4-oxo-4-(p-tolyl)butyric acid adduct with 4-ethylmorpholine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material was found to be non-mutagenic under the conditions of OECD 471.

The test material demonstrated no toxicologically significant or dose-related increases in the frequency of cells with aberrations in two seperate experiments. The test material was shown to be toxic to CHL cells in vitro, particularly in the 24 and 48-hour continuous exposure groups. The test material was shown to be non-clastogenic to CHL cells in vitro. (OECD 473)

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test item 4-Oxo-4-p-tolylbutyric acid, adduct with 4-ethylmorpholine (Halox 570) is considered to be “non-mutagenic under the conditions of the mouse lymphoma assay”. (OECD 490)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.02.1995 - 14.03.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Solvent: Dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene (with S9 only)

Details on test system and experimental conditions:

Tester Strains:
Salmonella typhimurium TA1535, TA1537, TA98 and TA100
Escherichia coli WP2uvrA
These strains were obtained from the British Industrial Biological Research Association on 14 August 1987 and were stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. Prior to being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa, R factors, uvr mutation and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 °C for approximately 10 hours.

Preparation of Test and Control Materials:
The test material was accurately weighed and approximate half-log dilutions in dimethyl sulphoxide prepared on the day of each experiment. Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined.
Vehicle and positive controls were used in parallel with the test material. A solvent treatment group was used as the vehicle control.
In addition, the material 2-Aminoanthracene (2AA), which is non-mutagenic in the absence of metabolising enzymes was used in the S9 series of plates at different concentrations.

Microsomal Enzyme Fraction:
S9 was prepared in-house on 17/01/95. It was prepared from the livers of male Sprague-Dawley rats weighing ~ 200 g. These had each received a single i. p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation.
The S9 was stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

S9-Mix and Agar:
The S9-Mix was prepared at 4 °C as follows:
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
Top agar was prepared using 0.6% Difco Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mK histidine/1.0 mO biotin and 1.0 mM tryptophan solution added to each 100 mL of top agar. Base agar plates were prepared using 1.2% Oxoid Agar Technical No. 3 with Vogel-Bonner Medium E and 20 mg/mL D-glucose.

Preliminary Toxicity Study:
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. A mixture of 0.1 mL of bacterial suspension (TA100 or WP2uvrA-), 0.1 mL of test solution, 0.5 mL phosphate buffer and 2 mL of molten, trace histidine/tryptophan supplemented media was overlaid onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Five doses of the test material and a vehicle control (dimethyl sulphoxide) were tested in duplicate. After approximately 48 hours incubation at 37 °C the plates were scored for revertant colonies and examined for a thinning of the background lawn.

Mutation Study - Experiment 1 (Range-finding Study):
Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.
Test Material and Vehicle Controls:
Known aliquots (0.1 mL) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 mL of molten trace histidine/tryptophan supplemented top agar at 45 °C, 0.1 mL of the appropriately diluted test material or vehicle control and either 0.5 mL of the S9 liver microsome mix or 0.5 mL of pH 7.4 buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material with and without S9-mix.
Positive Controls:
Without Activation: A known aliquot (0.1 mL) of one of the positive control solutions (ENNG, 9AA or 4NQ0) was added to a test tube containing 2.0 mL of molten, trace histidine/tryptophan supplemented top agar and 0.1 mL of the appropriate bacterial suspension. Finally, 0.5 mL of pH 7.4 buffer was added to the tube, the contents mixed and poured onto an agar plate. This procedure was then repeated, in triplicate, for each tester strain.
With Activation: A known aliquot (0.1 mL) of 2 AA solution was added to a test tube containing 2.0 mL of molten, trace histidine/trpytophan supplemented top agar and 0.1 mL of the appropriate bacterial suspension. Finally, 0.5 mL of S9-mix was added to the tube, the contents mixed and poured onto an agar plate. This procedure was then repeated, in triplicate, for each tester strain.
The plates were incubated at 37 °C for approximately 48 hours and the number of revertant colonies counted.

Mutation Study - Experiment 2 (Main Study):
The secon experiment was performed using methodology as described for experiment 1, using fresh bacterial cultures, test material and control solutions in triplicate.

Interpretation of Results:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained, then a third experiment may be used to confirm the correct response. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 μg/plate. In this case the limiting factor was the maximum recommended dose.

Key result

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:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Study:
The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 μplate. The test material was non-toxic in the strains of bacteria used (TA100 and WP2uvrA-).
Mutation Study:
The results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
No toxicity was exhibited to any of the strains of bacteria used.
No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material either with or without metabolic activation.
All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Conclusions:

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

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA^- were treated with the test material using the Ames plate incorporation method at five 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 MITI, MOL and MAFF. It also meets the requirements of the OECD, EC, and USE, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate in the first experiment. The experiment was repeated on a seperate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh chemical formulations.

The vehicle (dimethyl sulphoxide) control plates produced counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation, it was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06.03.1995 - 10.08.1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The Chinese hamster lung (CHL) cell line, isolated by Koyama et al. 1970 and cloned by Ishidate and Sofuni (1985) was used. The CHL cell line has an average generation time of approximately 11 hours when grown in the conditions described below.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Concentration range in the preliminary cytotoxicity test: 0, 19.5, 39, 78.1, 156.25, 312.5, 625, 1250, 2500, 5000 µg/ml
Concentration range in the main test (Experiment 1): 0, 156.25, 312.5, 625, 1250, 2500, 5000 µg/ml
Concentration range in the main test (Experiment 2): 0, 625, 1250, 2500, 5000 µg/ml

Vehicle / solvent:

Solvent: dimethyl sulphoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cell Culture:
Cells were grown in Eagle's Minimal Essential medium with Earle's Salts (MEM), supplemented with 10% foetal bovine serum and antibiotics, at 37 °C with 5% CO2 in air.

Preparation of Test and Control Materials:
The test material was accurately weighed and prepared in dimethyl sulphoxide (DMSO) and appropriate dilutions made. Analysis for concentration, homogeneity and stability of the test material preparations was not a requirement of the test guidelines and therefore was not performed.
The initial purity of the test material was 99%, therefore an allowance for test material purity was not made when dosing solutions were prepared. There was no observable change in pH when the test material was dosed into media and the osmolality was within the 50mOsm limiting range.
Vehicle and positive controls were used in parallel with the test material. Solvent treatment groups were used as the vehicle controls and the positive control materials were as follows:
In the absence of metabolic activation, Mitomycin C (MMC) (Sigma Batch No. 53H2521), was used at 0.075 μg/ml in Experiment 1 and 0.075 μg/ml for the 12-hour cultures and at 0.05 μg/ml for the 24 and 48-hour cultures in Experiment 2.
Cyclophosphamide (CP) 10 μg/ml (Sigma Batch No. 73H0846) for cultures treated for 6(18)-hour both with and without S9 mix, and the 4(8)-hour culture with S9 mix in Experiment 2.

Preliminary Cytotoxicity Test:
A preliminary cytotoxicity test was performed on cell cultures using 12, 24 and 48-hour continuous exposure times without metabolic activation and a 6(18)-hour and 4(8)-hour exposure period both with and without metabolic activation. The 6(18)-hour exposure group both with and without metabolic activation were exposed for 6 hours and the 4(8)-hour exposure group with metabolic activation were exposed for 4 hours followed by an 18-hour culture period and an 8-hour culture period respectively, in treatment-free media. The dose range used was 19.5 to 5000 μg/ml. Growth inhibition was estimated by counting the number of cells at the end of the culture period on an electronic cell counter (Coulter) and expressing the cell count as a percentage of the concurrent vehicle control value. Slides were also prepared from the cells in order to check for the presence of cells in metaphase.

Microsomal Enzyme Fraction:
Lot No. Aro. S9/17/1/95 was prepared 'in-house' on 17/1/95. It was prepared from the livers of male Sprague-Dawley rats weighing ~ 200 g. These had received a single i.p injection of Aroclor 1254 at 500 mg/kg, up to 5 days before S9 preparation. The S9 was stored at -196 °C in a liquid nitrogen freezer.

Culture Conditions:
Cultures were established approximately 24 hours prior to treatment, 0.4 to 0.5E6 cells were seeded per flask for the 6, 12 and 24-hour treatment cultures and 0.5E6 cells were seeded per flask for the 48-hour treatment cultures. The cells were exposed to up to six doses of the test material, vehicle and positive controls, both with and without metabolic activation. All treatments were performed in duplicate (A+B). Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 air.

Experiment 1:
a) Without Metabolic Activation
12 hours continuous exposure to the test material prior to cell harvest.
b) With Metabolic Activation
4 hours exposure to the test material S9 mix (0.5 ml per 4.5 ml culture medium, of 50% S9 in standard co-factors). A phosphate buffered saline wash and then a further 8 hours in treatment-free media prior to cell harvest.

Experiment 2:
a) Without Metabolic Activation
i) 24 hours continuous exposure to the test material prior to cell harvest.
ii) 48 hours continuous exposure to the test material prior to cell harvest.
iii) 6 hours exposure to the test material, a phosphate buffered saline wash and then a further 18 hours culture in treatment-free media prior to cell harvest. This group acts as a control group for b) i).
iv) 12 hours continuous exposure to the test material prior to cell harvest.
b) With Metabolic Activation
i) 6 hours exposure to the test material and S9 mix (0.5 ml per 4.5 ml culture medium of 10% S9 in standard co-factors). A phosphate buffered saline wash and then a further 18 hours in treatment-free media prior to cell harvest.
ii) 4 hours exposure to the test material and S9 mix (0.5 ml per 4.5 ml culture medium of 50% S9 in standard co-factors). A phosphate buffered saline wash and then a further 8 hours in treatment-free media prior to cell harvest.

Cell Harvest:
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were trypsinised to detach them from the tissue culture flask and suspended in 5 ml of culture medium. A sample of the cell suspension from each culture was counted to measure growth inhibition at each concentration. The cells were centrifuged, the culture medium drawn off and discarded, and the cells resuspended in 0.075 M hypotonic KCl. After fifteen minutes (including five minutes centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KCl suspension into 3 ml fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed several times and the cells stored at 4 °C for sufficient time to ensure complete fixation.

Preparation of Metaphase Spreads:
The cells were resuspended in fresh fixative prior to slide preparation. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining:
When the slides were dry they were stained in 5% Gurrs Giemsa R66 for 5 minutes, rinsed, dried and coverslipped using Depex mountant.

Coding:
After checking that the slide preparations were of good quality, the slides were coded using a computerised random number generator.

Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 23 to 27 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 (Appendix III). Abberations recorded by the slide scorer were checked by a senior cytogeneticist. Cells with 28 to 31 chromosomes were scored as aneuploid cells. Cells with greater than 31 chromosomes were classified as polyploid cells and the % incidence of polyploid cells reported. The percentage of cells showing structural chromosome aberrations (gaps, breaks and exchanges) were calculated and reported as both including and excluding those with gaps.

Mitotic Index:
A total of 1000 cells were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 5000 μg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 5000 μg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 5000 μg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 625 μg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test:
It can be seen that the test material showed evidence of a dose-related increase in cell toxicity in every exposure case. A greater toxicity response was observed in the continuous exposure groups. Microscopic assessment of the slides prepared from the preliminary cytotoxicity test showed metaphases present up to 5000 μg/ml (the maximum recommended dose) in the 6-hour with and without S9-mix treatment cases and the 12-hour with S9-mix treatment; at up to 2500 μg/ml in the 12-hour without S9-mix treatment case and the 24-hour continuous exposure treatment case. The maximum dose level with metaphases present in the 48-hour exposure case was 312.5 μg/ml.

Chromosome Aberration Test - Experiment 1:
It can be seen that the test material showed the expected level of toxicity similar to that seen in the preliminary toxicity study.
The results of the mitotic index also show a clear dose-related increase in toxicity. However, in the 12-hour group without S9 5000 μg/ml was toxic in the preliminary toxicity study, whereas in Experiment 1 there were scorable metaphases present, therefore this dose was selected for analysis of chromosome aberrations. The mitotic indices generally give a measure of toxicity greater than that of the cell counts.
The vehicle control cultures gave values of chromosome aberrations within the expected range.
Both positive control cultures gave statistically significant increases in the frequency of aberrations indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected. Both responses were quite weak but this is typical for an early cell harvest time-point and are considered to result from toxicity induced cell cycle delay.
The test material was seen to induce a statistically significant increase in the frequency of cells with aberrations at 2500 μg/ml in the 12-hour treatment with metabolic activation. There was no dose-related increase observed at the higher dose level, 5000 μg/ml, and there was poor replication of the response between the duplicate cultures and it was therefore considered to be spurious.
The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in either treatment cases.

Chromosome Aberration Test - Experiment 2:
It can be seen that the test material showed the expected level of toxicity, similar to that seen in the preliminary cytotoxicity study and Experiment 1.
The 5000 μg/ml dose level in the 12-hour without S9 treatment group demonstrates similar toxicity to Experiment 1 but was not as toxic as observed in the preliminary cytotoxicity study. As in Experiment 1 the mitotic indices generally indicate a higher level of toxicity than shown by the cell counts.
The vehicle control cultures gave values of chromosome aberrations within the expected range.
All positive control cultures, except the cyclophosphamide without S9 from the 6(18)-hour treatment and MMC without S9 in the 12-hour treatment, gave statistically significant increases in the frequency of cells with aberrations indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected. It was considered, as in Experiment 1, that the toxicity of the positive controls induced cell cycle cell delay and that this was respionsible for the weak response seen in both of the 12-hour treatment cases.
The test material was seen to induce a statistically significant increase in the frequency of cells with aberrations at 625 μg/ml in the 24-hour continuous exposure group. The increase was not dose-related but was subject to inter-culture variation and was therefore considered to be spurious.
An isolated increase in the frequency of polypoid cells was observed at 5000 μg/ml in the 6(18)-hour treatment group without S9. It was considered to be spurious because it was not dose-related and no similar increases were seen in any other treatment group.

Remarks on result:

other: 6-hour exposure, 18-hour recovery

Conclusions:

The test material demonstrated no toxicologically significant or dose-related increases in the frequency of cells with aberrations in two seperate experiments. The test material was shown to be toxic to CHL cells in vitro, particularly in the 24 and 48-hour continuous exposure groups. The test material was shown to be non-clastogenic to CHL cells in vitro.

Executive summary:

Duplicate cultures of Chinese hamster lung (CHL) cells were treated with the test material at a minimum of four dose levels, in each treatment case, together with vehicle and positive controls in each of two experiments. In Experiment 1 a single cell-harvest time-point at 12 hours, both with and without metabolic activation, was used. In Experiment 2 six treatment regimes were used: 6 hours exposure both with and without the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 18 hours treatment-free incubation); 4 hours exposure both with the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 8 hours treatment-free incubation); 12 hours continuous exposure and 48 hours continuous exposure.

The dose range for metaphase analysis was selected from a series of at least four dose levels chosen on the basis of the results of a preliminary toxicity test. The test material was evaluated at doubling dose levels between 78.1 and 5000 μg/ml depending on the particular treatment regime used, the 24 and 48-hour continuous exposures were more toxic than the pulse exposures.

The vehicle (solvent) controls gave frequencies of aberrations within the range expected for the CHL cell line.

All the positive control treatments, except cyclophosphamide without S9 from the 6(18)-hour treatment and MMC in the 12-hour treatment without S9 gave statistically significant increases in the frequency of aberrations indicating that the satisfactory performance of the test and of the activity of the metabolising system. The weak responses seen in the 12-hour treatment cases were typical for this early cell harvest time-point and were probably due to cell cycle delay resulting from treatment toxicity.

The test material induced no toxicologically significant or dose-related increases in the frequency of cells with aberrations in Experiment 1 or Experiment 2 in any of the treatment cases. The test material was shown to be toxic to CHL cells in vitro. An isolated increase in the frequency of polyploid cells was observed at 5000 μg/ml in the 6(18)-hour treatment group without S9. It was considered to be spurious because it was not dose-related and no similar increases were seen in any other treatment group. The test material was shown to be non-clastogenic to CHL cells in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23.05.2016 - 22.12.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell forward mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 41505042
- Expiration date of the lot/batch: 14.09.2018
- Purity test date: not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: cool, dry environment, No temperature requirements
- Stability under test conditions: not stated
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO, RPMI and Aceton up to 10 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: 20 mg/ml (nominal)
- Final preparation of a solid: n/a

Target gene:

thymidine kinase enzyme (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC (Wesel, Germany)
- Suitability of cells: cell line described in Guideline
- Cell cycle length, doubling time or proliferation index: 10-12 hours
- Sex, age and number of blood donors if applicable: n/a
- Whether whole blood or separated lymphocytes were used if applicable: n/a
- Number of passages if applicable: n/a
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: 40
- Normal (negative control) cell cycle time: 10-12 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1650 in 5 +- 0.5 % CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not stated
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

2000, 1000, 500, 250, 125, 63, 31 mg/ml

Vehicle / solvent:

RPMI 1640 medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 4*10^3

DURATION
- Preincubation period: none
- Exposure duration: 4h (with/without metabolic activation) 24 h (without metabolic activation)
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): not stated

Selection agent: Trifluorothymidine

NUMBER OF REPLICATIONS: 2


DETERMINATION OF CYTOTOXICITY
- Method: relativecloning efficiency (RCE)
- Any supplementary information relevant to cytotoxicity: determined also in a Pre-test

Rationale for test conditions:

standard test conditions comparable to guideline

Evaluation criteria:

total suspension growth (TSG)
relative suspension growth (RSG)
relative cloning efficiency (RCE)
relative total growth (RTG)
Colonies were counted manually under a binocular magnifying glass. In accordance with
their size, the colonies were classified into two groups:
Less than 25 % of the well’s diameter = small colony
More than 25 % of the well’s diameter = large colony

Statistics:

no statistics performed

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in one experiment for the highest dose observed after 24 hour treatment. All other concentrations were similar to the negative control

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect on pH observed
- Effects of osmolality: no effect on osmolality observed
- Precipitation: only in highest concentration did precipitation occur

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test
item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using
the cell line L5178Y in the absence and presence of metabolic activation.
Therefore, the test item 4-Oxo-4-p-tolylbutyric acid, adduct with 4-ethylmorpholine (Halox
570) is considered to be “non-mutagenic under the conditions of the mouse lymphoma
assay”.

Executive summary:

This study was performed to investigate the potential of 4-Oxo-4-p-tolylbutyric acid, adduct

with 4-ethylmorpholine (Halox 570) to induce mutations at the thymidine kinase locus (Tk1)

on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma

L5178Y Tk+/- cells.

The assay was performed in a pre-test and two independent experiments (experiment I

and II). The pre-test was performed to detect a potential cytotoxic effect of the test item.

Based on the results of this test the concentrations for the two experiments were determined.

Experiment I was performed with and without metabolic activation (liver enzyme S9 fraction

/ “liver S9 mix from male rats, treated with Aroclor 1254”) and a treatment period of

4 h. Experiment II was performed with a treatment period of 24 h in the absence of metabolic

activation.

The highest nominal concentration (2000 μg/mL) applied was chosen with regard to the

solubility of the test item in organic solvents and aqueous media and the cytotoxicity.

Appropriate reference mutagens were used as positive controls and showed a distinct increase

in induced mutant colonies, indicating that the tests were sensitive and valid.

The evaluated experimental points and the results are summarised in table 8-a, page 22.

No substantial and reproducible dose dependent increase in mutant colony numbers was

observed in both experiments. No relevant shift of the ratio of small versus large colonies

was observed up to the maximal concentration of the test item.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)