3-(isodecyloxy)propiononitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ethernitrile-C10iis not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. Further information based on molecular profiling, QSAR evaluations and read-across to data available on fatty nitriles do not indicate a genotoxic potential.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 September 2017 - 27 September 2017

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 03 November 2015

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The test item is not irritant or corrosive.

Target gene:

Salmonella typhimurium: histidine gene
Escherichia coli: tryprophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9; 5 and 10%), prepared from male Sprague Dawley rats injected with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

Dose range finding test (TA100 and WP2uvrA, with and without 5% S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of experiment 1)
First experiment (TA1535, TA 1537 and TA98, with and without 5% S9): 5.4, 17, 52, 164, 512 and 1600 μg/plate
Repeated first experiment (TA1535 and TA98, with and without 5% S9): 512, 1600 and 5000 μg/plate
Second experiment (all strains, with and without 10% S9): 275, 492, 878, 1568, 2800 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was dissolved in DMSO and the vehicle was according to OECD guideline 471.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Remarks:

See 'any other information on materials and methods' for details

Details on test system and experimental conditions:

Two individual mutation experiments were performed: the first experiment was performed with 5% S9 mix, the second experiment was performed with 10% S9 mix.
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 +/- 4 hours

NUMBER OF REPLICATIONS: 3

PLATE PREPARATION: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in dimethyl sulfoxide and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h.

NUMBER OF CELLS EVALUATED: 10^9 cells/mL

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

COLONY COUNTING:
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Acceptability criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at this laboratory.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: at and above 512 μg/plate. Experiment 2: at and above 275 μg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Remarks:

Experiment 1: at and above 5.4 μg/plate (without S9) and at and above 52 μg/plate (with S9). Experiment 2: at and above 275 μg/plate (without S9) and at and above 878 μg/plate (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 2: at and above 2800 μg/plate (without S9) and at and above 1568 μg/plate (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Remarks:

Experiment 1A: at and above 1600 μg/plate (with and withou S9) Experiment 2: at and above 429 μg/plate (without S9) and a slight reduction at and above 492 μg/plate (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Since no precipitate or toxicity was observed in tester strains TA1535 and TA98, these strains were tested in a repeated experiment at a dose range of 512 to 5000 μg/plate.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
* Experiment 1: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 μg/plate and upwards in tester strains TA100, WP2uvrA (absence and presence of S9-mix) and TA1537 (absence of S9-mix). Precipitation of the test item on the plates in tester strain TA1535, TA98 (absence and presence of S9-mix) and TA1537 (presence of S9-mix) was observed at the start of the incubation period at the concentration of 1600 μg/plate and no precipitate was observed at the end of the incubation period.
In the repeat experiment precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 μg/plate and upwards.
* Experiment 2: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period in tester strains TA1535, TA1537 and WP2uvrA at concentrations of 2800 μg/plate and upwards whereas in tester strains TA98 and TA100 precipitate was already observed at the concentration of 1568 μg/plate at the end of the incubation period.

CYTOTOXICITY:
- Experiment 1: Cytotoxicity as evidenced by a reduction of the bacterial background lawn and/or a biologically relevant decrease in the number of revertants was observed in tester strain TA100 and TA1537, with and without S9.
- Experiment 2: In the second mutation assay, cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in tester strains TA1535, TA1537, TA98 and TA100 in the absence of S9-mix and in tester strains TA1537 and TA100 in the presence of S9-mix.

MUTAGENICITY: in both experiments and at all dose levels, no increase in he number of revertants was observed upon treatment with the test item, with and without S9.

HISTORICAL CONTROL DATA
- Positive historical control data: The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
- Solvent historical control data: The negative control values were within the laboratory historical control data ranges, except the response for TA100 in the presence of S9-mix. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (53 revertant colonies) when compared against relevant historical control data (63 revertant colonies), the validity of the test was considered to be not affected.

Table 1 Historical control data of the solvent control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	3 - 27	3 – 20	3 – 23	8 - 41	8 - 55	63 – 176	54 - 160	10 – 59	9 - 69
Mean	10	11	6	7	16	23	108	107	25	32
SD	3	4	2	3	5	7	19	20	7	8
n	2356	2336	2264	2235	2319	2360	2341	2336	2075	2078

SD = Standard deviation; n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Table 2 Historical control data of the positive controls

	TA1535		TA1537		TA98
S9-mix	-	+	-	+	-	+
Range	125 – 1248	73 – 1206	55 – 1353	54 – 1051	365 – 1995	250 – 1977
Mean	846	219	787	353	1406	887
SD	146	119	345	162	258	349
n	2348	2229	2003	2234	2200	2276

	TA100		WP2uvrA
S9-mix	-	+	-	+
Range	439 – 1848	408 - 2651	93 – 1951	111 - 1359
Mean	901	1232	1094	437
SD	168	343	477	149
n	2335	2327	2021	2085

SD = Standard deviation; n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Conclusions:

The results of an Ames test, performed according to OECD guideline 471 and GLP principles, showed that Propanenitrile, 3-(isodecyloxy)- is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of Propanenitrile, 3-(isodecyloxy)- and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Propanenitrile, 3-(isodecyloxy)- was a hazy light yellow liquid with a purity of 100%. The test item was dissolved in dimethyl sulfoxide. 

In the dose-range finding test, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP₂uvrA. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in tester strain TA100 at dose levels of 164 and 512 μg/plate and upwards in the absence and presence of S9-mix, respectively. In tester strain WP₂uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 5.4 to 1600 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at a dose level of 1600 μg/plate in tester strain TA1537 in the absence of S9-mix only. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in tester strain TA1537 at dose levels of 512 and 1600μg/platein the absence of S9-mix and at a dose level of 1600 µg/plate in the presence of S9-mix. 

Since in tester strain TA1535 and TA98 no precipitate or toxicity was observed at the highest dose level tested, a repeat experiment was performedat a concentration range of 512 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate in the absence and presence of S9-mix. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 492 to 5000 µg/plate in the absence and presence of 10% (v/v)
S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA. The test item was tested up to or beyond a precipitating dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in tester strains TA1535, TA1537, TA98 and TA100in the absence of S9-mix and in tester strains TA1537 and TA100 in the presence of S9-mix. 

The test item did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in the tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that Propanenitrile, 3-(isodecyloxy)- is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No data available from an in vivo study.

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Ethernitrile-C10i like other fatty-nitriles used as intermediate in the production of fatty amine surfactants, are not genotoxic. None of the available test data, as well as molecular profiling and QSAR evaluations, provide an alert suggesting genotoxicity hazards.

Additional information

Ethernitrile-C10i was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA in accordance to OECD guideline 471 and under GLP. Adequate toxicity was observed, and appropriate reference mutagens produced significant increases in the number of revertant colonies, demonstrating the sensitivity of the assay. In both the presence and in the absence of the metabolic activation system. Ethernitrile-C10i did not result in relevant increases in the number of revertants in any of the bacterial strains. These results were confirmed in a follow-up experiment. It was therefore concluded that Ethernitrile-C10i is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Molecular profiling does not show alerts that could indicate a concern for possible hazards for genotoxicity. (QSAR Toolbox v.4.2)

DNA alerts for AMES by OASIS	No alert found
DNA alerts for CA and MNT by OASIS	No alert found
Oncologic Primary Classification	Not classified
Protein binding alerts for Chromosomal aberration by OASIS	No alert found
in vitro mutagenicity (Ames test) alerts by ISS	No alert found
in vivo mutagenicity (Micronucleus) alerts by ISS	No alert found
DNA binding by OASIS	No alert found
DNA binding by OECD	No alert found

 

Also there QSAR predictions do not indicate specific concerns:

ACD - Ames:	No hazourdous fragments found (P=0.077, Moderate riliability: RI = 0.58)
TOPKAT - Ames mutagenicity:	Non-Mutagen (No unknown or out-range features)
DEREK - Genotox (Ames):	Mutagenicity in vitro in bacterium is INACTIVE (No misclassified or unclassified features)
VEGA Mutagenicity (Ames test) CONSENSUS model (version 1.0.2):	NON-Mutagenic (Consensus score: 0.82)
VEGA Mutagenicity (Ames test) model (CAESAR) (version 2.1.13):	NON-Mutagenic (good reliability)
VEGA Mutagenicity (Ames test) model (SarPy/IRFMN) (version 1.0.7):	NON-Mutagenic (good reliability)
VEGA Mutagenicity (Ames test) model (ISS) (version 1.0.2):	NON-Mutagenic (moderate reliability)
VEGA Mutagenicity (Ames test) model (KNN/Read-Across) (version 1.0.0):	NON-Mutagen (good reliability)

Justification for classification or non-classification

Ethernitrile-C10iis not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. Further information based on molecular profiling, QSAR evaluations and read-across to data available on fatty nitriles do not indicate a genotoxic potential.

Consequently, Ethernitrile-C10i does not need to be classified for genotoxicity.