Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenicity was observed in an Ames test, a mouse lymphoma test and an in vitro micronucleus test using human lymphocytes.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-conducted study performed prior to GLP regulations. Documentation is less complete than current practice but sufficient for the study to be used and rated. Test method used is similar to current guideline.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Remarks:
Study performed prior to GLP regulations
Type of assay:
bacterial forward mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S-9 induced with Aroclor 1245
Test concentrations with justification for top dose:
0, 0.8, 4, 20, 100, 500 µg/plate
Vehicle / solvent:
acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
methylmethanesulfonate
other: N-methyl-N-nitro-N-nitrosoguanidine, 4-aminobiphenyl
Details on test system and experimental conditions:
The Salmonella typhimurillin mutants used in this study, viz. S. typh. TA 1535, TA 1531, TA 1538, TA 98 and TA 100, a gift or Dr. B.N. Ames, Berkeley,California, USA, are stored as frozen cultures at - 80 °C. To obtain fresh cultures for mutagenesis testing, nutrient broth is inoculated with the frozen culture and grown up overnight with shaking at 37 °C. The reversion properties of each strain are regularly checked, using the mutagens: MMS; MNNG; 9-AA and 4-ABP. In addition, the strains are checked for histidine requirement, for sensitivity to crystal violet, deoxycholate and for resistance to ampicillin.

The procedures used in this mutagenic assay are described in detail by Ames et al. (1975). Briefly, the procedure was as follows: to 2.5 ml molten soft agar vere added 0.1 ml of a fully grown culture of one of the tester strains, 0.1 ml of an appropriate dilution/suspension of the test compound and the liver microsome system if indicated. The ingredients were thoroughly mixed and immediately poured onto minimal glucose agar plates. After the top agar had been allowed to harden, the plates vere incubated at 37 °C for three days. Then the colonies (revertants vhich are histidine-independent) were counted, and the background lawn of bacterial growth examined. Based on the results of preliminary toxicity tests, the test materials were examined at levels up to 1000 µg/plate except for TMPH which was tested at levels up to 500 µg/plate. All determinations were carried out in triplicate and appropriate controls were included in each assay.
Key result
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:
cytotoxicity
Remarks:
at 100 and 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Additional information on results:
Background lawn of bacterial growth less dense than in control plates.
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Not mutagenic
Executive summary:

The mutagenic activity of THPH was examined in the Salmonella/ microsome mutagenicity test, using a set of five histidine requiring mutants of Salmonella typhimurium (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and liver homogenate of Aroclor induced rats.

Incorporation of TMPH up to non-inhibitory levels did not induce an increase of the reversion rate to his+ prototrophy with any of the five tester strains. At higher levels, chemical toxicity interfered with the mutagenicity testing. It was concluded that the present results did not reveal any mutagenic activity of TMPH in the Salmonella/microsome mutagenicity test.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 February 2012 to 9 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.
Qualifier:
according to
Guideline:
other: OECD 487
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date GLP of inspection Date of signature on GLP form:
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbecco's modified Eagle's medium and Ham's F-12 (1:1) supplemented with GlutaMax 200 mM, penicillin/streptomycin 100 U/ml/100 µg/ml respectively, phytohemagglutinin 3 µg/ml, 10% fetal bovine serum, 10 mM HEPES and heparin 125 USP units/ml

Blood samples were obtained from healthy, non-smoking donors not receiving medication. For this study, blood was collected from a 33 year-old female donor for Experiments I and IIC, from a 34 year-old female donor for Experiment IIA and from a 23 year-old female donor for Experiment IIB. All donors have a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to Harlan CCR to initiate cell cultures within 24 hrs after blood collection. The blood was stored at 4 °C until used.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Experiment I (4 hours exposure, with and without metabolic activiation):
10.4, 18.2, 31.8, 55.7, 97.5, 170.6, 298.5, 522.4, 914.3 and 1600 µg/mL

Experiment IIA (20 hours exposure, with and without metabolic activiation):
10.4, 18.2, 31.8, 55.7, 97.5, 170.6, 298.5, 522.4, 914.3 and 1600 µg/mL.

Experiment IIB (20 hours exposure without metabolic activiation):
6.3, 12.5, 25.0 ,50.0, 60.0, 700, 80.0, 90.0, 100.0 and 200 µg/mL.

Experiment IIB (20 hours exposure with metabolic activiation)
10.4, 18.2, 31.8, 55.7, 97.5, 170.6, 298.5, 522.4, 914.3 and 1600 µg/mL.

Experiment IIC (20 hours exposure without metabolic activiation):
25.0, 50.0, 100.0, 200.0, 250.0, 300.0, 350.0, 400.0, 450.0, 500.0, 550.0, 600.0, 700.0 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Solvent (DMSO) treatment groups were used as the vehicle controls.

- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: colcemid
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 metabolic activation 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 REPLICATES: 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 106 cells above the corresponding solvent control.
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 the solvent con¬trols of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth 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.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation .
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
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
- Effects of osmolality: not increased
- Evaporation from medium: not examined
- Precipitation: Not observed
- 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 25.0 µg/mL and 3200 µg/mL were used. Relevant toxic effect (relative suspension growth, RSG below 50% of the solvent control) occurred at 200 µg/mL and above following 4 h treatment with and at 100.0 µg/mL and above without metabolic activation. After 24 h treatment without metabolic activation cytotoxic effects as described above were noted at 25 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 h) before the test item was removed. Phase separation was observed by the unaided eye at 1600 µg/mL and above in the parts following 4 hours and 24 hours treatment with and without metabolic activation.
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:
A single and isolated increase of the mutation frequency exceeding the historical range of solvent controls occurred at 150.0 µg/mL in the first culture of the first experiment with metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: strain/cell type: in vitro gene mutation assay with L5178Ycells
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 DMSO - 100.0  99 225 100.0  85 211
Pos. control with MMS  19.5 -  23.9 353 225  15.5 322 211
Test item   6.3 - culture was not continued# culture was not continued#
Test item  12.5 - 121.4  91 225  95.4  90 211
Test item  25.0 -  90.6  94 225  52.8  80 211
Test item  50.0 -  57.8  72 225  61.5  60 211
Test item  75.0 -  37.9 112 225  32.9  63 211
Test item  100.0 -  16.9 104 225  16.8  90 211
       
Solv. control with DMSO + 100.0 131 257 100.0  88 214
Pos. control with CPA   3.0 +  60.1 247 257  78.9 138 214
Pos. control with CPA   4.5  +   48.8 304 257  48.5 266 214
Test item  12.5  +  106.8  87 257 123.0  60 214
Test item  25.0  +  113.8 129 257 124.9  84 214
Test item  50.0  +   85.6 163 257  80.0  92 214
Test item  100.0  +   70.1 148 257  58.9  78 214
Test item  150.0  +   14.1 285 257  16.1  88 214
Test item  200.0  +  culture was not continued## culture was not continued##
Experiment II / 24 h treatment   culture I culture II
Solv. control with DMSO - 100.0 110 236 100.0  81 207
Pos. control with MMS  13.0 -  25.0 319 236  15.8 695 207
Test item   3.1 - culture was not continued# culture was not continued#
Test item   6.3 -  83.8  95 236  75.6 113 207
Test item  12.5 -  91.4  72 236  62.4 132 207
Test item  25.0 -  61.6  64 236  25.6 123 207
Test item  37.5 -  12.4  73 236  5.5 141 207
Test item  50.0 -  4.9  65 236  1.1 228 207
Experiment II / 4 h treatment   culture I culture II
Solv. control with DMSO + 100.0 101 227 100.0 110 236
Pos. control with CPA   3.0 +  34.8 380 227  39.7 380 236
Pos. control with CPA   4.5 +  24.9 464 227  32.1 411 236
Test item  12.5 +  73.1  99 227 123.7  93 236
Test item  25.0 +  74.3 105 227  59.4 192 236
Test item  50.0 +  89.2  76 227 107.0 111 236
Test item  100.0 +  73.7  97 227  83.2 149 236
Test item  150.0 +  15.4 109 227  14.1 195 236
Test item  200.0 + 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

The values printed in bold are judged as invalid, since the acceptance criteria (page22) are not met (RTG < 10% in both parallel cultures).

Conclusions:
Interpretation of results
negative Non-mutagenic

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 of 1,1,3,3-tetramethylbutyl hydroperoxide (CAS 5809-08-5) 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 second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

Relevant cytotoxic effects indicated by a relative total growth of less than 50% were observed in the first experiment at 150 µg/mL with metabolic activation and at 75 µg/mL and above without metabolic activation. In the second experiment cytotoxic effects as described above were noted at 150 µg/mL and above with metabolic activation and at 25.0 µg/mL and above without metabolic activation. The data generated at 50.0 µg/mL in the second experiment without metabolic activation are not considered valid since the relative total growth fell short of the 10% limit in both parallel cultures.

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. A single and isolated increase of the mutation frequency exceeding the threshold of 126 plus the solvent control count and the historical range of solvent controls, occurred at 150.0 µg/mL in the first culture of the first experiment with metabolic activation. This increase however, was judged to be an irrelevant cytotoxic artefact as it was not reproduced in the parallel culture or in the second experiment with metabolic activation at comparable levels of cytotoxicity.

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 solely determined in experiment I culture I with metabolic activation. This increase was judged to be based on a cytotoxic artefact as discussed above.

The viability measured in the solvent control of the first culture of the first experiment without metabolic activation slightly exceeded the acceptance criteria (127% compared to 120%). This deviation was judged as irrelevant since it was rather minor and the parallel culture met the acceptance criteria under identical conditions.

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 in general an increase of the relative quantity of small versus large induced colonies with at least one of the concentrations. Although the positive control induced a substantial increase of total colonies in the first culture of the second experiment without metabolic activation (319 versus 110 colonies of the corresponding solvent control), the criterion of at least 150 induced small colonies was not quite met as many large colonies were induced. This deviation had no impact on the validity of the data however, as the parallel culture easily met this criterion under identical conditions.

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:
25 January 212 and 19 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.
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
GLP compliance:
yes (incl. certificate)
Remarks:
Date GLP of inspection Date of signature on GLP form:
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640
- 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 3.7.2.C
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 6.3; 12.5; 25.0; 50.0; 75.0; and 100 µg/mL
with metabolic activation: 12.5; 25.0; 50.0; 100; 150; and 200 µg/mL
Experiment II:
without metabolic activation: 3.1; 6.3; 12.5; 25.0; 37.5; and 50.0 µg/mL
with metabolic activation: 12.5; 25.0; 50.0; 100; 150; and 200 µg/mL

Following the expression phase of 48 hours the cultures at lowest concentrations of 6.3 µg/mL (experiment I) and 3.1 mg/ml (experiment II) without metabolic activation were not continued since minimum of only four analysable concentrations is required by the guidelines. The cultures at the maximum concentration of 200 µg/mL in experiment I and II with metabolic activation were not continued due to exceedingly severe cytotoxic effects.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Solvent (DMSO) treatment groups were used as the vehicle controls.

- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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 metabolic activation 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 REPLICATES: 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 106 cells above the corresponding solvent control.
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 the solvent con¬trols of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth 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.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation .
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
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
- Effects of osmolality: not increased
- Evaporation from medium: not examined
- Precipitation: Not observed
- 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 25.0 µg/mL and 3200 µg/mL were used. Relevant toxic effect (relative suspension growth, RSG below 50% of the solvent control) occurred at 200 µg/mL and above following 4 h treatment with and at 100.0 µg/mL and above without metabolic activation. After 24 h treatment without metabolic activation cytotoxic effects as described above were noted at 25 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 h) before the test item was removed. Phase separation was observed by the unaided eye at 1600 µg/mL and above in the parts following 4 hours and 24 hours treatment with and without metabolic activation.
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:
A single and isolated increase of the mutation frequency exceeding the historical range of solvent controls occurred at 150.0 µg/mL in the first culture of the first experiment with metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: strain/cell type: in vitro gene mutation assay with L5178Ycells
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 DMSO - 100.0  99 225 100.0  85 211
Pos. control with MMS  19.5 -  23.9 353 225  15.5 322 211
Test item   6.3 - culture was not continued# culture was not continued#
Test item  12.5 - 121.4  91 225  95.4  90 211
Test item  25.0 -  90.6  94 225  52.8  80 211
Test item  50.0 -  57.8  72 225  61.5  60 211
Test item  75.0 -  37.9 112 225  32.9  63 211
Test item  100.0 -  16.9 104 225  16.8  90 211
       
Solv. control with DMSO + 100.0 131 257 100.0  88 214
Pos. control with CPA   3.0 +  60.1 247 257  78.9 138 214
Pos. control with CPA   4.5  +   48.8 304 257  48.5 266 214
Test item  12.5  +  106.8  87 257 123.0  60 214
Test item  25.0  +  113.8 129 257 124.9  84 214
Test item  50.0  +   85.6 163 257  80.0  92 214
Test item  100.0  +   70.1 148 257  58.9  78 214
Test item  150.0  +   14.1 285 257  16.1  88 214
Test item  200.0  +  culture was not continued## culture was not continued##
Experiment II / 24 h treatment   culture I culture II
Solv. control with DMSO - 100.0 110 236 100.0  81 207
Pos. control with MMS  13.0 -  25.0 319 236  15.8 695 207
Test item   3.1 - culture was not continued# culture was not continued#
Test item   6.3 -  83.8  95 236  75.6 113 207
Test item  12.5 -  91.4  72 236  62.4 132 207
Test item  25.0 -  61.6  64 236  25.6 123 207
Test item  37.5 -  12.4  73 236  5.5 141 207
Test item  50.0 -  4.9  65 236  1.1 228 207
Experiment II / 4 h treatment   culture I culture II
Solv. control with DMSO + 100.0 101 227 100.0 110 236
Pos. control with CPA   3.0 +  34.8 380 227  39.7 380 236
Pos. control with CPA   4.5 +  24.9 464 227  32.1 411 236
Test item  12.5 +  73.1  99 227 123.7  93 236
Test item  25.0 +  74.3 105 227  59.4 192 236
Test item  50.0 +  89.2  76 227 107.0 111 236
Test item  100.0 +  73.7  97 227  83.2 149 236
Test item  150.0 +  15.4 109 227  14.1 195 236
Test item  200.0 + 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

The values printed in bold are judged as invalid, since the acceptance criteria (page22) are not met (RTG < 10% in both parallel cultures).

Conclusions:
Interpretation of results (migrated information):
negative Non-mutagenic

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 of 1,1,3,3-tetramethylbutyl hydroperoxide (CAS 5809-08-5) 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 second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

Relevant cytotoxic effects indicated by a relative total growth of less than 50% were observed in the first experiment at 150 µg/mL with metabolic activation and at 75 µg/mL and above without metabolic activation. In the second experiment cytotoxic effects as described above were noted at 150 µg/mL and above with metabolic activation and at 25.0 µg/mL and above without metabolic activation. The data generated at 50.0 µg/mL in the second experiment without metabolic activation are not considered valid since the relative total growth fell short of the 10% limit in both parallel cultures.

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. A single and isolated increase of the mutation frequency exceeding the threshold of 126 plus the solvent control count and the historical range of solvent controls, occurred at 150.0 µg/mL in the first culture of the first experiment with metabolic activation. This increase however, was judged to be an irrelevant cytotoxic artefact as it was not reproduced in the parallel culture or in the second experiment with metabolic activation at comparable levels of cytotoxicity.

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 solely determined in experiment I culture I with metabolic activation. This increase was judged to be based on a cytotoxic artefact as discussed above.

The viability measured in the solvent control of the first culture of the first experiment without metabolic activation slightly exceeded the acceptance criteria (127% compared to 120%). This deviation was judged as irrelevant since it was rather minor and the parallel culture met the acceptance criteria under identical conditions.

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 in general an increase of the relative quantity of small versus large induced colonies with at least one of the concentrations. Although the positive control induced a substantial increase of total colonies in the first culture of the second experiment without metabolic activation (319 versus 110 colonies of the corresponding solvent control), the criterion of at least 150 induced small colonies was not quite met as many large colonies were induced. This deviation had no impact on the validity of the data however, as the parallel culture easily met this criterion under identical conditions.

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

Genetic toxicity in vivo

Description of key information

In vivo testing is waived

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

No mutagenicity was observed in three in vitro assays: an Ames test, a mouse lymphoma test and an in vitro micronucleus test using human lymphocytes. Because no mutagenicity was observed in three separate in vitro assays, no in vivo genetic toxicity studies are planned or proposed.