2-Propenamide, N-(2-hydroxyethyl)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of HEAA is assessed with Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA/pKM101, according to OECD Guideline 471, under GLP.

Under the test conditions employed, it is concluded that HEAA showed no evidence of mutagenic activity in this bacterial system.

 

HEAA was tested for mutagenic potential in an in vitro mammalian cell mutation assay with mouse lymphoma L5178Y cells, according to OECD Guideline 476, under GLP.

It was concluded that HEAA did demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May 2006 to 3 July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PAD Notification No. 444.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Tests for Pharmaceuticals. PMSB/ELD Notification No. 544.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Batch number: 050804
Purity: >99%

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: R0 RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.
R10p R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R30p R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no

Metabolic activation:

with and without

Metabolic activation system:

S9 mix.

Test concentrations with justification for top dose:

39.06, 50, 78.13, 100, 156.25, 200, 300, 312.5, 400, 600, 625, 700, 800, 900, 1250 µg / ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: vehicle chosen is purified water

- Justification for choice of solvent/vehicle: Purified water was chosen as the vehicle because test substance is water soluble.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

in presence of S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

in absence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 24 and 48 hours

Evaluation criteria:

The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6. The assay was considered valid in accordance with the assay acceptance criteria.
The test agent was regarded as negative if:
the mean concurrent solvent control mutant frequency and the GEF.
If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.
Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Main mutation test - 3 hour treatment in the absence of S9 mix:

In the 3 hour exposure, cultures were exposed to HEAA at concentrations from 39.06 to 1250 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to HEAA at concentrations from 39.06 to 1250 μg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 104 to 86% were obtained relative to the solvent control. Data are presented for concentrations tested up to the maximum concentration, in accordance with current guidelines (approximately 10 mM). There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent solvent control mutant frequency and the Global Evaluation Factor, (GEF), within acceptable levels of toxicity.

The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Main mutation test - 3 hour treatment in the presence of S9 mix:

In the 3 hour exposure, cultures were exposed to HEAA at concentrations from 50 to 1250 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to HEAA at concentrations from 50 to 600 μg/mL were assessed for determination of mutation frequency. RTG values from 97 to 16% were obtained relative to the solvent control. There was a clear increase in the mean mutant frequency of the cells when exposed to HEAA at 600 μg/mL that exceeded the sum of the mean concurrent solvent control mutant frequency plus the GEF, within acceptable levels of toxicity. At 400 μg/mL the mean mutant frequency was marginally below the GEF. This dose-dependent increase in mutant frequency was accompanied by a decrease in toxicity which was reduced to an acceptable level of 16% RTG.

This dose-response showed a significant positive linear trend (p<0.001). The increases in mean mutant frequency were predominantly due to an increase in small colony formation, when compared to the concurrent solvent control. These increases fulfilled the criteria for a positive response, with a continuous exposure to the test substance not deemed to be necessary.

The positive control, 3-methylcholanthrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Conclusions:

It was concluded that HEAA did demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

HEAA was tested for mutagenic potential in an in vitro mammalian cell mutation assay with mouse lymphoma L5178Y cells, according to OECD Guideline 476, under GLP.

It was concluded that HEAA did demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May 2000 to 5 June 2000

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:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, NohSan No. 4200

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Joint Directives of J EPA, J MHW and J MITI. (31 October 1997) Kanpoan No. 287, Eisei No. 127 and Kikyoku No. 2 (31 October 1997) and Official Notice of J MOL (8 February 14999)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Lot number: MDB-329
Purity: >99%

Target gene:

S. typhimurium: histidine;
E. coli: tryptophan

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

Test concentrations with justification for top dose:

5000, 1500, 500, 150 and 50 µg / plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sodium azide, 9-aminoacridine, 2-nitrofluorene, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide, 2-aminoanthraceneand benzo(a)pyrene. The solvent used was purified water.
- Justification for choice of solvent/vehicle: purified water, obtained by reverse osmosis, was used as the solvent because the test substance is soluble in water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

for strains TA1535 and TA100 in absence of S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for strain TA1537 in absence of S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

for strain TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide

Remarks:

for strain WP2uvrA/pKM101 in absence of S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

for strains TA1535, WP2uvrA/pKM101 in presence of S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

for strains TA1537, TA98 and TA100 in presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar
DURATION
- Preincubation period: 10 hours at 37°C
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony numbers and background lawn of non-revertant cells.

Evaluation criteria:

Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to HEAA at any concentration in either the presence or absence of S9 mix.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to HEAA.

Conclusions:

Under the test conditions employed, it is concluded that HEAA showed no evidence of mutagenic activity in this bacterial system.

Executive summary:

The mutagenic potential of HEAA is assessed with Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA/pKM101, according to OECD Guideline 471, under GLP.

Under the test conditions employed, it is concluded that HEAA showed no evidence of mutagenic activity in this bacterial system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The study was performed to assess the potential induction of micronuclei by HEAA in bone marrow cells of CD-1 mice, according to OECD Guideline 474 under GLP.

HEAA did not show any evidence of causing an increase in the induction of micronucleated immature erythrocytes or bone marrow cell toxicity in CD-1 mice, when administered orally by gavage in the in vivo test procedure.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 June 2004 to 17 August 2004

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Batch number: 7646-67-5
Purity: >99%

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at study initiation:
- Weight at study initiation: males 28-32 g, females 22-26 g
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: each group was kept with the sexes separated, in cages and maintained in a controlled environment
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): adlibitum
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 55±15%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours per day

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of the test substance were freshly prepared on the day of the test in purified water.
All animals in all groups were dosed orally by gavage with the standard volume of 20 mL/kg bodyweight.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

once daily

Post exposure period:

24 hours

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control(s): mitomycin C
- Route of administration: oral
- Doses / concentrations: 0.6 mg/mL, 12 mg/kg

Tissues and cell types examined:

The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. One smear per animal was examined and the remaining smears were held temporarily in reserve in case of technical problems with the first smear.
Micronuclei are identified by the following criteria:
Large enough to discern morphological characteristics;
Should possess a generally rounded shape with a clearly defined outline;
Should be deeply stained and similar in colour to the nuclei of other cells - not black;
Should lie in the same focal plane as the cell;
Lack internal structure, ie they are pyknotic;
There should be no micronucleus-like debris in the area surrounding the cell.
The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept.

Details of tissue and slide preparation:

The animals were killed by cervical dislocation following carbon dioxide inhalation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 mL of pre-filtered foetal calf serum. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner. Three smears were made from each animal. The prepared smears were fixed in methanol. After air-drying the smears were stained for 10 minutes in 10% Giemsa. Following rinsing in purified water and differentiation in buffered purified water, the smears were rinsed in purified water, air dried and mounted with coverslips using DPX.

Evaluation criteria:

A positive response is normally indicated by a statistically significant increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent vehicle control group.
A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent vehicle control group.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decreases in the proportion of immature erythrocytes were observed in mice treated with test substance, compared to vehicle control values.
The positive control compound mitomycin C producted significant increases in the frequency of micronucleated immaturer erythrocytes.

Conclusions:

HEAA did not show any evidence of causing an increase in the induction of micronucleated immature erythrocytes or bone marrow cell toxicity in CD-1 mice, when administered orally by gavage in the in vivo test procedure.

Executive summary:

The study was performed to assess the potential induction of micronuclei by HEAA in bone marrow cells of CD-1 mice, according to OECD Guideline 474 under GLP.

HEAA did not show any evidence of causing an increase in the induction of micronucleated immature erythrocytes or bone marrow cell toxicity in CD-1 mice, when administered orally by gavage in the in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

HEAA showed no evidence of mutagenic activity in the Bacterial Reverse Mutation Test and only demonstrated mutagenic potential in the in vitro cell mutation assay. The in vivo micronucleus test also showed negative result.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, the test substance should not be classified as germ cell mutagens.