Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

TODI was examined in three in vitro genetic toxicity studies (AMES, Mouse Lymphoma Assay, Chromosome Aberration Test) with and without S9 mix and two different in vivo genetic toxicity (MNT and UDS assay) studies.

The results demonstrated that TODI is mutagenic in bacteria (without S9 mix) and mammalian cells (with and without S9 mix) and increased structural chromosome aberrations with and without S9 mix.

No genotoxicity potential of TODI was observed in vivo.

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-01-01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reference:
Composition 1
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Species / strain:
S. typhimurium TA 102
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Species / strain:
S. typhimurium TA 1538
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Species / strain:
other: TA104
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Species / strain:
E. coli WP2 uvr A pKM 101
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
10, 20, 50, 100, 200, 500, 1000, 2000 µg/plate
Vehicle:
DMSO
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -2-(2-furyl)-3-(5-nitro-2furyl)acrylamide
Remarks:
without activation: TA100, WP2uvrA and WP2uvrA/pKM101
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without activation: TA1585
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without activation: TA1537
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without activation: TA1538
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without activation: TA1538
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: bleomycin
Remarks:
without activation: TA102
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: pyruvic aldehyde
Remarks:
without activation: TA104
Negative controls:
not specified
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-amino anthracene
Remarks:
with activation: all cited strains (S. typhimurium TA 1535, TA 1537, TA1538, TA 98, TA 100, TA 102, TA 104, WP2uvrA and WP2uvrA/pKM101)
Evaluation criteria:
Two-fold rule criteria was used for data evaluation (Ames et al., 1975). The chemicals are considered to be mutagenic when a dose- related increase in revertant colonycount is observed and the number of the revertant colonies per plate with the test substance is more than twice that of the negative control (solvent control) and when the reproducibility of the test result is observed.
Mutagenic potency was calculated by the following equation and maximum value of mutagenic potency was expressed as the specific activity on the data sheet:
mutagenic potency (induced revertants / mg test substance) = (number of induced revertants on the dose X - number of revertant on the solvent control) / mg test chemical on the dose X.
Statistics:
Determination of Mean and Standard deviation
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
other: TA 104
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
Mutagenicity (frame-shift) was observed for S. typhimurium TA 98 and TA 1538 at concentrations of 10 to 1000 µg/plate. An evaluation of 2000 µg/plate was not possible due to growth inhibition.
The specific mutagenicity in case of TA 98 was 5900 revertants /mg, in case of TA 1538 13900 revertants /mg.
Conclusions:
The reported data of this mutagenicity assay according to AMES shows, that after metabolic activation, the test item induced gene mutations by frameshift in the genome of 2 of 9 strains (S. typhimurium TA 98 and TA 1538) used. Therefore, TODI is considered mutagenic in this bacterial reverse mutation assay.
Executive summary:

A study was performed to assess the mutagenicity potency of TODI. The method was similar to OECD Guideline 471 (Bacterial Reverse Mutation Assay and EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria) of Commission Directive 92/69/EEC (which constitues Annex V of Council Directive 67/548/EEC). The test strains S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100, TA 102, TA 104 and E. coli WP2 uvr A as well as E. coli WP2 uvr A pKM 101 were examined at 10, 20, 50, 100, 200, 500, 1000, 2000 µg TODI/plate. Mutagenicity (frame-shift) was observed for S. typhimurium TA 98 and TA 1538 at concentrations of 10 to 1000 µg/plate. An evaluation of 2000 µg/plate was not possible due to growth inhibition. The reported data of this mutagenicity assay according to AMES shows, that after metabolic activation, the test item induced gene mutations by frameshift in the genome of 2 of 9 (S. typhimurium TA 98 and TA 1538) strains used. Therefore, TODI is considered mutagenic in this bacterial reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1993
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In vitro gene mutation test in mammalian cells
Test material information:
Composition 1
Target gene:
Thymidine kinase (TK) locus L5178Y 3.7.2c mouse lymphoma cell line
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK (cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time)
- Cell cycle length, doubling time or proliferation index: 12 hours

MEDIA USED
- Type and identity of media including CO2 concentration: RPMI 1640 medium supplemented with 10% donor horse serum and 20 mM Hepes buffer (R10), 5% CO2
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 from the liver of male Sprague-Dawley rats
Test concentrations with justification for top dose:
1. experiment: 2, 4, 8, 12, 16 µg/mL without and with S9
2. experiment: 4, 8, 16, 20, 24 µg/mL without S9 and 4, 8, 12, 14, 16 µg/mL with S9
3. experiment: 4, 6, 8, 10, 12 µg/mL without S9 and 6, 8, 10, 12, 14 µg/mL with S9
Vehicle:
- Vehicle used: acetone
- Justification for choice of solvent: test item hydrolytically unstable
Negative controls:
yes
Solvent controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in presence of metabolic activation (S9)
Negative controls:
yes
Solvent controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
in absence of metabolic activation (S9)
Details on test system and conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 1 x 10E+6 cells/mL (for 3 hours exposure), 0.3 x 10E+5 cells/mL (for 24 hours exposure)

DURATION
- Exposure duration:
- 1. Experiment: 3 hours (+/- S9)
- 2. Experiment: 3 hours (+ S9) and 24 hours (without S9)
- 3. Experiment: 3 hours (+/- S9)
- Expression time (cells in growth medium): not specified
- Selection time: not specified
- Fixation time (start of exposure up to fixation or harvest of cells): not specified

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 2000 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: colony formation


Evaluation criteria:
Significant result, if two or more of the following criteria are met:
- Statistically significant increase in mutant frequency
- Greater than three-fold increase in the mutant frequency per survivor over the negative control value
- Dose-related increase in the mutant frequency per survivor
- Increase in the absolute number of mutants
Statistics:
Calculation of plating efficiency (viability) by using the zero term of the Poisson distribution method.
Calculation of mutation frequency using a dedicated computer program which followis the statistical guidelines recommended by the UKEMS.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed in a concentration range of 0.63 – 40 µg/mL with and without S9 mix. Toxicity was observed with the test material at and above 20 µg/mL in the absence and presence of S9 mix. A rapid onset of test material toxicity was observed such that very few cells were observed in the cultures treated with test material at and above 20 µg/mL in the pulse treatments with and without S9, and at 40 µg/mL in the 24- hour continuous exposure in the absence of S9.

HISTORICAL CONTROL DATA
- Positive historical control data: 362.33 – 3108.19 (- S9); 226.33 – 1559.97 (+ S9)
- Negative (solvent/vehicle) historical control data: 31.93 – 194.07 (- S9); 54.34 – 194.86 (+ S9)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Plating efficiency
- For more detailed information on cytotoxicity please refer to "Attached background material"
Conclusions:
TODI induced statistically significant increases in the mutant frequency in the absence and presence of metabolic activation at the TK +/- locus in L5178Y cells and is therefore considered to be mutagenic under the conditions of the test.
Executive summary:

The mutagenic potential of the test substance was investigated in a Mouse Lymphoma Assay according to OECD 476 and EU method B.17.

L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to five dose levels, in duplicate, together with vehicle (solvent) and positive controls. Three-hour pulse exposures were used, both with and without activation, in Experiment 1. A second experiment was performed in which the exposure time without activation was increased to 24 hours. Small but statistically significant increases in mutant frequency were observed in Experiments 1 and 2. A third, confirmatory, experiment was performed using a three-hour pulse treatment both with and without S9.

The dose range used was selected on the results of a preliminary toxicity test and was 2, 4, 8, 12 and 16 μg/mL for the first experiment. For the second experiment the dose range used was 4 to 24 μg/mL in the absence of S9 and 4 to 16 μg/mL with S9. The dose range used in the third experiment was 4 to 12 μg/mL without S9 and 6 to 14 μg/mL with S9.

The vehicle (solvent) controls gave acceptable levels of mutant frequencies for the L5178Y cell line at the TK +/- locus. The positive control treatments, both in the absence and in the presence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

TODI induced small but statistically significant, increases in mutant frequency in each of three experiments. TODI was shown to be mutagenic to L5178Y cells under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information
TODI was further evaluated with two in vivo tests which consider systemic metabolic activation (Unscheduled DNA synthesis UDS assay in the rat liver and Micronucleus test in the mouse). Even if tested at systemically toxic doses, TODl did not show any significant increase in the incidence of micronucleated polychromatic erythrocytes or genotoxic potential. Thus based on the negative results observed in the in vivo tests, TODI is considered to be non-mutagenic.
Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-10-29 to 1997-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian somatic cell cytogenicity assay
Test material information:
Composition 1
Species:
mouse
Strain:
other: Albino Crl:CD-1TM (ICR) BR
Details on species / strain selection:
The mouse has been shown to be a suitable model for this type of study was recommended in the test method.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (IUK) Limited, Margate, Kent
- Age at study initiation: five to eight weeks
- Weight at study initiation: 25 to 30 g (male); 20 to 26 g (female)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in groups up to five in solid-floor polypropylene cages with woodflakes bedding
- Diet (e.g. ad libitum): free access to food (Rat and Mouse Expanded Diet No. 1, Special Diets Services Limited, Wtiham, Essex, UK)
- Water (e.g. ad libitum): free access to drinking water
- Acclimation period: five days at minimum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22°C
- Humidity (%): 59-64 %
- Air changes (per hr): approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours darkness
Route of administration:
intraperitoneal
Vehicle:
- Vehicle/solvent used: arachis oil
- Justification for choice of solvent/vehicle: The test substance is hydrolytically unstable.
- Concentration of test material in vehicle: 12.5, 25, and 50 mg/mL (main study)
- Amount of vehicle: 10 mL/kg
- Lot/batch no.: T30
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil.

Duration of treatment / exposure:
single exposure
Frequency of treatment:
Once
Post exposure period:
24 hours after dosing (first group); 48 hours after dosing (second group)
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
5 animals per sex and dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s): guideline suggestion
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A range-finding study was conducted to determine a suitable dose level. The level selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 2000 mg/kg bw.

TREATMENT AND SAMPLING TIMES: Animals were dosed once only via the route with the test material at 125, 250 or 500 mg/kg bw. Sampling was done 24 or 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: 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-Grünwald/Giemsa.

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined using light microscopy at x 1000 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.


Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythorcytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.

A positive mutagenic response was demonstrated when a statistically significant increase in the number of imcronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.

If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a √ (X + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250 – 2000 mg/kg bw (ip), 2000 mg/kg bw (oral)
- Clinical signs of toxicity in test animals: none (oral route); premature deaths were observed at and above 1000 mg/kg and clinical signs were observed at and above 250 mg/kg, as follows: hunched posture, lethargy, ptosis, dehydration, ataxia, splayed gait and decreased respiratory rate (ip administration)

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No statistically significant increases in the frequency of micronucleated PCEs compared to the vehicle control were observed.
- Ratio of PCE/NCE: A statistically significant decreases in the PCE/NCE ratio in the 24-hour 125 and 500 mg/kg bw test material groups compared to the vehicle control were observed.
- Appropriateness of dose levels and route: The maximum tolerated dose (MTD) of the test material selected for use in the main study was 500 mg/kg bw, with 125 and 250 mg/kg bw as the lower dose levels. The intraperitoneal route of administration was selected for use based on the results of the range-finding study.


Conclusions:
The test material did not produce a significant in crease the frequency of micornuclei in polychromatic erythrocytes of mice under the conditions of the test. Thus, the test material was considered to be non-genotoxic.
Executive summary:

A study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method used has been 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 EEC Commission Directive 92/69/EEC, the US, EPA, TSCA and FIFRA guidelines and the Japanese Ministry of International Trade and Industry guidelines for the testing of new chemical substances. Sampling of bone marrow did not occur after 48 hours, the need for the later sampling time (72 hours) is now considered to be unnecessary.

A range-finding study was performed to find suitable dose levels for the test material and route of administration. The micronucleus study was conducted via the intraperitoneal route in groups of ten mice (five males and five females) at the maximum tolerated dose (MTD) 500 mg/kg with 125 and 250 mg/kg as the two lower dose levels. Animals were killed 24 and 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single intraperitoneal dose of arachis oil or dosed orally with cyclophosphamide, to serve as vehicle and positive controls respectively. 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. A statistically significant decrease in the PCE/NCE ratio was observed in the 24-hour test material dose groups when compared to their concurrent control groups. This observation and the presence of clinical signs at and above the 125 mg/kg dose level and the premature deaths in the 24 and 48 -hour 500 mg/kg test material dose groups was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes. The test material, TODI, was considered to be non-genotoxic under the conditions of the test.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-08-11 to 1998-02-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Qualifier:
according to
Guideline:
other: UKEMS Sub-Committee on Guidelines for Mutagenicity Testing: Report, Part II revised (supplementary mutagenicity tests: UKEMSrecommended procedures, 1993)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
unscheduled DNA synthesis
Test material information:
Composition 1
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Rats are commonly used.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK, Margate, Kent
- Age at study initiation: six to nine weeks old
- Weight at study initiation: 225 to 277 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in groups of up to four in solid-floor polypropylene cages with woodflake bedding
- Diet: ad libitum (Rat and Mouse Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK)
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 53 to 66
- Air changes (per hr): ~ 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: arachis oil
- Justification for choice of solvent/vehicle: The test item is hydrolytically unstable.
- Concentration of test material in vehicle: 100 mg/mL and 200 mg/mL (range-finding); 70 mg/mL and 200 mg/mL (experiment 1, 2);
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Lot/batch no.: 1 (experiment 1); T7 (experiment 2)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test material was freshly prepared as required as suspension at the appropriate concentrations in arachis oil.

Duration of treatment / exposure:
single exposure
Frequency of treatment:
once
Post exposure period:
16 hours (experiment 1); 2 hours (experiment 2)
Dose / conc.:
700 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
Range-finding study: 2 male per dose
Experiment 1, 2: 4 male per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
2-acetylaminofluorene (16-hour positive control; experiment 1); N,N'-Dimethylhydrazine dihydrochloride (2-hour positive control; experiment 2)
- Route of administration: oral administration
- Doses / concentrations: 50 mg/kg bw (2-acetylaminofluorene); 20 mg/kg bw (N,N'-Dimethylhydrazine dihydrochloride)
Tissues and cell types examined:
Hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Range-finding test (1000, 2000 mg/kg): Maximum recommended dose 2000 mg/kg; lower dose level: 700 mg/kg

TREATMENT AND SAMPLING TIMES: Sampling was done 16 hours and 2 hours after dosing, respectively.

DETAILS OF SLIDE PREPARATION:
The isolation of the hepatocytes was conducted using a two stage in situ method. The post-aortic vena cava was ligatured, and then cannulae were introduced into the hepatic portal vein and superior vena cava. The liver were first perfused with a buffered medium containing chelating agents to remove metal ions, this was followed by a digest medium containing collagenase and calcium containing the liver to diassociate into a single population.
The liver was removed from the body and the capsule opened and the liver cells were suspended in attachment medium. The suspended cells were then passed through nylon gauze to removed large particles and debris. The cells were centrifuged and washed three times using a buffered medium and finally suspended in attachment medium at 1.5 x 10E+5 viable cells/mL. They were then seeded onto coverslips in six well plates at 3 mL/well (six coverslips per animal) and subsequently incubated at 37°C in 5% CO2 for 1.5 to 2 hours.
Radiolabelling of cells was done as follows: Hepatocytes were washed with serum-free medium which was replaced with 2 mL of serum-free medium containing 10 µL/mL (370 kBq/mL) of [3H] thymidine. Incubation took place for 3 hours at 37°C and 5% CO2. To remove excess radiolabel from cultures, they were washed with serum-free medium containing 0.25 mM unlabelled thymidine solution (‘cold-chase’ procedure).
Fixation of cells was done in freshly prepared 1:3 acetic acid: methanol. After washing and drying of coverslips, they were coated with Ilford K2 autoradiographic emulsion and incubated at 4°C for 7 to 14 days in a sealed light proof container. Following the exposure period the slides were processed using photographic developer and fixative, and then the cells were stained using haematoxylin/eosin. When the cells were dry they were coverslipped using a mounting medium.

METHOD OF ANALYSIS:
Automated image analysis system linked to a computer program (Grain) which followed the UKEMS guidelines for statistical analysis.The method used to score the slides was an area counting method which complies with the UKEMS guidelines. Ideally a minimum of three slides for each animal were scored with a maximum of 50 cells per slide giving an accumulative total of 150 cells per animal.
The number of silver grains within the nucleus were first observed and recorded as the Nuclear Count (N). Three cytoplasmic areas (each equal to the nuclear area) were also scored to give the Mean Cytoplasmic Count (C). A net Nuclear Grain Count (N-C), was then calculated. Cells in 'S'-phase were not scored for grain counts, these were easily recognisable due to the dense 'graining' appearance of the nucleus. Mean values of (N), (C), (N-C) and percentage cells in repair (% R) were calculated. Values of (N-C) are typically in the range of -6 to -2 for vehicle controls, although variations in experimental conditions can give values outside of this range.




Evaluation criteria:
The UKEMS guidelines suggest that in positive responses at least 20 % of all cells assessed should be in repair, i.e. undergoing unscheduled DNA synthesis, having a (N-C) value of +5 or greater.
Statistics:
None
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 mL/kg, 2000 mL/kg
- Clinical signs of toxicity in test animals: No clinical signs were observed.
- Rationale for exposure: The maximum recommended dose and one lower dose was selected.
- Other: No premature deaths were observed.

Conclusions:
The test material, TODI, did not induce any marked or toxicologically significant increases in the incidence of cells undergoing unscheduled DNA synthesis in isolated rat hepatocytes following in vivo exposure for 2 and 16 hours. Therefore, TODI, was considered to be non-genotoxic under the conditions of this study.
Executive summary:

A study was performed to assess the potential of the test material to induce DNA repair in isolated rat hepatocytes following oral administration to rats. The method used has been designed to comply with the OECD Guidelines for Testing of Chemicals No. 486 and follows the recommendations of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing: Report, Part II revised (Supplementary Mutagenicity Tests: UKEMS recommended procedures, 1993).

Following a preliminary range-finding study to confirm toxicity, the UDS assay was conducted using the test material at the maximum recommended dose (MRD) of 2000 mg/kg with 700 mg/kg as the lower dose level. The study was performed in two parts, in Experiment 1 the livers were perfused approximately 16 hours after dosing, and in Experiment 2, perfusion was performed approximately 2 hours after dosing. Following perfusion the liver hepatocytes were processed to give stained slides which were then scored using a microscope and an automated image analysis system. The method used for scoring the hepatocytes was an area counting method which complies with the UKEMS guidelines.

Further groups of rats were given a single oral dose of arachis oil, 2 -Acetylamniofluorene (2AAF) at 16 hours or N,N'-dimethylhydrazine dihydrochloride (NDHC) at 2 hours to serve as vehicle and positive controls respectively. There was no increase in the incidence of unscheduled DNA synthesis in animals dosed with test material at either time point. The positive controls both produced marked increases in the incidence of cells in repair. The test material, TODI, was considered to be non-genotoxic under the conditions of the test.

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

Additional information

Additional information from genetic toxicity in vivo:

In vitro mutagenicity tests

(1) AMES test

The method followed OECD Guideline 471 (Bacterial Reverse Mutation Assay) and EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria) of Commission Directive 92/69/EEC (which constitues Annex V of Council Directive 67/548/EEC). The test strains S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100, TA 102, TA 104 and E. coli WP2 uvr A as well as E. coli WP2 uvr A pKM 101 were examined at 10, 20, 50, 100, 200, 500, 1000, 2000 µg TODI/plate. Mutagenicity (frame-shift) was observed for S. typhimurium TA 98 and TA 1538 at concentrations of 10 to 1000 µg/plate. An evaluation of 2000 µg/plate was not possible due to growth inhibition. The reported data of this mutagenicity assay according to AMES shows, that after metabolic activation, the test item induced gene mutations by frameshift in the test strains TA 98 and TA 1538. Therefore, TODI is considered mutagenic in this bacterial reverse mutation assay.

(2) Mouse lymphoma assay

TODI was tested for gene mutation according to EU Method B.17 / OECD Guideline 476. At the assay L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to five dose levels, in duplicate, together with vehicle (solvent) and positive controls. Three-hour pulse exposures were used, both with and without activation, in Experiment 1. A second experiment was performed in which the exposure time without activation was increased to 24 hours. Small but statistically significant increases in mutant frequency were observed in Experiments 1 and 2. A third, confirmatory, experiment was performed using a three-hour pulse treatment both with and without S9.

The dose range used was selected on the results of a preliminary toxicity test and was 2, 4, 8, 12 and 16 μg/mL for the first experiment. For the second experiment the dose range used was 4 to 24 μg/mL in the absence of S9 and 4 to 16 μg/mL with S9. The dose range used in the third experiment was 4 to 12 μg/mL without S9 and 6 to 14 μg/mL with S9.The vehicle (solvent) controls gave acceptable levels of mutant frequencies for the L5178Y cell line at the TK +/- locus.The positive control treatments, both in the absence and in the presence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.
TODI induced small but statistically significant, increases in mutant frequency in each of three independent experiments. TODI was shown to be mutagenic to L5178Y cells under the conditions of the test.

(3) Chromosome Aberration Test

The method followed OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) and EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test) of Commission Directive 92/69/EEC (which constitues Annex V of Council Directive 67/548/EEC). The test substance was examined with and without metabolic activation up to 0.6 mg/mL. The reported data of this Chromosome Aberration Test shows, that under the experimental conditions described, the test item induced chromosome aberration after metabolic activation at 0.6 mg/mL. The D20 value was calculated to be 0.79 (structural chromosomal aberrations). The D20 value is the concentration (mg/mL) required to induce any aberration in 20% of metaphases.Therefore, TODI is considered mutagenic in this Chromosome Aberration Test.

 

In vivo mutagenicity tests

(4) Unscheduled DNA synthesis (UDS) assay

A study was performed to assess the potential of the test material to induce DNA repair in isolated rat hepatocytes following oral administration to rats. The method used has been designed to comply with the OECD Guidelines for Testing of Chemicals No. 486 and follows the recommendations of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing: Report, Part II revised (Supplementary Mutagenicity Tests: UKEMS recommended procedures, 1993).

Following a preliminary range-finding study to confirm toxicity, the UDS assay was conducted using the test material at the maximum recommended dose (MRD) of 2000 mg/kg with 700 mg/kg as the lower dose level. The study was performed in two parts, in Experiment 1 the livers were perfused approximately 16 hours after dosing, and in Experiment 2, perfusion was performed approximately 2 hours after dosing. Following perfusion the liver hepatocytes were processed to give stained slides which were then scored using a microscope and an automated image analysis system. The method used for scoring the hepatocytes was an area counting method which complies with the UKEMS guidelines.

Further groups of rats were given a single oral dose of arachis oil, 2 -Acetylamniofluorene (2AAF) at 16 hours or N,N'-dimethylhydrazine dihydrochloride (NDHC) at 2 hours to serve as vehicle and positive controls respectively.
There was no increase in the incidence of unscheduled DNA synthesis in animals dosed with test material at either time point.
The positive controls both produced marked increases in the incidence of cells in repair. The test material, TODI, was considered to be non-genotoxic under the conditions of the test.

(5) Micronucleus test

A study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method used has been 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 EEC Commission Directive 92/69/EEC, the US, EPA, TSCA and FIFRA guidelines and the Japanese Ministry of International Trade and Industry guidelines for the testing of new chemical substances. Sampling of bone marrow did not occur after 48 hours, the need for the later sampling time (72 hours) is now considered to be unnecessary.

A range-finding study was performed to find suitable dose levels for the test material and route of administration. The micronucleus study was conducted via the intraperitoneal route in groups of ten mice (five males and five females) at the maximum tolerated dose (MTD) 500 mg/kg with 125 and 250 mg/kg as the two lower dose levels. Animals were killed 24 and 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single intraperitoneal dose of arachis oil or dosed orally with cyclophosphamide, to serve as vehicle and positive controls respectively.

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. A statistically significant decrease in the PCE/NCE ratio was observed in the 24-hour test material dose groups when compared to their concurrent control groups. This observation and the presence of clinical signs at and above the 125 mg/kg dose level and the premature deaths in the 24 and 48 -hour 500 mg/kg test material dose groups was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

The test material, TODI, was considered to be negative in this micronucleus test.

 


Justification for classification or non-classification

Based on results of the two in vivo genetic toxicity studies, TODI, was not classified and labelled as genotoxic according to Regulation 1272/2008/EC (CLP).