3-((C12-18)-acylamino)-N-(2-((2-hydroxyethyl)amino)-2-oxoethyl)-N,N-dimethyl-1-propanaminium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline, GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Purity: 94%
Lot number: 1514MP
Description: orange solid gel
Storage conditions: room temperature in the dark

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The ceIl-cycle time for the lymphocytes from the donor used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 14 hours under optimal growth conditions.

Metabolic activation:

with and without

Metabolic activation system:

Lot No. Aro. S9/22/08/98 was prepared, in-house, from the livers of male Sprague-Dawley rats weighing 250g. These had received a single injection of Aroclor 1254 at 500 mg/kg, five days before S9 preparation.

Test concentrations with justification for top dose:

Experiment 1
Final concentration of the test item (ug/mL)
(4) 16h -S9 0, 39.07, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000, EMS 750
(4) 16h +S9 0, 39.07, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000, CP 25

EMS: Ethyl methanesulphonate
CP: Cyclophosphamide

Experiment 2
Final concentration of the test item (pg/ml)
20h -S9 0, 2.44, 4.88, 9.77, 19.54, 39.07, 78.13, 156.25, 312.5, EMS 500
(4) 16h +S9 0, 4.88, 9.77, 19.54, 39.07, 78.13, 156.25, 312.5, 625, CP 25

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

CelI Culture
CeIls were grown in Eagle’s minimal essential media, (supplemented with sodium bicarbonate, HEPES buffer, L-glutamine, pen iciIin/streptomycin, amphotericin B and 15% foetal calf serum) at 37 °C with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at 90 ug/mL final concentration.

The test material was accurately weighed, dissolved in Minimal Essential Media (MEM) and serial two-fold dilutions prepared. The maximum dose level tested was 5000 ug/mL which was the maximum recommended dose level. There was no observable change in pH when the test material was dosed into media and the osmolality did not increase by more than 50 mOSM. Chemical analysis of the test material formulations was not performed because it is not a requirement of the test method.

Culture Conditions - Experiment 1
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05 to 9.05 ml MEM, 15% (FCS)
0.1 ml Li-heparin
0.1 ml phytohaemagglutinin
0.75 ml heparinised whole blood

Treatment With-activation:
After approximately 48 hours incubation at 37°C, 5% C02 in humidified air, the with-S9 cultures were centrifuged after transfer into tubes and approximately 9 ml of the culture medium removed, reserved and replaced with MEM (including serum) and 0.1 ml of the appropriate solution of positive control or 1 ml of appropriate solution of vehicle or test material was added to each culture. The final concentrations of the test material were 39.07, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 ug/ml. 1 ml of 10% S9 (je 1 % final concentration of S9) in standard co-factors was added and the cultures returned to the incubator.
After 4 hours at 37 °C the with-S9 cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 16 hours.

Treatment Without-activation:
After approximately 48 hours incubation at 37°C, 5% C02 in humidified air the cultures were decanted into centrifuge tubes and centrifuged, as for the treatment with-activation. Most of the culture media was drawn off and stored. The cells were then resuspended with fresh MEM (including serum).
The total volume for each culture was a nominal 9.9 ml for non-aqueous vehicles and 9.0 mIs for aqueous vehicles. The final concentrations were the same as for the with-activation treatments.

Culture Conditions - Experiment 2
These were as in previous section, except that there was continuous exposure in the absence of metabolic activation. The exposure in the presence of activation was for 4 hours, followed by a 16-hour expression period as performed in Experiment 1. The final concentration of S9 was increased, 1 ml of 20% S9 (ie 2% final concentration) in standard co-factors was added and the cultures returned to the incubator. The final concentrations of the test material were 2.44, 4.88, 9.77, 19.54, 39.07, 78.13, 156.25, 312.5 and 625 ug/ml.

Evaluation criteria:

Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. CeIls with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations (excluding gaps) in either the presence or absence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system (S9) with cell harvest after a 16-hour expression period and a 4-hour exposure in the absence of activation with a 16-hour expression period, this was Experiment 1. In Experiment 2 the 4-hour exposure with addition of S9 was repeated (using 2% final S9 concentration), whilst in the absence of activation the exposure time was increased to 20 hours.

The method used followed that described in the OECD Guidelines for the Testing of Chemicals (1997) No. 473 “Genetic Toxicology: Chromosome Aberration Test” and Method B10 of Commission Directive 92/69/EEC.

 

Cytotoxicity was observed with the tested concentrations.

 

All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes.

 

AIl the positive control treatments gave statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either of two separate experiments. The test material was shown to be non-clastogenic to human lymphocytes in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, OECD guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0.5 to 1500 ug/plate

Vehicle / solvent:

sterile water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

3 ug/plate for TA100, 5 ug/plate for TA1535 and 2 ug/plate for WP2uvrA-

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 ug/plate for TA1537

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 ug/plate for TA98

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

1 ug/plate for TA100 and 10 ug/plate for WP2uvrA-

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

2 ug/plate for TA1535 and TA1537

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

5 ug/plate for TA98

Details on test system and experimental conditions:

Salmonella typhimurium TA1535, TA1537, TA98 and TA100
Escherichia coli WP2uvrA-
The Salmonella strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 whilst the Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association on 17 August 1987. AIl of the strains were stored at -196 °C in a Stateboume Iiquid nitrogen freezer, model SXR 34. Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

Evaluation criteria:

The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statisticaliy (Dunnett’s method of linear regression) significant increase in the revertant count in at least one strain of bacteria. If a greater than two fold increase in revertant count is observed in two experiments then this is taken as evidence of a positive response.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including M1T1, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and ranged between 0.5 and 1500 pg/plate in the first experiment depending on bacterial strain type and presence or absence of S9-mix. The experiment was repeated on a separate day using doses ranging between 0.5 and 500 ug/plate, fresh cultures of the bacterial strains and fresh test material formulations. An extra dose level was included in both experiments to allow for the toxicity of the test material to the bacterial strains used and to ensure there were at least four non-toxic doses plated out.

2. The vehicle (sterile distiiled water) control plates gave counts of revertant colonies within the normal range.

3. AIl of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

4. The test material caused a visible reduction in the growth of the bacterial lawn to all of the tester strains both with and without metabolic activation. The first indication of a toxic response was observed at 50 ug/plate to tester strain TA98 without S9-mix. The test material was, therefore, tested up to its toxic limit.

5. No signifiant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline, GLP study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sufficient albino Crl:CD-1TM(ICR)BR strain mice were supplied by Charles River (UK) Limited, Margate, Kent. At the start of the main study the mice weighed 26 to 30g and were approximately five to eight weeks old. After a minimum acclimatisation period of seven days the animals were selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card.

The animals were housed in groups of up to seven in solid-floor polypropylene cages with woodflakes bedding. Free access to mains drinking water and food (Certified Rat and Mouse Diet 5LF2, IPS Product Supplies Ltd, P.O. Box 6655, Weliingborough, Northants) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

distilled water
The vehicle was supplied by IVAX Healthcare Ltd, as follows:
Supplier's identification : water for irrigation
Supplier's lot number : A2044
Safepharm serial number : V-2443
Date received : 15 March 2002
Description : clear colourless liquid
Storage conditions : room temperature

Duration of treatment / exposure:

once

Remarks:

Doses / Concentrations:

Basis:
nominal conc.
2000 mg/kg

Remarks:

Doses / Concentrations:

Basis:
nominal conc.
1500 mg/kg

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate

Tissues and cell types examined:

Bone Marrow:
- Polychromatic erythrocytes (PCE)
- Normochromatic erythrocytes (NCE)

Details of tissue and slide preparation:

Immediately following termination (je. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grûnwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

Evaluation criteria:

Stained bone marrow smears were coded and examined blind using light microscopy at xl000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Premature deaths were observed at 1500 mg/kg in the 48-hour group. Clinical signs were observed in animals dosed with the test material at 1500 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: diarrhoea, hunched posture, pilo-erection and distended abdomen. It was considered that the loss of the two animals due to premature death did not affect the integrity of the study, because at least five analysable animals were availabie per group, as recommended in the OECD guidelines.

Conclusions:

Interpretation of results (migrated information): negative
The test material was considered to be non-genotoxic under the conditions of the test.

Executive summary:

Introduction.

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to comply with the UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990). The study design also complies with the revised OECD Guidelines for Testing of Chemicals No.474 “Micronucleus Test”, Method B12 of the EEC Commission Directive 2000/32/EC, the USA EPA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods.

A range-finding study was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes, therefore the main study was performed using only male mice. The micronucleus study was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) of 1500 mg/kg with 750 and 375 mg/kg as the two lower dose levels.

Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single oral dose of distilled water (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectiveiy. Vehicle control animals were killed 24 or 48 hours later, and positive control animais were killed after 24 hours.

Results.

Marked decreases in PCE/NCE ratios were observed in both the 24 and 48-hour 1500 mg/kg test material dose groups, and a clear dose-related response was seen within the 24- hour test material dose groups. Although decreases in the PCE/NCE ratio at 1500 mg/kg, and a dose-related response within the 24-hour groups were observed this did not result in any statistically significance. It was considered that the relatively high standard deviation value of the 24-hour exposure resulted in the absence of a statistical significance. However, the reductions in PCE/NCE ratios, the occurrence of premature deaths and clinical signs was taken to indicate that systemic absorption had occurred. Whilst premature deaths were recorded in the 48-hour1500 mg/kg test material group, the minimum number of animals required for analysis stated in the OECD Guideline was achieved. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

Conclusion.

The test material was considered to be non-genotoxic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The notified chemical shows no genotoxic activity in a bacterial reverse mutation assay and in a chromosome aberration test.

Short description of key information:
A bacterial reverse mutation assay was available for evaluation
An in vitro chromosome aberration study was available for evaluation
An in vivo micronucleus test was available for evaluation

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data do not lead to classification for genotoxicity according to DSD and CLP.