Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was negative in 3 Ames tests, a Mouse Lymphoma Assay, an in vitro chromosomal aberration assay and an in vivo Micronucleus test.

Endpoint Conclusion:No adverse effect observed (negative)

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:
weight of evidence
Study period:
4 November 1981 - 21 November 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well-conducted and reasonably documented study performed under GLP. - The test material is described by trade name, physical appearance and lot number; there is no CoA. - E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 have not been included and therefore with this test you may not detect certain oxidising mutagens, cross-linking agents and hydrazines. - An independent repeat was not performed to confirm negative results and no justification is given for not repeating it.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
- E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 have not been included and therefore with this test you may not detect certain oxidising mutagens, cross-linking agents and hydrazines.
- An independent repeat was not performed to confirm negative results and no justification is given for not repeating it.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S-9 prepared from male rat liver following enzyme induction by a single IP dose of Aroclor 1254, 500 mg/kg as a 20% w/v solution in soya bean oil.
Test concentrations with justification for top dose:
2.0,0.67, 0.22, 0.074, 0.025 and 0 mg per plate
Vehicle / solvent:
ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See "Any other information..."
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 days
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells):not applicable

SELECTION AGENT (mutation assays): histidine
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: colonies (revertants which are histidine independent) are counted.

DETERMINATION OF CYTOTOXICITY
- Method: other: The background lawn of bacterial growth is examined microscopically.
Evaluation criteria:
No data
Statistics:
No data
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: seen at 1mg/plate
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: The range finding study showed no toxicity but precipitation was seen at 1mg/plate and above.

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Because of inconclusive results, the data of the first experiment. (November 4) with strain TA 1538 have been omitted from this report.

Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that the substance did not show any mutagenic activity in the Ames test.
Executive summary:

The substance was examined for mutagenic activity. In the Ames test using the histidine requiring S. typhimurium mutants TA 1535, TA 1537, TA 1538, TA 98 and TA 100 as indicator strains and a liver microsome fraction of Aroclor-induced rats for metabolic activation. It was concluded that it did not show any mutagenic activity in the Ames test.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well-conducted and reasonably documented study. The results data was retrieved from the NTP website. Not all required strains were included in the study. No data on GLP. No study report avialable. Results are available online and published. - The test material is described by trade name, physical appearance and lot number; there is no CoA. - E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 have not been included and therefore with this test you may not detect certain oxidising mutagens, cross-linking agents and hydrazines.
Qualifier:
according to
Guideline:
other: Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E (1983): Salmonella mutagenicity results for 250 chemicals. Environ Mutagen 5(Suppl 1):3-142.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
- E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 have not been included and therefore with this test you may not detect certain oxidising mutagens, cross-linking agents and hydrazines.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
S. typhimurium, other: TA98, TA97, TA100, TA1535
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from rat and hamster
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9-mix: sodium azide (TA1535 and TA100), 9-aminoacridine (TA97) and 4-nitro-o-phenylenediamine (TA98). +S9-mix: 2-aminoanthrace
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min.
- Exposure duration: 48 hours
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent):Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): histidine
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable

NUMBER OF REPLICATIONS: At least five doses of each chemical were tested in triplicate. Experiments were repeated at least one week following the initial trial.

NUMBER OF CELLS EVALUATED: Plates were machine counted (New Brunswick, Edison, NJ; Artek, Farmingdale, NY) unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the background agar. At the discretion of the investigators, plates with low numbers of colonies were counted by hand.

DETERMINATION OF CYTOTOXICITY
- Method: detection of background lawn
Evaluation criteria:
A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible dose-related reponse over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a "+W" response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or nonmutagenicity.
Statistics:
None
Species / strain:
S. typhimurium, other: TA98, TA97, TA100, TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
no data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The sustance did not show any mutagenic activity in the Ames test.
Executive summary:

The substance was examined for mutagenic activity in the Ames test. Histidine requiring S. typhimurium mutants TA1535, TA97, TA98 and TA 100 were used as indicator strains and a liver microsome fraction of rats and hamsters for metabolic activation. It was concluded that it did not show any mutagenic activity in the Ames test.

Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
According to OECD 476 Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
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)
Deviations:
no
Principles of method if other than guideline:
first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase Locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: Clone 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
Experiment I (4 hours treatment)
without S9 mix : 1.5, 2.9, 5.9, 11.8, 23.5, 47.0, 70.5, 94.0 µg/mL
with S9 mix: 2.9, 5.9, 11.8, 23.5, 47.0, 94.0 (only culture II analysed), 141.0, 188.0 µg/mL

Experiment II (24 hours treatment)
without S9: 2.9, 5.9, 11.8, 23.5, 47.0, 70.5, 94.0 µg/mL

Experiment II (4 hours treatment)
with S9 mix: 2.9, 5.9, 11.8, 23.5, 47.0, 70.5, 94.0 µg/mL
Following the expression phase of 48 hours the cultures at the maximum concentration of 70.5 and 94.0 µg/mL in experiment I without metabolic activation and at 94.0 (in culture I only), 141.0, and 188.0 µg/mL with metabolic activation were not continued due to exceedingly severe cytotoxic effects. In experiment II the cultures at 70.5 and 94.0 µg/mL with and without metabolic activation were not continued for the same reason. The cultures at 1.5 µg/mL in experiment I without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the
corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Statistics:
Linear regression analysis (least squares) using SYSTAT(R) 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (solvent control: pH 7.26; test item at 1500 µg/mL: pH 7.28 )
- Effects of osmolality: (solvent control: osmolarity 379; test item at 1500 µg/mL: osmolarity 354)
- Evaporation from medium: Not examined
- Water solubility: --
- Precipitation:
Pre-experiment:
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed by the unaided eye at 187.5 µg/mL and above in the parts following 4 hours treatment. After 24 hours treatment precipitation occurred at the maximum concentration of 1500 µg/mL.

Main experiments: No precipitation occurred

- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 11.7 µg/mL and 1500 µg/mL were used. Relevant toxic effect (relative suspension growth, RSG below 50% of the solvent control) occurred at 11.7 µg/mL and above following 4h treatment without metabolic activation. Following 4h treatment with metabolic activation the RSG was reduced <50% at 23.4 µg/mL. After 24h treatment cytotoxic effects were noted at 46.9 µg/mL and above without metabolic activation.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed.
Precipitation was observed by the unaided eye at 187.5 µg/mL and above in the parts following 4 hours treatment. After 24 hours treatment precipitation occurred at the maximum concentration of 1500 µg/mL.
Therefore, the maximum concentration of the main experiments was adjusted according to the toxicity data of the pre-experiment. To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations. The individual concentrations were spaced by a factor of 2. Narrower spacing was used at high concentrations to cover the cytotoxic range more closely.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects indicated by a relative total growth of less than 50% were observed in the first experiment at 23.5 µg/mL and above with and without metabolic activation. In the second experiment cytotoxic effects as described were noted at 47.0 µg/mL with and without metabolic activation. The recommended cytotoxic range of approximately 10-20% relative total growth was covered with and without metabolic activation.
Remarks on result:
other: strain/cell type: in vitro gene mutation assay with L5178Y cells
Remarks:
Migrated from field 'Test system'.
Summary Table
    relative mutant   relative mutant  
  conc. µg S9 total colonies/   total colonies/  
  per mL mix growth 106cells threshold growth 106cells threshold
Column 1 2 3 4 5 6 7 8
Experiment I / 4 h treatment   culture I culture II
Solv. control with ethanol - 100.0  68 194 100.0 112 238
Pos. control with MMS  19.5 -  47.4 304 194  46.5 415 238
Test item   1.5 - culture was not continued# culture was not continued#
Test item   2.9 - 115.3  72 194 152.3  73 238
Test item   5.9 - 125.1  67 194  94.2 119 238
Test item  11.8 - 125.6  64 194 149.4  47 238
Test item  23.5 -  55.3  56 194  34.6  97 238
Test item  47.0 -  10.8 123 194  10.6 102 238
Test item  70.5 - culture was not continued## culture was not continued##
Test item  94.0 - culture was not continued## culture was not continued##
       
Solv. control with ethanol + 100.0  72 198 100.0 117 243
Pos. control with CPA   3.0 +  51.3 146 198  53.2 294 243
Pos. control with CPA   4.5  +   21.0 302 198  21.5 779 243
Test item   2.9  +   90.2  46 198 culture was not continued#
Test item   5.9  +   62.1  93 198 147.1 156 243
Test item  11.8  +   61.0  62 198 333.0 103 243
Test item  23.5  +   47.2 140 198  77.3  87 243
Test item  47.0  +   9.3 150 198  60.3  83 243
Test item  94.0  +  culture was not continued##  47.8 102 243
Test item  141.0  +  culture was not continued## culture was not continued##
Test item  188.0  +  culture was not continued## culture was not continued##
Experiment II / 24 h treatment   culture I culture II
Solv. control with ethanol - 100.0  74 200 100.0 107 233
Pos. control with MMS  13.0 -  34.2 333 200  47.1 353 233
Test item   2.9 - 115.0  60 200  86.8 129 233
Test item   5.9 -  78.3  96 200 105.4  87 233
Test item  11.8 -  99.2  84 200 137.3  97 233
Test item  23.5 - 104.5  85 200  91.0 122 233
Test item  47.0 -  4.3 115 200  4.5  91 233
Test item  70.5 - culture was not continued## culture was not continued##
Test item  94.0 - culture was not continued## culture was not continued##
Experiment II / 4 h treatment   culture I culture II
Solv. control with ethanol + 100.0  48 174 100.0 117 243
Pos. control with CPA   3.0 +  45.4 126 174  47.8 228 243
Pos. control with CPA   4.5 +  23.9 248 174  36.1 336 243
Test item   2.9 + 106.7  65 174 105.5 107 243
Test item   5.9 +  91.8  68 174  93.3 122 243
Test item  11.8 +  72.4  80 174  94.6 120 243
Test item  23.5 +  73.2  52 174  78.4 146 243
Test item  47.0 +  8.7  61 174  7.6 170 243
Test item  70.5 + culture was not continued## culture was not continued##
Test item  94.0 + culture was not continued## culture was not continued##

threshold = number of mutant colonies per 106cells of each solvent control plus 126

#    culture was not continued since a minimum of only 4 analysable concentrations is required

##   culture was not continued due to exceedingly severe cytotoxic effects

 

Conclusions:
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.
Executive summary:

The study was performed to investigate the potential ofdi-tert-butyl 1,1,4,4 -tetramethyltetramethylene diperoxide (CAS 78 -63 -7) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The first main experiment was terminated prior to the generation of any data on mutagenicity since the cytotoxic range was not reached even at the highest concentrations. The dose range was adjusted and the experiment repeated under identical general experimental conditions. The data of the repeat experiment are reported as experiment I.

The second experiment was performed with a treatment period of 4 h with and 24 h without metabolic activation. The second experiment with metabolic activation was terminated prior to the generation of any data on mutagenicity since exceedingly severe cytotoxicity occurred already at low concentrations. Again, the dose range was adjusted and the experiment repeated with metabolic activation. The data of the repeat experiment are reported as experiment II with metabolic activation.

Relevant toxic effects indicated by a relative total growth of less than 50% were observed in the first experiment at 23.5 µg/mL and above with and without metabolic activation. In the second experiment cytotoxic effects as described were noted at 47.0 µg/mL with and without metabolic activation. The recommended cytotoxic range of approximately 10 -20% relative total growth was covered with and without metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control in any of the experimental parts.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTATâ11statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the first culture of the first experiment and the second culture of the second experiment with metabolic activation. However, both trends were judged as biologically irrelevant since the mutation frequency remained within the historical range of solvent controls.

In this study the range of the solvent controls was from 48 up to 117 mutant colonies per 106cells; the range of the groups treated with the test item was from 47 up to 170 mutant colonies per 106cells. In experiment II, culture I without metabolic activation the solvent control fell just short of the recommended 50 – 170 per 106control range as stated under paragraph 8.12,acceptability of the assay of this report. However, the deviation was rather minor (2 colonies per 106cells) and the spontaneous rate of the parallel culture and the mean value of both cultures was fully acceptable (48 and 117 colonies, equal to a mean of 82.5).

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies with at least one of the concentrations. The positive control of the second experiment, culture I with metabolic activation (at 4.5 µg/mL) and culture II without metabolic activation just failed to reach the lower limit of induced small colonies of 150 per 106cells. The data are acceptable however since the positive controls showed a substantial increase of induced total colonies compared to the corresponding solvent controls of 248 versus 48 colonies with and 353 versus 107 colonies without metabolic activation. Furthermore the positive controls of the parallel cultures exceeded the lower limit of 150 induced small colonies with and without metabolic activation.

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

Genetic toxicity in vivo

Description of key information

The in vivo micronucleus study is negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
genetic toxicity in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 April 1996 - 31 may 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-conducted and documented study performed under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: IRC
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Spr- Age at study initiation: 6-8 weeks
- Weight at study initiation:
Pilot study:
Males, 30.6 - 33.9 grams at randomization
Females, 25.0 - 28.4 grams at randomization
Micronucleus assay:
Males, 27.0 - 35.0 grams at randomization
Females, 24.0 - 29.9 grams at randomization
- Assigned to test groups randomly: yes, under following basis: equalization of group mean body weight.
- Fasting period before study: no data
- Housing: Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet (e.g. ad libitum): Mice had free access to certified laboratory rodent chow which had been analyzed for environmental contaminants (Harlan TEKLAD Certified Rodent Chow 7012C)
- Water (e.g. ad libitum): Mice had free access tap water (Washington Suburban Sanitary Commission, Potomac Plant).
- Acclimation period: no less than 5 days after receipt

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.3 ± 3.3
- Humidity (%): 50 ± 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 10 April 1996 - 31 may 1996
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: max 500 mg/ml
- Amount of vehicle (if gavage or dermal): 20ml/kg
- Type and concentration of dispersant aid (if powder): not applicable
- Lot/batch no. (if required): not applicable
- Purity: not applicable
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article-vehicle mixture, the vehicle alone and the positive control (CP) will be given as a single administration. The rate of administration will be 20 ml/kg to deliver the targeted dose. All mice in the experimental groups will be weighed and the dose volume will be based on individual body weight.
Duration of treatment / exposure:
single dose
Frequency of treatment:
once
Post exposure period:
Twenty-four, 48, and 72 hours after dose administration, five animals per sex per test article-treated and vehicle control groups will be sacrificed by carbon dioxide asphyxiation. The positive control group will be sacrificed 24 hours after dose administration.
Remarks:
Doses / Concentrations:
1250, 2500, 5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
Vehicle Control: 15
Low test dose (1250 mglkg): 15
Mid test dose (2500 mg/kg): 15
High test dose (5000 mg/kg): 20
Positive Control, 60 mg/kg: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): known positive
- Route of administration: oral gavage
- Doses / concentrations: 60 mg/kg bw
Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
For the pilot study, mice were randomly assigned to one group of five males and five females and to 4 groups of two males each. Each mouse was given a sequential number and identified by ear tag. All mice were weighed immediately prior to dose administration and the dose volume based on individual body weights. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of chemical effect. Body weights were recorded prior to dose administration and 1 and 3 days after dose administration.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test article-vehicle mixture, the vehicle alone, or the positive control was administered by oral gavage at a constant volume of 20 ml/kg body weight. All mice in the experimental and control groups were weighed immediately prior to dose administration and the dose volume was based on individual body weights. Mice were observed after dose administration for clinical signs of chemical effect.
Twenty-four, 48, and 72 hours after dose administration, five animals per sex per test article-treated and vehicle control groups will be sacrificed by carbon dioxide asphyxiation. The positive control group will be sacrificed 24 hours after dose administration.

DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 ml fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 115 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Evaluation criteria:
The test is valid when: The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1 000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p ≤ 0.05, Kastenbaum-Bowman Tables).
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970; Mackey and MacGregor, 1979). All analyses were performed separately for each sex and sampling time. In order to quantify the test article effect on erythropoiesis, as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
The test substance was considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p ≤ 0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Reductions of up to 21% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test substance-treated groups relative to their respective vehicle controls.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
For the pilot study the substance was administered by oral gavage to male mice at 1, 10, 100, or 1000 mg test article/kg and to male and female mice at 5000 mg/kg which was administered in a total volume of 20 ml test substance-vehicle mixture/kg body weight. Mortality was observed in 1/5 male mice at 5000 mg/kg. Clinical signs observed after dose administration included: lethargy in one male mouse at 1000 mg/kg and lethargy and diarrhea in male mice and female mice at 5000 mg/kg, and crusty eyes and prostration in one male mouse at 5000 mg/kg. All other animals appeared normal throughout the observation period. Due to less than 50% mortality in the pilot study, a toxicity study was not required. The highest dose level for the micronucleus study was set at 5000 mg/kg.

RESULTS OF DEFINITIVE STUDY
See tables
Conclusions:
Interpretation of results (migrated information): negative
The results of the assay indicate that under the conditions described in this report the substance did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. It was concluded to be negative in the mouse micronucleus assay.
Executive summary:

The test substance was tested in the mouse micronucleus assay.

The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot assay. The second phase, the micronucleus study, evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control articles were administered in a constant volume of 20 ml/kg body weight by a single oral gavage.

Corn oil was determined to be the solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals. The test substance was soluble in com oil at a concentration of 500 mg/ml, the maximum concentration tested. In the pilot assay, male mice were dosed with 1, 10, 100, or 1000 mg test article/kg body weight and male and female mice were dosed with 5000 mg/kg. Mortality occurred in 1/5 males at 5000 mg/kg. Clinical signs observed after dose administration included: lethargy in one male mouse at 1000 mg/kg and lethargy and diarrhea in male and female mice at 5000 mg/kg, and crusty eyes and prostration in one male mouse at 5000 mg/kg.

Due to less than 50% mortality at 5000 mg/kg in the pilot assay, the high dose for the micronucleus test was set at 5000 mg/kg.

In the micronucleus assay, male and female mice were dosed with 1250, 2500 or 5000 mg/kg body weight. No mortality occurred in male or female mice in the micronucleus study. Clinical signs observed after dose administration included diarrhea in male and female mice at all test substance dose levels, lethargy in male and female mice at 2500 and 5000 mg/kg and crusty eyes in one male mouse at 5000 mg/kg.

Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 21 %) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test substance treated groups relative to the vehicle control groups. No significant increase in micronucleated polychromatic erythrocytes in test subsatnce treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hours after dose administration (p>0.05, KastenbaumBowman).

The results of the assay indicate that under the conditions described in this report the substance did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. It was concluded to be negative in the mouse micronucleus assay.

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

Additional information

Justification for classification or non-classification

The substance was negative in 3 Ames tests, a Mouse Lymphoma Assay, an in vitro chromosomal aberration assay and in an in vivo Micronucleus test. Therefore the substance is not classified.