Reaction products of acrylonitrile and 2,2’-Iminodi(ethylamine), hydrogenated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10-12-2018 to 06-03-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of acrylonitrile and 2,2’-Iminodi(ethylamine), hydrogenated

EC Number:

810-418-2

Cas Number:

1703784-30-8

IUPAC Name:

Reaction products of acrylonitrile and 2,2’-Iminodi(ethylamine), hydrogenated

Test material form:

liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat; Arochlor 1254 induced)

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 3160 and 5000 μg/ plate

Vehicle / solvent:

highly purified water

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation: TA 1535; TA 100; 10 µg/plate

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation: TA 1537; 100 µg/plate

Controls 3

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation: TA 98, 10 µg/plate

Controls 4

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation: E.coli WP2 uvra pKM101, 5 µg/plate

Controls 5

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation: TA100, TA1535, E.coli WP2 uvra pKM101, 2 µg/plate

Controls 6

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation: TA 98, TA 1537, 10 µg/plate

Details on test system and experimental conditions:

The first experiment was carried out as the standard plate incorporation method whereas the second was carried out as the preincubation method.

Evaluation criteria:

A test substance producing no biologically relevant positive response in any one of the bacterial strains tested is considered to be non-mutagenic in this system.
A biologically relevant response is described as follows:
If the number of revertants is at least twice the spontaneous reversion rate for TA 98, TA 100, TA 1535, TA 1537 and E.coli WP2 and/or if there is a concentration related increasing number of revertants over the range tested.

Statistics:

yes, p <= 0.05, U-test according to MANN and WHITNEY

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: highly purified water was used as vehicle
- Precipitation: test item precipitation was noted at concentrations of 5000 µg / plate with E.coli WP2 uvra pK101 in both experiments.


COMPARISON WITH HISTORICAL CONTROL DATA: The results of the negative and positive control cultures were within the range of the historical data generated by LPT.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

The reported data show that the test item n did not induce gene mutations in the S. typhimurium and E.coli WP2 tester strains with and without mammalian metabolic activation. In conclusion the results of this bacterial reverse mutation assay were considered negative.

Executive summary:

Preliminary test

PU-2018-821 was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in the Salmonella typhimurium test strain TA100. Ten concentrations ranging from 0.316 to 5000 μg PU-2018-821/plate were tested. No signs of cytotoxicity were observed, neither in the experiment without nor in the experiment with metabolic activation. Hence, 5000 μg PU-2018-821/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 31.6 to 5000 μg PU-2018-821/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

Cytotoxicity in form of reduction of the number of revertants by more than 50% was noted in the E. coli strain WP2 uvrA [pKM101] in all experiments: in the experiments without metabolic activation at the concentrations of 3160 and 5000 μg/plate, in the experiments with metabolic activation at the concentration of 5000 μg/plate

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for PU-2018-821, tested up to the top concentration of 5000 μg/plate that led to cytotoxicity in E. coli strain WP2 uvrA [pKM101] in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system. The results of the negative and positive control cultures were within the range of the historical data . Hence, all acceptance criteria are met.

In conclusion, under the present test conditions, PU-2018-821 tested up to a concentration of 5000 μg/plate that led to cytotoxicity in the Escherichia coli strain WP2, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and in the Escherichia coli strain WP2 uvrA [pKM101], neither in the plate incorporation test nor in the preincubation test, each carried out without and with metabolic activation.