Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide

Genetic toxicity: in vitro

Administrative data

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:

guideline study

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, performed according to guideline and conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide

EC Number:

201-128-1

EC Name:

Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide

Cas Number:

78-63-7

Molecular formula:

C16H34O4

IUPAC Name:

2,5-bis(tert-butylperoxy)-2,5-dimethylhexane

Details on test material:

- Name of test material (as cited in study report): di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide
- Molecular weight (if other than submission substance): 290 g/mol
- Substance type: Industrial chemical
- Physical state: Liquid, colourless
- Analytical purity: 96.2% (dose calculation adjusted to purity)
- Composition of test material, percentage of components: --
- Purity test date: 2010-03-26
- Lot/batch No.: 1002516115
- Expiration date of the lot/batch: 2012-03-01
- Storage condition of test material: At room temperature (15-25 °C)
- Other:
On the day of the experiment (immediately before treatment), the test item was formulated in ethanol (purity 99.8 %). The final concentration of ethanol in the culture medium was 0.5 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cells.

Method

Target gene:

Thymidine Kinase Locus

Species / strain

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

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 2

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)

Results and discussion

Test results

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'.

Any other information on results incl. tables

Summary Table

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	10^6cells	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

 

Applicant's summary and conclusion

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. The test item is considered to be non-mutagenic in the in vitro mouse lymphoma assay.

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 SYSTAT^â11statistics 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.