(1S)-1-cyclopropylethanamine; (2R)-2-hydroxy-2-phenyl-acetic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type fo genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 October - 1 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 471

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1S)-1-cyclopropylethanamine; (2R)-2-hydroxy-2-phenyl-acetic acid

EC Number:

946-290-0

Cas Number:

2246580-61-8

Molecular formula:

C8H7O3.C5H12N

IUPAC Name:

(1S)-1-cyclopropylethanamine; (2R)-2-hydroxy-2-phenyl-acetic acid

Test material form:

solid: bulk

Specific details on test material used for the study:

IN 79066, batch number KLP04616, was a white solid. It was received on 24 October 2016
and stored at 15-25°C, protected from light. Purity was stated as 99.5% (by HPLC) and the
expiry date was given as 12 October 2017, see Certificate of Analysis.

Method

Target gene:

Four strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535 and TA1537) and
one strain of Escherichia coli bacteria (WP2 uvrA pKM101) were used in this study. Strains
TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strain TA100 was
derived from cultures originally obtained from Covance Laboratories Inc., USA. Strain WP2
uvrA pKM101 was obtained from MolTox Inc., USA.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9Mix

Test concentrations with justification for top dose:

Experiment 1 treatments of all the tester strains were performed in the absence and in the
presence of S-9, using final concentrations of IN 79066 at 5, 16, 50, 160, 500, 1600 and
5000 μg/plate, plus vehicle and positive controls. Following these treatments no evidence of
toxicity was observed, as would normally be manifest as a thinning of the background
bacterial lawn and/or a marked reduction in revertant numbers.
Experiment 2 treatments of all the tester strains were performed in the absence and in the
presence of S-9. The maximum test concentration of 5000 μg/plate was retained for all
strains. Narrowed concentration intervals were employed covering the range
160-5000 μg/plate, in order to examine more closely those concentrations of IN 79066
approaching the maximum test concentration and considered therefore most likely to provide
evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were
further modified by the inclusion of a pre-incubation step. In this way, it was hoped to
increase the range of mutagenic chemicals that could be detected using this assay system.
Following these treatments no evidence of toxicity was observed.
The test article was completely soluble in the aqueous assay system at all concentrations
treated, in each of the experiments performed.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene (AAN)

Details on test system and experimental conditions:

The test system was suitably labelled to clearly identify the study number, bacterial strain,
test article concentration (where appropriate), positive and vehicle controls, absence or
presence of S-9 mix.

Rationale for test conditions:

The assay was to be considered valid if the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges as defined
in Section 6.2
2. The positive control chemicals induced increases in revertant numbers of ≥2-fold (in
strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and The assay was to be considered valid if the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges as defined
in Section 6.2
2. The positive control chemicals induced increases in revertant numbers of ≥2-fold (in
strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and The assay was to be considered valid if the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges as defined
in Section 6.2
2. The positive control chemicals induced increases in revertant numbers of ≥2-fold (in
strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation
3. At least five analysable concentrations of the test article are available.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥2-fold (in strains TA98,
TA100 or, WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 or TA1537) the concurrent
vehicle control values
2. Any observed response is reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case
basis. Biological relevance was taken into account, for example consistency of response
within and between concentrations and (where applicable) between experiments.

Statistics:

Individual plate counts were recorded separately and the mean and standard deviation of the
plate counts for each treatment were determined. Control counts were compared with the
laboratory’s historical control ranges (Section 6.2 and Section 6.3). Data were considered
acceptable if the vehicle control counts fell within the calculated historical control ranges and
the positive control plate counts were comparable with the historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical
analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).
However, adequate interpretation of biological relevance was of critical importance.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

It was concluded that IN 79066 did not induce mutation in four histidine-requiring strains
(TA98, TA100, TA1535 and TA1537) of Salmonella typhimurium, and one tryptophanrequiring
strain (WP2 uvrA pKM101) of Escherichia coli when tested under the conditions of
this study. These conditions included treatments at concentrations up to 5000 μg/plate (the
maximum recommended concentration according to current regulatory guidelines), in the
absence and in the presence of a rat liver metabolic activation system (S-9).