment with Oxalonitrile, testing was performed on three occasions. Treatment of cultures for 21 hours at concentrations of 0.00000005% v/v and above resulted in overt toxicity. Therefore it was deemed to be impractical to accurately achieve concentrations which would obtain suitable cytotoxicity response for chromosome aberration analysis.

Summary and conclusion

 

Materials and methods

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test substance both in the absence and presence of exogenous metabolic activation (S9 mix). Vehicle and positive control cultures were also included where appropriate. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. Ethanedinitrile up to 5% v/v were tested. Ethanedinitrile was not tested at higher concentrations as they may result in an explosion hazard. Sterile air was used as the control.

The study consisted of a preliminary toxicity test and a main test. In both types of tests, the cells were treated for 3 and 21 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix. The mitotic index was assessed for all cultures to determine cytotoxicity. Based on the data from the preliminary toxicity test, test substance concentrations were selected for the main test.

 

In the main test, justification for the highest analyzed concentration was based on cytotoxicity.

 

The following test substance concentrations were selected for metaphase analysis:

 

In the absence of S9 mix, 3-hour treatment: 0.2, 0.3 and 0.4% v/v

In the presence of S9 mix, 3-hour treatment: 0.15, 0.25 and 0.4% v/v

 

Results and discussion

In the absence of S9 mix following 3-hour treatment, Ethanedinitrile caused statistically significant increases at 0.2 and 0.3% v/v (p<0.05 excluding gaps only) and at 0.4% v/v (p<0.001 excluding gaps and p<0.01 including gaps) in the proportion of metaphase figures containing chromosomal aberrations, when compared with the vehicle control. The mean values at 0.4% v/v were outside of the laboratory historical range for the vehicle control when taken at the 95% confidence limit. A reduction in the mitotic index to 49% (cytotoxicity of 51%) of the vehicle control value was seen at 0.4% v/v. The mean value at 0.2 and 0.3% v/v were within the laboratory historical range for vehicle controls when taken at the 95% confidence limit. The mean number of cells with aberrations, excluding gaps, in the concurrent vehicle control were 0% which is observed rarely (estimated to be only 5%).

 

In the presence of S9 mix following 3-hour treatment, Ethanedinitrile caused a statistically significant increase at 0.4% v/v (p<0.001 excluding and including gaps) in the proportion of metaphase figures containing chromosomal aberrations, when compared with the vehicle control. The mean values at 0.4% v/v were outside of the laboratory historical range for the vehicle control when taken at the 95% confidence limit. A reduction in the mitotic index to49% (cytotoxicity of 51%) of the vehicle control value was seen at 0.4% v/v.

 

A statistically significant increase in the proportion of polyploid metaphase cells was observed during metaphase analysis after 3-hour treatment in the absence of S9 mix (p<0.01) and in the presence of S9 mix (p<0.001) at 0.4% v/v when compared to the vehicle control.

The mitotic index is an indirect measure of cytotoxic/cytostatic effects (Galloway 2000), it is influenced by the time after treatment it is measured, the mitogen used and possible cell cycle disruption. However, the mitotic index is acceptable because other cytotoxicity measurements may be cumbersome and impractical and may not apply to the target population of lymphocytes growing in response to PHA stimulation. In the main test, exposure to Ethanedinitrile was seen to exhibit a steep dose-response curve in terms of toxicity. A steep dose response for some compounds is a factor that distinguishes them from truly DNA damaging agents that are active over a broad range (Galloway 2000).

Galloway (2000) also states that other information should be used to distinguish relevant from non-relevant positive response. These include evidence that the test item does not damage DNA directly, the shape of the dose relation for aberrations and toxicity, evidence for a secondary mechanism or the lack of genotoxicity in vivo along with evidence of exposure of the animals and with a reasonable safety margin over human exposure.

The increase in cells containing aberrations, excluding gaps, which was outside of the historical range of the vehicle control following exposure to Ethanedinitrile at 0.4% (v/v) was accompanied with a relative mitotic index of 49%. However, the OECD guideline 473 states that to avoid false positive results concentrations at the higher end of this 55 ± 5% cytotoxicity range should be interpreted with caution. When increases in aberrations are seen only at the higher end of the cytoxicity range the genotoxicity may be caused by an indirect mechanism related to cytotoxicity rather than direct genotoxicity caused by test item under investigation.

Ethanedinitrile is metabolic inhibitor with an identical mode of action as cyanide. Cyanide has been previously shown to be a metabolic inhibitor (Carlsen et al 1976, Carlsen at al 1977). Hilliard et al. (1998) discusses that several metabolic poisons (inhibitors) show false-positive resuts which are usually associated with cytotoxicity within the chromosome aberration test.

Although the conclusion of the study is that Ethanedinitrile has shown evidence of causing an increase in the frequency of both structural and numerical chromosome aberrations, this may be a non-direct effect on DNA.

 

Conclusion

It is concluded that the test substance Ethanedinitrile has shown evidence of causing an increase in the frequency of structural chromosome aberrations and numerical aberrations after 3-hour treatment in both the absence and presence of S9 mix, in this in vitro cytogenetic test system, under the experimental conditions described. The increases in structural and numerical aberrations was seen where a reduction in mitotic index to 49% (cytotoxicity of 51%) was observed. It should be noted that the chromosome aberration test is not specifically designed to measure numerical aberrations and is not routinely used for this purpose. However, the assay can give some indications of the potential of a test item to induce such aberrations.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch number 14/07
Purity in accordance with 5-batch analysis

Target gene:

Salmonella typhimurium strains TA100, TA1535, TA1537, and TA98 and E.coli strain WP2 uvrA (pKM101)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

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:

The five tester strains were exposed to the test substance in stainless steel vessels at seven concentrations: 0.005, 0.015, 0.05, 0.15, 0.5, 1.5 and 5% v/v (nominal).

Vehicle / solvent:

Untreated controls were treated under identical conditions to the plates exposed to the test substance. Appropriate plate-incorporated positive control compounds were also included.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

Administration/Exposure

Concentration of test substance
Concentrations of Ethanedinitrile up to 5 % v/v were tested. The vehicle controls were exposed to sterile air only.

Way of application
The seeded plates were placed in stainless steel vessels. The vessels were sealed and partially evacuated. Appropriate volumes of the test substance were injected via a valve. The vessels were warmed to 37°C and the contents equilibrated to atmospheric pressure, where necessary, by admitting sterile atmospheric air. The plates were incubated for ca 48 hours in the vessels at 37°C and then removed from the vessels under air extraction. The plates were incubated for a further period of ca 24 hours at 37°C to permit the growth of revertant colonies.

Examinations
After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer)

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

not applicable

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:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results:
Genotoxicity
Without metabolic activation Non-genotoxic
With metabolic activation Non-genotoxic
Cytotoxicity The maximum non-cytotoxic concentration tested was 0.05% v/v

Conclusions:

It was concluded that ethanedinitrile showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

Materials and Methods                               Test material                                Ethanedinitrile (Oxalonitrile)   Lot/Batch number                        14/07   Specification                                Specification in accordance with 5-batch analysis, see document J and                                                    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                                    Bacterial reverse mutation test Organism/cell type                       Salmonella typhimuriumstrains TA100, TA1535, TA1537, and TA98                                                     and E.colistrain WP2uvrA (pKM101)                 Metabolic activation system     S9 mix from rats treated with phenobarbital and 5,6-benzoflavone   Positive control                             Yes                    Administration/Exposure                 Concentration of test substance                          Concentrations of Ethanedinitrile up to 5 % v/v were tested. The vehicle                                                    controls were exposed to sterile air only.   Way of application                        The seeded plates were placed in stainless steel vessels. The                                                   vessels were sealed and partially evacuated. Appropriate volumes of                                                          the test substance were injected via a valve. The vessels were                                                    warmed to 37°C and the contents equilibrated to atmospheric                                                    pressure, where necessary, by admitting sterile atmospheric air. The                                                    plates were incubated forca48 hours in the vessels at 37°C and then                                                    removed from the vessels under air extraction. The plates were                                                    incubated for a further period ofca24 hours at 37°C to permit the                                                     growth of revertant colonies.   Examinations                                After this period, the appearance of the background bacterial lawn                                                    was examined and revertant colonies counted using an automated                                                     colony counter (Perceptive InstrumentsSorcerer) Results and Discussion   Genotoxicity   Without metabolic activation     Non-genotoxic               With metabolic activation          Non-genotoxic Cytotoxicity                             The maximum non-cytotoxic concentration tested was 0.05% v/v Summary and conclusion   Materials and methods   The five tester strains were exposed to the test substance in stainless steel vessels at seven concentrations: 0.005, 0.015, 0.05, 0.15, 0.5, 1.5 and 5% v/v (nominal). Untreated controls were treated under identical conditions to the plates exposed to the test substance. Appropriate plate-incorporated positive control compounds were also included. Separate tests were conducted in the absence and presence of S9 mix. S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 ml) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 ml minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. The seeded plates were placed in stainless steel vessels. The vessels were sealed and partially evacuated. Appropriate volumes of the test substance were injected via a valve. The vessels were warmed to 37°C and the contents equilibrated to atmospheric pressure, where necessary, by admitting sterile atmospheric air. The plates were incubated forca48 hours in the vessels at 37°C and then removed from the vessels under air extraction. The plates were incubated for a further period ofca24 hours at 37°C to permit the growth of revertant colonies. Further sets of plates were prepared for the liquid positive control compounds. Aliquots of 0.1 mL of the positive control solutions were added to the plates together with the bacteria, buffer or S9 mix and agar overlay. These plates were incubated at 37°C for 48–72 hours (not in stainless steel vessels). After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive InstrumentsSorcerer). An additional first test was performed due to severe toxicity seen in response to exposure to Ethanedinitrile. The maximum concentration tested was 0.05% v/v. Results and discussion First Test Toxicity, observed as a thin or absent background lawn of non-revertant colonies and/or reduction in the number of revertant colonies, was obtained in all strains following exposure to ethanedinitrile at concentrations of 0.015% v/v and greater. As an insufficient number of non-toxic concentrations were available to analyze, an additional test was performed. A maximum exposure concentration of 0.05% v/v was, therefore, selected for use in the additional first test.   Additional First Test Toxicity, observed as a thin or absent background lawn of non-revertant colonies and/or reduction in the number of revertant colonies, was obtained in all strains following exposure to ethanedinitrile at concentrations of 0.015% v/v and greater (except for strain TA98 in the presence of S9 mix where toxicity was seen at 0.005% v/v and above). A maximum exposure concentration of 0.05% v/v was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Ethanedinitrile at any concentration up to and including 0.05% v/v in either the presence or absence of S9 mix.   Second Test Toxicity, observed as a thin or absent background lawn of non-revertant colonies and/or reduction in the number of revertant colonies, was obtained in all strains following exposure to ethanedinitrile at concentrations of 0.015% v/v and greater. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to ethanedinitrile at any concentration up to and including 0.05% v/v in either the presence or absence of S9 mix. Conclusion

It was concluded that ethanedinitrile showed no evidence of mutagenic activity in this bacterial system under the test conditions employed

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Key study with rats:

 

No statistically significant increases in the frequency of chromosomal aberrations or changes in mitotic index compared with control values were found in bone marrow cells from four groups of 24 male and 24 female Sprague-Dawley rats administered a single dose of acetone cyanhydrin by oral gavage at levels of 0, 1.5, 5, or 15 mg/kg body weight with preparation intervals of 6, 12, and 24h post-administration (Monsanto Co. 1983b). This test is in full compliance with current mutagenicity test guidelines and is considered to be the key study for this endpoint.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study precedes the establishment of, but is similar to, OECD Guideline protocol for the in vivo Micronucleus assay.

Justification for type of information:

Ethanedinitrile, hydrogen cyanide (HCN), potassium cyanide and sodium cyanide can be considered as a chemical category, along with and acetone cyanohydrin (ACH, also known as 2-hydroxy-2-methylpropanenitrile), based on structural similarity, common breakdown/metabolic products in physical and biological systems, and similar physico-chemical properties. Particular attention is paid to the dissociation constant of HCN. Ethanedinitrile breaks down in aqueous solution into cyanide ion (CN-) and cyanate ion (OCN-) (Cotton and Wilkinson 1980). Ethanedinitrile due to its low log Kow (0.07) and relatively high solubility in water (2.34 g/L) needs to get dissolved in aqueous solutions in lungs to enter the body. The rate of hydrolysis of ethanedinitrile is very fast (Ajwa 2015). Also, in the vast majority of environmental and physiologic conditions, the cyanide salts will dissolve in water to form hydrogen cyanide. The physico-chemical hazards and toxicity therefore result from the activity of HCN. An ECETOC Task Force, in the 2007 ECETOC Joint Assessment of Commodity Chemicals (JACC) Report No. 53, “Cyanides of Hydrogen, Sodium and Potassium, and Acetone Cyanohydrin (CAS No. 74-90-8, 143-33-9, 151-50-8 and 75-86-5)” supports the development of the chemical category inclusive hydrogen cyanide, sodium and potassium cyanides. Hydrogen cyanide (Index No.006-006-00-X) and salts of hydrogen cyanides (Index No.006-007-00-5) are both listed in Annex VI, Table 3.1 of Regulation (EC) No. 1272/2008, entry 006-007-00-5, and are restricted in comparable ways taking into account physical characteristics. Thus, the assignment of ethanedinitrile to a chemical category does not result in a less protective regulatory status.

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Details on exposure:

approximates the LD 50 of 17 mg/kg bw, from previous studies with rats.

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

6, 12, and 24h post-administration

Dose / conc.:

1.5 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24 male
24 female

Control animals:

yes

Tissues and cell types examined:

bone marrow cells

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Additional information on results:

No statistically significant increases in the frequency of chromosomal aberrations or changes in mitotic index compared with control values were found in bone marrow cells from four groups of 24 male and 24 female Sprague-Dawley rats administered a single dose of acetone cyanhydrin by oral gavage at levels of 0, 1.5, 5, or 15 mg/kg body weight with preparation intervals of 6, 12, and 24h post-administration.

Conclusions:

No statistically significant increases in the frequency of chromosomal aberrations or changes in mitotic index compared with control values were found in bone marrow cells from four groups of 24 male and 24 female Sprague-Dawley rats administered a single dose of acetone cyanhydrin by oral gavage at levels of 0, 1.5, 5, or 15 mg/kg body weight with preparation intervals of 6, 12, and 24h post-administration.

Executive summary:

No statistically significant increases in the frequency of chromosomal aberrations or changes in mitotic index compared with control values were found in bone marrow cells from four groups of 24 male and 24 female Sprague-Dawley rats administered a single dose of acetone cyanhydrin by oral gavage at levels of 0, 1.5, 5, or 15 mg/kg body weight with preparation intervals of 6, 12, and 24h post-administration.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study is cited by the ECETOC Task Force on Cyanides (author of the ECETOC JACC No. 53) and the World Health Organization (WHO) in their review of cyanogenic glycosides, Food Additives Series No. 30. To the knowledge of the SIEF, this unpublished study is unavailable for review.

Justification for type of information:

Ethanedinitrile, hydrogen cyanide (HCN), potassium cyanide and sodium cyanide can be considered as a chemical category, along with and acetone cyanohydrin (ACH, also known as 2-hydroxy-2-methylpropanenitrile), based on structural similarity, common breakdown/metabolic products in physical and biological systems, and similar physico-chemical properties. Particular attention is paid to the dissociation constant of HCN. Ethanedinitrile breaks down in aqueous solution into cyanide ion (CN-) and cyanate ion (OCN-) (Cotton and Wilkinson 1980). Ethanedinitrile due to its low log Kow (0.07) and relatively high solubility in water (2.34 g/L) needs to get dissolved in aqueous solutions in lungs to enter the body. The rate of hydrolysis of ethanedinitrile is very fast (Ajwa 2015). Also, in the vast majority of environmental and physiologic conditions, the cyanide salts will dissolve in water to form hydrogen cyanide. The physico-chemical hazards and toxicity therefore result from the activity of HCN. An ECETOC Task Force, in the 2007 ECETOC Joint Assessment of Commodity Chemicals (JACC) Report No. 53, “Cyanides of Hydrogen, Sodium and Potassium, and Acetone Cyanohydrin (CAS No. 74-90-8, 143-33-9, 151-50-8 and 75-86-5)” supports the development of the chemical category inclusive hydrogen cyanide, sodium and potassium cyanides. Hydrogen cyanide (Index No.006-006-00-X) and salts of hydrogen cyanides (Index No.006-007-00-5) are both listed in Annex VI, Table 3.1 of Regulation (EC) No. 1272/2008, entry 006-007-00-5, and are restricted in comparable ways taking into account physical characteristics. Thus, the assignment of ethanedinitrile to a chemical category does not result in a less protective regulatory status.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

assumed

Principles of method if other than guideline:

information only from secondary sources

GLP compliance:

not specified

Remarks:

likely but no information

Type of assay:

chromosome aberration assay

Species:

hamster

Route of administration:

oral: gavage

Remarks:

Doses / Concentrations:
0.4 mg/kg bw
Basis:
nominal conc.

Genotoxicity:

negative

An in vivo mutagenicity study detecting chromosomal aberrations with HCN orally administered to Chinese hamsters was carried out. Preparations of metaphase cells were studied for structural chromosome aberrations after 6, 24 and 48 h after oral administration of 0.4 mg HCN/kg bw. The incidence of aberrations or gaps was within the spontaneous range. Neither multiple aberrations nor pulverised metaphases were found. There was no indication of mutagenic properties relative to structural chromatid or chromosome damage.

Conclusions:

Interpretation of results: negative
An in vivo mutagenicity study in Chinese hamsters detecting chromosomal aberrations with HCN orally administered to Chinese hamsters was carried out. Preparations of metaphase cells were studied for structural chromosome aberrations after 6, 24 and 48 h after oral administration of 0.4 mg HCN/kg bw. The incidence of aberrations or gaps was within the spontaneous range. Neither multiple aberrations nor pulverised metaphases were found. There was no indication of mutagenic properties relative to structural chromatid or chromosome damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)