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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an in vitro gene mutation study in mammalian cells (mouse lymphoma assay) the test item, Hydroxypropyl, 2-, trimethylammonium formate did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic.

In an in vitro cytogenicity / chromosome aberration study in mammalian cells (human peripheral blood lymphocytes) Hydroxypropyl, 2-, trimethylammonium formate was non-toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the maximum recommended dose level. The test item, Hydroxypropyl, 2-, trimethylammonium formate was considered to be non-clastogenic to human lymphocytes in vitro.

Link to relevant study records

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Identification: Hydroxypropyl, 2-, trimethylammonium formate
Physical state/Appearance: Colourless to very light blue liquid
Batch: Chernil.20160628.B
Purity: 95.25%
Expiry Date: 28 June 2017
Storage Conditions: Room temperature in the dark until 08 November
2016 and thereafter room temperature in the dark over silica gel
Intended use/Application: Not supplied
Formulated concentrations were adjusted to allow for the stated water/impurity content (4.75%) of the test item.
Species / strain / cell type:
lymphocytes: Human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a
non-smoking volunteer (aged 18-35) who had been previously screened for suitability. The
volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals
and had not knowingly recently suffered from a viral infection. Based on over 20 years
in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine)
incorporation to assess the number of first, second and third division metaphase cells to
calculate the average generation time (AGT) for human lymphocytes it is considered to be
approximately 16 hours. Therefore using this average the in-house exposure time for the
experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: female, aged 21 years
Main Experiment: female, aged 30 years
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0, 102, 204, 408, 816, 1020, 1224, 1632 µg/m

The dose range for the Preliminary Toxicity Test was 6.38 to 1632 µg/mL. The maximum dose was the maximum recommended dose level.
Vehicle / solvent:
Eagle's minimal essential medium with HEPES buffer (MEM)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA)

The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No’s. PB/βNF S9 25/08/16 (preliminary toxicity test) and PB/βNF S9 22/09/16 (main test) were used in this study. A copy of the S9 Certificates of Efficacy are presented in Appendix 2.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosedat a 10% volume of S9-mix into culture media, was 2%
Evaluation criteria:
The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a topconcentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
Key result
Species / strain:
lymphocytes:
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
Remarks on result:
other: Negative for genotoxicity
Conclusions:
The test item was non-toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the maximum recommended dose level.
The test item, Hydroxypropyl, 2-, trimethylammonium formate was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberratios at three dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of

an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of

metabolic activation. The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration was the maximum recommended dose level.

All vehicle (Eagle's minimal essential medium with HEPES buffer (MEM)) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the maximum recommended dose level.

The test item, Hydroxypropyl, 2-, trimethylammonium formate was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
With the exception stated below, the study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I consider the data generated to be valid.
• The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 3106, as amended by Statutory Instrument 2004 No. 994)
• OECD Principles of Good Laboratory Practice (as revised in 1997),
ENV/MC/CHEM(98) 17.
• EC Commission Directive 2004/10/EC of 11 February 2004 (Official Journal
No. L 50/44).

Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Identification: Hydroxypropyl, 2-, trimethylammonium formate
Physical state/appearance: Colourless to light blue liquid
Batch: chernil.20160628.B
CAS Number: 62314-25-4
Molecular Weight: 163.217
Purity: 95.25% active in water (purity correction applied)
Expiry Date: 28 June 2017
Storage Conditions: Room temp
Target gene:
thymidine kinase gene L5178Y TK+/- 3.7.2c
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the
MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were
originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and
were frozen in liquid nitrogen at that time.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item did not exhibit any marked toxicity in either of the 4-hour exposure groups. In the 24-hour exposure group moderate levels of test item induced toxicity can observed at the upper dose levels. No precipitate of the test item was observed in any of the exposure groups. Therefore the test item could be tested up the maximum recommended 10mM dose level in the main test. The dose levels plated for viability and expression of mutant colonies were as follows:
102, 204, 408, 816, 1224, 1632 µg/mL
Vehicle / solvent:
RO media
Solvent (R0 media) (Sigma batch 1813358 Expiry 03.10.2017*)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (R0 media)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
The stocks of cells are stored in liquid nitrogen at approximately -196°C. Cells were
routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM)
supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate
(1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at
approximately 37 o C with 5% CO2 in air. The cells have a generation time of approximately
12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20),
10% donor horse serum (R10), and without serum (R0), are used during the course of the
study. Master stocks of cells were tested and found to be free of mycoplasma.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Statistics:
The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear-trend events.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
Conclusions:
The test item, Hydroxypropyl, 2-, trimethylammonium formate did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in
this assay
Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels in duplicate, together with vehicle (RO media), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item did not exhibit any marked toxicity in either of the 4- hour exposure groups. In the 24-hour exposure group moderate levels of test item induced toxicity can observed at the upper dose levels. No precipitate of the test item was observed in any of the exposure groups. Therefore the test item could be tested up the maximum recommended 10mM dose level in the main test.

The maximum dose level used was the maximum recommended dose level (approximately 10mM) of 1632 µg/mL. No precipitate of the test item was observed throughout. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification