Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
FROM 29 APRIL 2021 to 21 JUNE 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Constituent 1
Chemical structure
Reference substance name:
Cerium gadolinium oxide
EC Number:
953-470-2
Molecular formula:
Gd2CeO5
IUPAC Name:
Cerium gadolinium oxide
Test material form:
solid: nanoform
Specific details on test material used for the study:
The test material was in the form of a white powder.

Method

Target gene:
TK
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS:
L5178Y/TK+/--3.7.2C mouse lymphoma cells
Source: American Type Culture Collection (ATCC, Manassas, USA).

MEDIA USED:
Horse serum :
- Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.

Basic medium :
- RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).

Growth medium :
- Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum.

Exposure medium :
- Cells were exposed to the test item in basic medium supplemented with 5% to 10% (v/v) heat-inactivated horse serum.

Selective medium :
- Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT) (Sigma).

Non-selective medium :
- Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and B-naphthoflavone (100 mg/kg body weight).
S9-mix was prepared immediately before use and kept refrigerated.
S9-mix components contained per mL physiological saline:
- 1.63 mg MgC12.6H20 (Merck);
- 2.46 mg KC1 (Merck);
- 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany);
- 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom);
- 4 µmol HEPES (Life technologies).
The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components, 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
The concentration of the S9-fraction in the exposure medium was 4% (v/v).
Test concentrations with justification for top dose:
Since the test item was not toxic and was difficult to dissolve in aqueous solutions, the highest concentration was determined by the solubility in the culture medium. The highest test item concentrations could show a slight to heavy precipitate in the exposure medium.

- First mutagenicity test:
0.04, 0.08, 0.4, 0.8, 4.0, 8.0, 20, 50, 75 and 100 µg/mL exposure medium.
This dose-range was based on the solubility test and range finding test.

- Second mutagenicity test:
0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 25, 50, 75 and 100 µg/mL exposure medium.
These dose levels were based on the dose range finding test and on the results of the first experiment.





Vehicle / solvent:
The vehicle for the test item was dimethyl sulfoxide (DMSO) (Merck Darmstadt, Germany).

No correction was made for the purity/composition of the test item.
A solubility test was performed based on visual assessment. The test item could not be dissolved or homogenously suspended in Milli-Q water, ethanol, acetone, hexane or tetrahydrofuran (THF).
The test item formed a white/beige homogenous suspension in DMSO at 10 mg/mL. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Test item concentrations were used within 1 hour after preparation.
The pH and the osmolarity of the culture medium containing the highest, non-precipitating concentration were recorded, by using a pH-meter and osmometer, respectively.
The final concentration of the solvent in the exposure medium was 1%
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (DMSO).
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: the solvent control was tested in duplicate. The test item concentrations and positive control were tested in single cultures.
- Number of independent experiments: 2.

TEST SYSTEM:
- Stock cultures of the cells were stored in the ultra-low freezer set to maintain -150°C. The cultures were checked for mycoplasma contamination.

ENVIRONMENTAL CONDITIONS:
- All incubations were carried out in a humid atmosphere (80 - 100%, actual range 54 - 100%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 32.7 — 38.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

CLEANSING:
- Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in growth medium containing 10E-4 M hypoxanthine (Sigma), 2 x 10E-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10E-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on growth medium containing hypoxanthine and thymidine only. After this period cells were returned to growth medium for at least 1 day before starting the experiment.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding : in the stock cultures, cell density was kept below 1 x 10E6 cells/mL.
- The test substance was added in the medium.

TREATMENT SCHEDULE:
- Exposure duration: in the first experiment, cell cultures were exposed for 3 hours to the test item in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test item in exposure medium for 24 hours in the absence of S9-mix.

FOR GENE MUTATION:
- Expression time: for expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10E6 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutant frequency (MF).
- Selection time: the microtiter plates for CEday2 and MF were incubated for 11 or 12 days.
- Method used: microwell plates.
- Selective agent: trifluorothymidine (TFT) in the selective medium which consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT) (Sigma).
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: per culture 8 x 10E6 cells (10E6 cells/mL for 3 hour treatment) or 6 x 10E6 cells (1.25 x 10E5 cells/mL for 24 hour treatment) were used. For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium. For determination of the mutant frequency (MF) a total number of 9.6 x 10E5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10E5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL of 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye.

- Criteria for small (slow growing) and large (fast growing) colonies: the small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony was classified as one small colony. A well containing more than one large colony was classified as one large colony. A well containing one small and one large colony was classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG).
- Any supplementary information relevant to cytotoxicity:
Relative Suspension Growth (RSG) = SG (test)/ SG (controls) x 100.
The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. The value obtained is the P(0), the zero term of the Poisson distribution: P(0) = number of empty wells/total number of wells.
The cloning efficiency (CE) was then calculated as follows: CE = -ln P(0)/number of cells plated per well.
The relative cloning efficiency (RCE) at the time of mutant selection = CE (test) /CE (controls) x 100.
The Relative Total Growth (RTG) was also calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture: RTG = RSG x RCE/100.


METHODS FOR MEASUREMENTS OF GENOTOXICIY
- The mutant frequency was expressed as the number of mutants per 10E6 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutant frequency (MF) was calculated as follows:
MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 10E6
Small and large colony mutation frequencies were calculated in an identical manner.
Rationale for test conditions:
SOLUBILITY: The test item was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test item concentration for the dose-range finding test was 100 µg/mL.
After 3 hours, the test item precipitated in the exposure medium at the concentration of 100 µg/mL. After 24 hours, the test item precipitated in the exposure medium at concentrations of 10 µg/mL and above.
The highest concentration which did not precipitate in the exposure medium in the 30 mL
centrifuge tubes was 10 µg/mL.

CYTOTOXICITY:
In the dose-range finding test, L5178Y cells were treated with a test item concentration range of 0.001 to 100 µg/mL in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
After 3 hours of treatment, both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 100 µg/mL compared to the solvent control.
After 24 hours of treatment, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 100 µg/mL compared to the solvent control.



Evaluation criteria:
Acceptability criteria to consider the test as acceptable:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%.
b) The spontaneous mutant frequency in the solvent control is > 50 per 10E6 survivors and < 170 per 10E6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutant frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10E-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10E-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10E-6)

Analysis:
Any increase of the mutant frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) +126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutant frequency of MF(controls) +126.

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: the pH at a concentration of 10 µg/mL was 7.178 compared to 7.193 in the solvent control.
- Data on osmolarity: the osmolarity at a concentration of 10 µg/mL was 0.468 Osm/kg compared to 0.469 Osm/kg in the solvent control.
- Water solubility: the test item could not be dissolved or homogenously suspended in Milli-Q water.

RANGE-FINDING STUDY (See Table 1 and Table 2 hereafter):
- In the dose-range finding test, L5178Y cells were treated with a test item concentration range of 0.001 to 100 µg/mL in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
After 3 hours of treatment, both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 100 µg/mL compared to the solvent control.
After 24 hours of treatment, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 100 µg/mL compared to the solvent control.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The mutant frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The suspension growth over the two-day expression period for cultures treated with DMSO was between 13 and 20 (3 hour treatment) and 64 and 66 (24 hour treatment).

Criteria for data analysis and interpretation:
- GEF criteria: the global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Gene mutation tests in mammalian cells (see Table 3 and Table 4 hereafter):
- Results from cytotoxicity measurements:
In the first mutagenicity test, after 3 hours, the test item precipitated in the exposure medium at concentrations of 75 and 100 µg/mL (the two highest dose levels) in the presence and absence of metabolic activation. The dose levels selected to measure mutant frequencies at the TK-locus were: 0.08, 0.4, 0.8, 4.0, 8.0, 20, 50 and 75 µg/mL exposure medium.
In the absence of S9-mix, the relative total growth of the highest test item concentration was 37% compared to the total growth of the solvent controls. No toxicity was observed at the highest test item concentration in the presence of S9-mix.

In the second mutagenicity test, without S9 mix, after 24 hours, the test item precipitated in the exposure medium at concentrations of 25 µg/mL and upwards. The dose levels selected to measure mutant frequencies at the TK-locus were: 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10 and 25 µg/mL.
No toxicity was observed at the highest test item concentration.

- Genotoxicity results:
First mutagenicity test:
No biologically relevant increase in the mutant frequency (i.e. none of the tested concentrations reaching a mutant frequency of MF(controls) + 126) at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix.

Second mutagenicity test:
No biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test item

HISTORICAL CONTROL DATA
- Negative (solvent/vehicle) historical control data: see Table 5 hereafter.
- Positive historical control data: see Table 6 hereafter.

Any other information on results incl. tables

Table 1: 


Dose-range Finding Test: Cytotoxicity of Cerium Gadolinium Oxide (3-Hour Treatment)






















































































































Dose (µg/mL)

cell count after 24 hours of
subculture (105 cells/mL)


cell count after 48 hours of
subculture (105 cells/mL)
SGRSG (%)
 Without metabolic activation

SC



4.1



9.9



20



100



0.001



4.2



9.9



21



102



0.01



4.2



9.5



20



98



0.1



4.8



8.0



19



95



1



4.1



10.9



22



110



10



5.0



9.7



24



119



100 (1)



3.3



10.8



18



88



 


With metabolic activation

SC



3.3



10.5



17



100



0.001



3.2



10.7



17



99



0.01



3.2



11.1



18



103



0.1



3.3



10.8



18



103



1



3.1



10.4



16



93



10



3.4



11.3



19



111



100 (1)



2.3



12.3



14



82



All calculations were made without rounding off.


SC = solvent control


SG = suspension growth


RSG = relative suspension growth


(1) the test item precipitated in the exposure medium.


SG = Suspension growth = [Cell count after 24 hour of subculture (Day 1) /1.6 x 105] x [Cell count after 48 hours of subculture (Day 2)/1.25 x 105]


RSG = [SG(test)/SG(control)] x 100


 


Table 2: 


Dose-range Finding Test: Cytotoxicity of Cerium Gadolinium Oxide (24-Hour Treatment)


































































Dose (µg/mL)



cell count after 24 hours of
subculture (105 cells/mL)



cell count after 48 hours of
subculture (105 cells/mL)



SG



RSG (%)


 Without metabolic activation

SC



10.1



9.2



59



100



0.001



11.1



8.9



63



106



0.01



11.6



8.5



63



106



0.1



12.2



8.7



68



115



1



11.3



8.9



65



109



10



10.7



8.7



59



100



100 (1)



10.4



8.9



59



100



All calculations were made without rounding off.


SC = solvent control


SG = suspension growth


RSG = relative suspension growth


(1) the test item precipitated in the exposure medium


SG = Suspension growth = [Day 0 Cell count /1.25 x 105] x [Day 1 Cell count /1.25 x 105]


RSG = [SG(test)/SG(control)] x 100


 


Table 3:  


Experiment 1: Cytotoxic and Mutagenic Response of Cerium Gadolinium Oxide in the Mouse Lymphoma L5178Y Test System














































































































































































































































Dose (µg/mL)RSG (%)CEday2 (%)RCE (%)RTG (%)Mutant frequency per 106 survivors
total( smalllarge )
Without metabolic activation - 3-hour treatment

SC


100107100100

88


(1175)

SC


94

98


(2372)

0.08



84



118


11899

101


(2374)

0.4



98



86


8685

107


(2776)

0.8



106



84


8489

121


(4570)

4.0



95



107


106101

76


(1658)

8.0



112



85


8595

87


(1767)

20



102



108


108110

93


(2664)

50



68



125


12584

90


(2165)

75 (1)



56



67


6737

137


(5675)

MMS



66



38


3825

1852


(1032523)
With metabolic activation - 3-hour treatment

SC


10098100100

94


(1083)

SC


101

99


(1087)

0.08



146



85


86125

131


(20107)

0.4



105



110


110116

105


(1685)

0.8



87



85


8674

144


(25113)

4.0



121



102


103124

82


(1862)

8.0



117



76


7689

174


(27140)

20



99



89


8989

127


(2597)

50



81



113


11492

160


(46102)

75 (1)



89



88


8878

133


(4383)
CP

46



24


2411

2929


(1510934)

Note: all calculations were made without rounding off


RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth;


SC = Solvent control= DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamide


(1) = the test item precipitated in the exposure medium.


 


Table 4 :


Experiment 2: Cytotoxic and Mutagenic Response of Cerium Gadolinium Oxide in the Mouse Lymphoma L5178Y Test System

































































































































Dose (µg/mL)



RSG (%)



CEday2 (%)



RCE (%)



RTG (%)



Mutant frequency per 106 survivors



total



( small



large )



Without metabolic activation - 24-hour treatment



SC



100



115



100



100



128



(78



41)



SC



110



128



(81



40)



0.01



95



102



91


87

136



(81



47)



0.05



101



104


9293

111



(57



47)



0.1



103



111


99103

104



(73



26)



0.5



93



98


8781

123



(75



41)



1.0



108



90


8086

122



(70



46)



5.0



115



101


90104

136



(93



35)



10



108



113


101108

103



(55



43)



25 (1)



110



98


8796

135



(80



47)



MMS



83



83


7461

823



(377



310)



Note: all calculations were made without rounding off


RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth;


SC = Solvent control= DMSO; MMS = Methylmethanesulfonate; 


(1) = the test item precipitated in the exposure medium.


 


Table 5:


Historical Control Data of the Spontaneous Mutant Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

















































 



Mutant frequency per 106 survivors



-S9 Mix



+S9 mix



3 hour treatment



24 hour treatment



3 hour treatment



Mean



100



99



100



SD



28



28



28



n



96



87



93



Lower Control Limit
(95% Control Limits)



45



44



46



Upper Control Limit
(95% Control Limits)



154



153



154



SD = Standard deviation
n = Number of observations


Distribution historical negative control data from experiments performed between May 2018 and May 2021.


 


Table 6:


Historical Control Data of the Mutant Frequencies of the Positive Controls for the Mouse Lymphoma Assay

















































 



Mutant frequency per 106 survivors



-S9 Mix



+S9 mix



3 hour treatment



24 hour treatment



3 hour treatment



Mean



961



776



1154



SD



408



236



648



n



94



89



93



Lower Control Limit
(95% Control Limits)



162



313



-116



Upper Control Limit
(95% Control Limits)



1761



1239



2425



SD = Standard deviation
n = Number of observations


Distribution historical negative control data from experiments performed between May 2018 and May 2021.

Applicant's summary and conclusion

Conclusions:
In conclusion, the test substance is not mutagenic in the TK mutation test system under the experimental conditions described in this study.
Executive summary:

The objective of this study, performed according to OECD guideline 490 and in accordance with GLP, was to evaluate the mutagenic potential of the test substance by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix).


The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The vehicle of the test item was dimethyl sulfoxide (DMSO).


In the first experiment, the test item was tested up to a concentration of 75 µg/mL in the absence and presence of S9-mix. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 37% in the absence of S9-mix. No toxicity was observed at this dose level in the presence of S9-mix. The test item precipitated in the culture medium at this dose level.


In the second experiment, the test item was tested up to concentrations of 25 µg/mL in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level. The test item precipitated in the culture medium at this dose level.


The mutant frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.


Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.


In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutant frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.


In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutant frequency.


In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this study.