Di-tert-butyl peroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Di-tert-butyl peroxide (DTBP) has been tested for potential genetic toxicity in a variety of in vitro assays. In general, the results of these tests are negative. DTBP was negative in a number of Ames Assays, in the mouse lymphoma assay, in the SOS chromotest, in the Neurospora back-mutation assay and did not transform Pneumococcus DNA. Taken together, DTBP is not genotoxic in vitro.

DTBP was negative in an in vivo MN test in rats following 90 days of inhalation exposure to a high level of 1000 mg/m3; the test did not reveal chromosomal damage and/or damage to the mitotic apparatus of bone marrow erythrocytes. DTPA was also negative in an in vivo spermatogonal assay via the intraperitoneal route. However, results were equivocal in anin vivooral micronucleus assay and gave a weak positive response in an in vivo mouse micronucleus study in which the test material was administered by intraperitoneal injection. In these two latter studies, (weakly) positive results were only seen at high levels. In addition, as the ip route of administration by-passes the normal absorption, distribution, metabolism and excretion pathways, the relevance of ip treatment to hazard assessment is questionable.

Therefore, DTBP is not a germ cell mutagen and was negative in an in vivo micronucleus assay by the most relevant route of exposure, inhalation. Furthermore, the closely related tert-butylhydroperoxide (TBHP) was recently (2016) negative in an inhalative Comet Assay in nasal tissue by the most relevant route of exposure, inhalation.

Justification for selection of genetic toxicity endpoint
An in vivo MN test was carried out in rats exposed for 90 days via inhalation indicating a very long period of exposure.

Link to relevant study records

Reference

Reference 1

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:

2010 -02-22 till 2010-04-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-conducted GLP study performed according to established guideline OECD 471 with no deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:THF
- Justification for choice of solvent/vehicle: chosen because of its solubility properties

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no relevant

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used in experiment I. In experiment II reduced background growth was observed at 2500 µg/plate and 5000 µg/plate in all strains with and without metabolic activation
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. Only in experiment II a minor reduction in the number of revertants (below the indication factor of 0.5) was observed in strain TA 98 at 5000 µg/plate in the absence of metabolic activation.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Summary of Results Pre-Experiment and Experiment I

Study Name: 1294201	Study Code: Harlan CCR 1294201
Experiment: 1294201 VV Plate	Date Plated: 24/02/2010
Assay Conditions:	Date Counted: 02/03/2010

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	THF			27 ± 3	13 ± 3	38 ± 4	141 ± 11	393 ± 23
	Untreated			20 ± 6	11 ± 3	39 ± 1	140 ± 26	388 ± 21
	di-tert-butyl	3 µg		23 ± 5	12 ± 3	36 ± 10	137 ± 12	392 ± 12
	peroxide	10 µg		23 ± 4	12 ± 2	38 ± 8	134 ± 13	380 ± 15
		33 µg		20 ± 7	15 ± 1	33 ± 6	132 ± 18	394 ± 23
		100 µg		30 ± 5	15 ± 2	40 ± 5	148 ± 17	378 ± 42
		333 µg		19 ± 10	12 ± 2	40 ± 3	126 ± 13	386 ± 27
		1000 µg		16 ± 3	14 ± 5	40 ± 12	116 ± 5	388 ± 12
		2500 µg		23 ± 4	10 ± 0	41 ± 1	119 ± 12	419 ± 7
		5000 µg		19 ± 1	12 ± 2	28 ± 2	132 ± 3	416 ± 11
	NaN3	10 µg		1779 ± 90			2242 ± 24
	4-NOPD	10 µg				266 ± 26
	4-NOPD	50 µg			103 ± 9
	MMS	3.0 µL						3642 ± 772
With Activation	THF			27 ± 7	25 ± 4	52 ± 3	163 ± 5	575 ± 8
	Untreated			23 ± 9	25 ± 2	49 ± 4	158 ± 24	620 ± 21
	di-tert-butyl	3 µg		22 ± 1	21 ± 3	63 ± 6	168 ± 4	586 ± 21
	peroxide	10 µg		23 ± 1	22 ± 1	56 ± 11	155 ± 11	581 ± 29
		33 µg		25 ± 9	23 ± 1	50 ± 2	171 ± 19	602 ± 20
		100 µg		19 ± 4	23 ± 7	58 ± 8	169 ± 14	611 ± 32
		333 µg		27 ± 4	26 ± 3	57 ± 3	162 ± 16	590 ± 31
		1000 µg		26 ± 3	25 ± 6	53 ± 7	173 ± 10	561 ± 33
		2500 µg		28 ± 4	25 ± 3	54 ± 6	154 ± 5	557 ± 37
		5000 µg		18 ± 4	25 ± 3	64 ± 4	154 ± 9	570 ± 19
	2-AA	2.5 µg		464 ± 54	582 ± 14	2334 ± 141	3605 ± 355
	2-AA	10.0 µg						2563 ± 222

Key to Positive Controls
NaN3 2-AA MMS 4-NOPD	sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine

Summary of Results Experiment II

Study Name: 1294201	Study Code: Harlan CCR 1294201
Experiment: 1294201 HV2 pre	Date Plated: 01/04/2010
Assay Conditions:	Date Counted: 07/04/2010

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	TA 102
Without Activation	THF			14 ± 10	11 ± 1	29 ± 4	119 ± 5	371 ± 22
	Untreated			10 ± 4	15 ± 4	26 ± 2	109 ± 16	329 ± 33
	di-tert-butyl	33 µg		15 ± 5	9 ± 2	27 ± 4	113 ± 9	354 ± 15
	peroxide	100 µg		17 ± 5	13 ± 1	32 ± 2	115 ± 32	385 ± 28
		333 µg		17 ± 5	11 ± 5	30 ± 4	111 ± 4	384 ± 19
		1000 µg		13 ± 2	10 ± 5	23 ± 1	115 ± 9	354 ± 11
		2500 µg		14 ± 11^R	7 ± 1^R	28 ± 3^R	108 ± 22^R	375 ± 33^R
		5000 µg		11 ± 3^R	5 ± 4^R	7 ± 2^R M	89 ± 15^R	345 ± 31^R
	NaN3	10 µg		2133 ± 59			2255 ± 104
	4-NOPD	10 µg				309 ± 7
	4-NOPD	50 µg			74 ± 11
	MMS	3.0 µL						3077 ± 243
With Activation	THF			21 ± 4	23 ± 4	42 ± 7	132 ± 10	530 ± 28
	Untreated			23 ± 2	18 ± 5	43 ± 8	126 ± 9	501 ± 24
	di-tert-butyl	33 µg		21 ± 7	23 ± 4	49 ± 13	122 ± 26	514 ± 66
	peroxide	100 µg		21 ± 3	22 ± 3	45 ± 14	124 ± 30	590 ± 21
		333 µg		24 ± 5	25 ± 4	47 ± 2	124 ± 5	541 ± 45
		1000 µg		23 ± 1	22 ± 7	39 ± 5	116 ± 8	511 ± 49
		2500 µg		18 ± 3^R	13 ± 3^R	54 ± 2^R	132 ± 2^R	508 ± 31^R
		5000 µg		17 ± 4^R	11 ± 3^R	40 ± 8^R	130 ± 7^R	563 ± 27^R
	2-AA	2.5 µg		308 ± 25	332 ± 18	2623 ± 269	2681 ± 63
	2-AA	10.0 µg						3533 ± 206

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA MMS 4-NOPD	sodium azide 2-aminoanthracene methyl methane sulfonate 4-nitro-o-phenylene-diamine	R M	Reduced background growth Manual count

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item di-tert-butyl peroxide (CAS No. 110-05-4) was assessed for its potential to induce gene mutations ac­cording to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:    3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                        33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used in experiment I. In experiment II reduced background growth was observed at 2500 µg/plate and 5000 µg/plate in all strains with and without metabolic activation.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol­ic activation. Only in experiment II a minor reduction in the number of revertants (below the indication factor of 0.5) was observed in strain TA 98 at 5000 µg/plate in the absence of metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with di-tert-butyl peroxide (CAS No. 110-05-4) at any concentration level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowled­ged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

Reference 2

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: Well-conducted GLP study performed according to established guideline OECD 476 with no deviations.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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
without S9 mix 46.3; 92.5; 185; 370; 740; 1480 µg/mL
with S9 mix 46.3; 92.5; 185; 370; 740; 1480 µg/mL
Experiment II
without S9 mix 46.3; 92.5; 185; 370; 740; 1480 µg/mL
with S9 mix 46.3; 92.5; 185; 370; 740; 1480 µg/mL
Following the expression phase of 48 hours the cultures at 46.3 µg/mL in experiment I and II were not continued since a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF (tetrahydrofuran)
- 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 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:

no 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
- Water solubility: --
- 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 11.6 µg/mL and 1480 µg/mL were used. The highest concentration in the pre-experiment was chosen with regard to the purity (99.0 %) and the molecular weight (146 g/mol) of the test item.
No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 and 24 hours of treatment.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. No precipitation was observed by the unaided eye up to the maximum concentration.
Therefore, the maximum concentration of the main experiment was again 1480 µg/mL or approximately 10 mM. The lower concentrations were spaced by a factor of 2. To overcome problems with possible deviations in toxicity or solubility both main experiments


COMPARISON WITH HISTORICAL CONTROL DATA: complies


ADDITIONAL INFORMATION ON CYTOTOXICITY: none

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	10^6cells	threshold	growth	10^6cells	threshold
Experiment I / 4 h treatment			culture I			culture II
Solv. control with THF		-	100.0	194	320	100.0	117	243
Pos. control with MMS	19.5	-	27.1	417	320	85.1	291	243
Test item	46.3	-	culture was not continued^#			culture was not continued^#
Test item	92.5	-	98.4	159	320	180.4	195	243
Test item	185.0	-	101.8	141	320	125.7	177	243
Test item	370.0	-	114.4	123	320	148.8	125	243
Test item	740.0	-	79.6	165	320	159.2	128	243
Test item	1480.0	-	69.9	157	320	210.6	161	243
Experiment I / 4 h treatment			culture I			culture II
Solv. control with THF		+	100.0	194	320	100.0	139	265
Pos. control with CPA	3.0	+	32.2	321	320	47.2	237	265
Pos. control with CPA	4.5	+	50.7	388	320	41.4	313	265
Test item	46.3	+	culture was not continued^#			culture was not continued^#
Test item	92.5	+	136.0	170	320	104.7	122	265
Test item	185.0	+	123.2	179	320	104.1	141	265
Test item	370.0	+	132.7	184	320	114.4	184	265
Test item	740.0	+	94.1	212	320	131.5	109	265
Test item	1480.0	+	126.8	172	320	71.5	226	265
Experiment II / 24 h treatment			culture I			culture II
Solv. control with THF		-	100.0	182	308	100.0	197	323
Pos. control with MMS	13.0	-	25.8	529	308	37.1	564	323
Test item	46.3	-	culture was not continued^#			culture was not continued^#
Test item	92.5	-	92.5	145	308	150.6	98	323
Test item	185.0	-	51.1	279	308	120.9	79	323
Test item	370.0	-	84.5	176	308	181.9	76	323
Test item	740.0	-	73.6	210	308	151.3	107	323
Test item	1480.0	-	69.7	241	308	177.8	95	323
Experiment II / 4 h treatment			culture I			culture II
Solv. control with THF		+	100.0	151	277	100.0	160	286
Pos. control with CPA	3.0	+	51.0	240	277	56.7	262	286
Pos. control with CPA	4.5	+	34.6	302	277	47.9	379	286
Test item	46.3	+	culture was not continued^#			culture was not continued^#
Test item	92.5	+	90.0	163	277	124.8	154	286
Test item	185.0	+	96.1	143	277	84.4	224	286
Test item	370.0	+	90.6	214	277	76.5	206	286
Test item	740.0	+	111.8	152	277	128.4	111	286
Test item	1480.0	+	66.6	273	277	105.8	216	286

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

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

 

Conclusions:

In conclusion it can be stated that 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 of di-tert-butyl peroxide (CAS No. 110-05-4) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

This study was conducted according to the procedures indicated by the following internationally accepted guidelines and recommendations:

Ninth Addendum to the OECD Guidelines for Testing of Chemicals, February 1998, adopted, Guideline No. 476 “In vitro Mammalian Cell Gene Mutation Test”.

Commission Regulation (EC) No. 440/2008, B17: “Mutagenicity – In vitro Mammalian Cell Gene Mutation Test“, dated May 30, 2008.

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 4 h with and 24 h without metabolic activation. The maximum tested concentration was equal to about 10 mM.

Both main experiments were evaluated at the following concentrations:

without S9 mix:          92.5; 185; 370; 740; and 1480 µg/mL
with S9 mix:    92.5; 185; 370; 740; and 1480 µg/mL

No relevant toxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed up to the maximum concentration with and without metabolic activation, following 4 and 24 hours of treatment.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments. The threshold of 126 plus each solvent control count was not exceeded 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 SYSTATâ11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups.

In this study the range of the solvent controls was from 117 up to 197 mutant colonies per 10⁶cells; the range of the groups treated with the test item was from 76 up to 279 mutant colonies per 10⁶cells.The highest solvent control values (182, 194, and 197 colonies per 10⁶cells) exceeded the recommended range of 50 – 170 x 10⁶cells. The data are judged as acceptable however, since the range of up to 200 cultures per 10⁶cells recommended by the IWGT in 2003 was covered.

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.

Reference 3

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

Genetic toxicity in vivo

Description of key information

Di-tert-butyl peroxide (DTBP) has been tested for potential genetic toxicity in a variety ofin vitroassays. In general, the results of these tests are negative. DTBP was negative in a number of Ames Assays, in the mouse lymphoma assay, in the SOS chromotest, in the Neurospora back-mutation assay and did not transform Pneumococcus DNA. Taken together, DTBP is not genotoxicin vitro.

DTBP was negative in an in vivo MN test in rats following 90 days of inhalation exposure to a high level of 1000 mg/m3; the test did not reveal chromosomal damage and/or damage to the mitotic apparatus of bone marrow erythrocytes. DTPA was also negative in anin vivospermatogonal assay via the intraperitoneal route. However, results were equivocal in anin vivooral micronucleus assay and gave a weak positive response in anin vivomouse micronucleus study in which the test material was administered by intraperitoneal injection. In these two latter studies, (weakly) positive results were only seen at high levels. In addition, as the ip route of administration by-passes the normal absorption, distribution, metabolism and excretion pathways, the relevance of ip treatment to hazard assessment is questionable.

Therefore, DTBP is not a germ cell mutagen and was negative in an in vivo micronucleus assay by the most relevant route of exposure, inhalation. Furthermore, the closely related tert-butylhydroperoxide (TBHP) was recently (2016) negative in an inhalative Comet Assay in nasal tissue by the most relevant route of exposure, inhalation.

Justification for selection of genetic toxicity endpoint
An in vivo MN test was carried out in rats exposed for 90 days via inhalation indicating a very long period of exposure.

Link to relevant study records

Reference

Reference 1

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:

November 2012 - March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study conducted according to modern standards of internationally accepted protocol and Good Laboratory Practice. Test Material analytically confirmed for purity

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Animals were exposed for 90 days via inhalation.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

The study was conducted with albino rats. Young adult, male and female Wistar Hannover outbred rats (RccHan®:WIST) were obtained from a colony maintained under specific pathogenfree (SPF) conditions at Harlan Laboratories, The Netherlands (for the in vivo MN test, only male rats were used). On the day of randomization (shortly before the first exposure day), the age of the rats was about 7-8 weeks, and the initial body weight variation did not exceed ± 20% of the mean weight for each sex. Mean body weights at the start of treatment were 264 and 176 grams for male and female animals, respectively.

Upon arrival, the rats were taken to a quarantine room and checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from five randomly selected animals. Two days after arrival, the
results of serological tests were passed on by telephone and indicated an acceptable microbiological status. Subsequently, the animals were released for experimental use and moved to their definitive room. The duration of the acclimatization period to the conditions in the experimental room
prior the first exposure was 10 days (males) or 11 days (females). Shortly before initiation of exposure (study days -4 and -5 for males and females, respectively), the animals were allocated to the various groups by computer randomization proportionally to body weight (males and females separately). The surplus animals were kept in reserve to serve as sentinels (four/sex). These animals were discarded at the end of the in-life phase of the study.

From their arrival, the rats were housed under conventional conditions in one room separated by sex. No other test system was housed in the same room during the study. Lighting was artificial (fluorescent tubes) with a sequence of 12 hours light and 12 hours dark. The room was ventilated with about 10 air changes per hour. The temperature and relative humidity in the room were 22 ± 2°C and 45-65%, respectively, with a few exceptions.

The animals were housed in groups of five, separated by sex, in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment (Lillico, Betchworth, England). During exposure, the animals were kept individually in the exposure unit. Immediately after each exposure, the animals were returned to their home cages. After treatment with the mutagen Mitomycin C, the five animals of the positive control group were kept in smaller Makrolon® cages with filter tops (one or two animals per cage; bedding: wood shavings; enrichment: strips of paper) until sacrifice the next day.

Feed and drinking water were provided ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and during the fasting period before scheduled sacrifice. The animals received a proprietary cereal-based rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Whitham, England). Each batch of RM3 diet is analysed by the supplier for nutrients and contaminants. The feed was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The feed in the feeders was replaced with fresh portions once weekly and filled as needed. Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of the test facility for a limited number of variables.

Route of administration:

inhalation: vapour

Vehicle:

Air

Details on exposure:

The animals were exposed to the test atmosphere in a nose-only inhalation chamber (Institute’s design) consisting of a cylindrical PVC column with a volume of about 75 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column and was exhausted at the top. Each column included three rodent tube levels of 20 ports each. The animals were placed at the top level. Empty ports were used for measurement of temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column. Only the nose of the rats protruded into the interior of the column. The remaining ports were closed. Male and female rats were placed in alternating order. Animals were rotated weekly with respect to the position in the column. From 25 February 2013, larger sized animal holders were used for the male rats because these animals had reached a size for which the standard holders were too small.
The animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing
a positive pressure in the central column and a slightly negative pressure in the outer hood, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose. This way, dilution of test atmosphere at the animals’ noses was avoided.
The units were illuminated externally by normal laboratory fluorescent tube lighting. The total airflow through the unit was at least 1 litre/min per animal. The air entering the unit was maintained between 22 ± 3˚C and the relative humidity between 30% and 70%.

The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. To generate the test atmospheres, a liquid flow of test material, controlled by a peristaltic pump (Gilson France SA, Villiers le Bel, France), was evaporated in a glass evaporator. The temperature of the evaporator was controlled at 22.5˚C (exceptions: 22.7 or 22.8˚C on a few occasions) using a temperature controlled flow of circulating water. The vapour was transported in a stream of humidified compressed air, the flow of which was controlled by a mass flow controller (Bronkhorst, Hi Tec, Ruurlo, The Netherlands). All three test atmospheres (target concentrations 0.1, 0.3 and 1 g/m3) were obtained by diluting a pre-mixture containing about 3 g/m3 of the test material in humidified compressed air. First, mass flow controlled streams of the pre-mixture were supplemented with a mass flow controlled stream of humidified compressed air via an eductor to obtain the low- and mid-concentration. Next, the remaining stream of the pre-mixture was diluted with a mass flow controlled stream of humidified compressed air to obtain the high-concentration. The generated test atmospheres were directed to the bottom inlets of the exposure units. The exposure unit for the control animals was supplied with a measured stream of humidified compressed air only. The animals were placed in the exposure unit after stabilization of the test atmosphere.

The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (Sick Maihak EuroFID total hydrocarbon analyser; Sick Instruments Benelux, Hedel, the Netherlands). The response of the analyser was recorded on a PC every minute using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute. Representative test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser (TCA) through a sample line.
Prior to the first exposure, the output of the flame ionization detector of the TCA was calibrated using PET sample bags with known volumes of clean dry air and known amounts (by weighing) of test material. For each target concentration three calibration concentrations were prepared, at least in duplicate, and analysed (approximately 80, 100 and 120% of the target concentration). The calibrations were checked weekly by measuring the concentration in a sample bag with a theoretical concentration close to the target concentration. If the measured concentration deviated more than 5% from the theoretical concentration and this was confirmed with a second sample bag, the TCA was recalibrated.

The nominal concentration was determined, for each exposure day, by dividing the total amount of test material used (by weight) by the total volume of air passed through the exposure unit. The nominal concentration was calculated for the low-, mid- and high-concentration as well as for the pre-mixture which was diluted to obtain the test atmospheres. The generation efficiency was calculated from the actual and the nominal concentration (efficiency = actual concentration as percentage of nominal concentration).

The chamber airflow of the test atmospheres was recorded about hourly using a Rotameter (group 1) or a mass flow controller (groups 2-4). The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). For group 4 of the sub-chronic study the temperature and relative humidity were additionally measured about hourly by means of a RH/T device (TESTO 635-1, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany).

The overall mean actual concentrations (+/- standard deviation) of the test material in the test atmospheres as measured by total carbon analysis were 101 (± 3), 299 (± 3) and 993 (± 10) mg/m3 for the low-, mid- and high-concentration, respectively. These actual concentrations were very close to the target concentrations (100, 300 and 1000 mg/m3).

The overall mean nominal concentrations, calculated from the daily consumption of test material, the airflow and the duration of test atmosphere generation, were 96, 290 and 1036 mg/m3 for the low-, mid- and high-concentration, respectively. The corresponding generation efficiencies were close to the expected 100%, namely 105, 103 and 96%, respectively.

The overall mean (± standard deviation) chamber airflows were 27.8 (± 0.0), 26.2 (± 0.2), 27.6 (± 0.3) and 24.3 (± 0.2) L/min for exposure chambers 1 (control), 2 (low), 3 (mid) and 4 (high), respectively.

The air temperature in the exposure chambers during exposure was within the target range of 19 – 25°C. The overall mean temperature was about 23°C for each chamber. The relative humidity during exposure was within the target range of 30-70%. The overall mean relative humidity was 46, 40, 39 and 44% in exposure chambers 1 (control), 2 (low), 3 (mid) and 4 (high), respectively.

Duration of treatment / exposure:

90 days (65 exposure days)

Frequency of treatment:

6 h/day, 5 days/week

Post exposure period:

Not applicable

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/m3
Basis:
other: target concentrations

Remarks:

Doses / Concentrations:
0, 101, 299, and 993 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

5 males/concentration
5 males positive control

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C was administered by a single intraperitoneal injection at a dose level of 1.5 mg/kg body weight as a solution in physiological saline (dose volume 10 ml/kg body weight; concentration 0.15 mg/ml).

Tissues and cell types examined:

At sacrifice, bone marrow cells of one of the femurs (left femur) were collected from five male animals per group.

Details of tissue and slide preparation:

The bone marrow cells were immediately collected into foetal calf serum and processed into glass drawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grünwald-Giemsa solution. The other fixed unstained smear was kept in reserve and discarded after completion of analysis.

The bone marrow smears of the males of groups 1-5 were examined microscopically. The slides were randomly coded by a person not involved in the
scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines, taking care that areas selected for evaluation were evenly distributed over the whole smear.
The following criteria were used for the scoring of cells:
˗ A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
˗ A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
˗ A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain. The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal. If micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. The incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.

Evaluation criteria:

The test was considered valid if the positive controls showed a statistically significant increase in the mean number of MPE/2000 PE and the negative controls were within the historical range.
A test material was considered to cause chromosomal damage and/or damage to the mitotic apparatus if it showed a dose-related positive response or a clear increase of micronucleated cells in a single dose group.
A test material was considered to be negative in the micronucleus test if it did not produce a positive response at any of the dose levels analysed.
The test material or its metabolites were considered to be cytotoxic to the bone marrow via the general circulation, if the test material statistically significantly reduced the mean number of PE.
Both statistical significance and biological relevance were considered together in the evaluation.

Statistics:

Statistical tests were performed using GraphPad Prism®, Version 5.03, Copyright © 1992-2010 GraphPad Software, Inc., CA, USA.. In all tests a significance level of 5% was used (α = 0.05). Data on PE and MPE (PE/200 E and MPE/2000 PE) were analysed by one-way analysis of variance [Anova]. Prior to Anova, it was checked if the Anova assumptions were met (i.e. variances equal). In case assumptions were not met non-parametric testing was performed using the Mann-Whitney test (positive control compared with negative control) or Kruskal-Wallis analysis of variance (test substance groups compared with negative control). Two Anova models were applied for both PE/200 E and MPE/2000 PE. In the first Anova model it was tested if the positive control differed from the negative control (t-test). In the second Anova model (including Dunnett’s test as post-hoc test) it was tested if the test material (different doses) differed from the negative control.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MPE results (see Table 5 below)
The mean number of MPE/2000 PE in the negative control (group 1) was within the historical range. The mean number of MPE/2000 PE in the positive control group treated with mitomycin C (group 5) was within the historical positive control range and statistically significantly increased (p value: 0.0097) compared to the concurrent negative control (group 1). This indicates that the positive control substance mitomycin C reached the bone marrow and induced damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells under the conditions of this study. These results, together with the normal MPE/PE ratio in the negative control group, demonstrate the validity of the test system.
The mean numbers of MPE/2000 PE in the groups exposed to the test material (groups 2-4) did not differ statistically significantly from the mean MPE/2000 PE in the negative control group (group 1). This indicates that treatment with the test material under the conditions of this study did not result in damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells.

PE results (see Table 5 below)
Compared with the negative control group (group 1), positive controls treated with mitomycin C showed a statistically significant decrease (p value: 0.0001) in the number of PE/200 E, indicating that mitomycin C was cytotoxic to the bone marrow. The mean numbers of PE/200 E in the groups exposed to the test material did not differ statistically significantly from the mean PE/200 E in the negative control (group 1). This indicates that treatment with the test material under the conditions of this study did not result in cytotoxicity to the bone marrow.

Clinical signs and mortality

All animals survived until scheduled sacrifice. No treatment-related clinical signs were observed in animals exposed to the test material or treated with the positive control substance. The few signs observed were incidental findings unrelated to treatment.

Body weight

Mean body weights of animals exposed to the test material showed no biologically or statistically significant differences from controls.

Organ weights

The organ weight results for male animals showed the following statistically significant differences between animals exposed to the test material and controls:

- Higher relative liver weight at the high-concentration (relative difference from control 9%).

- Higher relative kidney weight at the high-concentration (relative difference from control 11%).

Conclusions:

Interpretation of results: negative
The results of this micronucleus test incorporated in a sub-chronic (13-week) toxicity study did not provide any indication of chromosomal damage or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats exposed via inhalation to di-tert-butyl peroxide CAS# 110-05-4 for 6 hours/day, 5 days/week (total of 65 exposure days) at concentrations of 101 (± 3), 299 (± 3) or 993 (± 10) mg/m3 (mean actual concentrations ± standard deviation, determined by total carbon analysis). As treatment-related systemic effects were observed in male rats of the high-concentration group (increased weights of the liver and kidneys), there is no reason to assume that the negative bone marrow response was due to lack of systemic exposure.

Executive summary:

The purpose of this mammalian in vivo micronucleus test was to examine the potential of di-tert-butyl peroxide CAS# 110-05-4 to cause damage to the chromosomes and/or the mitotic apparatus of erythroblasts (micronuclei). This micronucleus test was part of a sub-chronic (13-week) inhalation toxicity study in which Wistar Hannover rats were exposed nose-only to target concentrations of 0 (control, clean air), 100, 300 and 1000 mg/m3 of the test material 6 hours/day, 5 days/week for 13 consecutive weeks (resulting in 65 exposure days in total).

The micronucleus test was conducted in accordance with the OECD Guideline for the Testing of Chemicals 474. Mammalian Erythrocyte Micronucleus Test, adopted 21st July 1997. At scheduled necropsy at the end of the 13-week study period, bone marrow cells of one of the femurs of five male rats per group (negative control, low, mid and high concentration) were collected, processed into smears and examined microscopically. The study included a positive control group of five male rats treated with the mutagen Mitomycin C (single intraperitoneal injection; 1.5 mg/kg body

weight) and sacrificed 24 hours after administration of the mutagen.

The target concentrations were accurately achieved as demonstrated by the results of total carbon analysis of the test atmospheres. The overall mean actual concentrations (± standard deviation of the daily mean concentration) were 101 (± 3), 299 (± 3) and 993 (± 10) mg/m3 for the low-, mid- and highconcentration level respectively.

Di-tert-butyl peroxide CAS# 110-05-4 did not adversely affect the general health, appearance or body weight development of the animals. Microscopic examination of bone marrow smears of male rats revealed no signs of toxicity to the bone marrow and no evidence of chromosomal damage and/or

damage to the mitotic apparatus of bone marrow erythrocytes. There was no reason to assume that the negative bone marrow response was due to lack of systemic exposure because treatment-related systemic effects (including increases in liver and kidney weight) occurred in male rats of the high-concentration group. Positive controls (five male rats treated with the mutagen Mitomycin C) showed the expected bone marrow response (cytotoxicity and increased number of micronucleated polychromatic erythrocytes).

Under the conditions of this study exposure to di-tert-butyl peroxide CAS# 110-05-4 did not induce chromosomal damage or damage to the mitotic apparatus of bone marrow erythrocytes of male rats.

Reference 2

Endpoint:

genetic toxicity in vivo, other

Remarks:

Comet assay (inhalation)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

December 3 2015- May 3 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Apparently well conducted GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vivo Alkaline Comet

Specific details on test material used for the study:

tert-Butyl hydroperoxide
Lot no. 15101A1630
Exp. date: 23-Feb-2016
CAS no. 75-91-2

Clear, colorless liquid

The purity of the test substance was 69.2%.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Hsd:SD rats

Sex:

male

Details on test animals or test system and environmental conditions:

Seventy-one male Hsd:SD rats were received in good health from Envigo RMS, Inc.,
Indianapolis, IN, on 14-Dec-2015. The animals were approximately 36 days old at receipt.
All animals were housed individually in clean, solid bottom cages containing ground corncob
bedding material (Bed O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). The
cages were cleaned and changed routinely at a frequency consistent with maintaining good
animal health. The bedding material is periodically analyzed by the manufacturer for
contaminants. Analyses of the bedding material were provided by the manufacturer. No
contaminants were present in the bedding at concentrations sufficient to interfere with the
objectives of this study. The results of these analyses are maintained at WIL Research. The
animals were single-housed in this short-term study (less than 2 weeks from receipt to necropsy)
due to the stress associated with establishing a social hierarchy (cost benefit for the animal).
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory
Animals (National Research Council, 2011). The animal facilities at WIL Research are
accredited by AAALAC International. Enrichment devices were provided to all animals as
appropriate throughout the study for environmental enrichment and to aid in maintaining the
animals’ oral health, and were sanitized weekly.


All animals were housed throughout acclimation and during the study in an environmentally
controlled room. The room temperature and relative humidity controls were set to maintain
environmental conditions of 71 ± 5°F (22 ± 3°C) and 50 ± 20%, respectively. Room temperature
and relative humidity data were monitored continuously and were scheduled for automatic
collection on an hourly basis. These data are summarized in Appendix E. Actual mean daily
temperature ranged from 69.9°F to 71.4°F (21.1°C to 21.9°C) and mean daily relative humidity
ranged from 48.7% to 58.2% during the study. Fluorescent lighting provided illumination for a
12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off)
was recorded once every 15 minutes. Air handling units were set to provide a minimum of
10 fresh air changes per hour.

The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent
LabDiet® 5002 (meal), is a certified feed with appropriate analyses performed by the
manufacturer and provided to WIL Research. Reverse osmosis-treated (on-site) drinking water,
delivered by an automatic watering system, and the basal diet were provided ad libitum
throughout the study, except during acclimation to the nose-only restraint (approximately
1-6 hours) and the exposure periods (approximately 6 hours) , when food and water were
withheld. Municipal water supplying the facility was analyzed for contaminants according to
SOPs. The results of the diet and water analyses are maintained in the facility records. No
contaminants were present in animal feed or water at concentrations sufficient to interfere with
the objectives of this study.

Route of administration:

inhalation

Vehicle:

filtered air

Details on exposure:

Animal exposures were conducted using a two-tier 7.9-L stainless steel conventional nose-only
exposure system (CNOS; designed and fabricated by WIL Research) with grommets in exposure
ports to engage the nose-only tubes. One system was dedicated for each group (Groups 1-4) for
the duration of the study. Air supplied to the CNOS was provided by the WIL Research
Inhalation Department breathing-quality, in-house compressed air source and a HEPA- and
charcoal-filtered, temperature- and humidity controlled supply air source. Exposure system
exhaust passed through the facility exhaust system, which consisted of redundant exhaust
blowers preceded by activated-charcoal- and HEPA-filtration units. The exposure period was
6 hours per day for 3 consecutive days. Animals were housed in a normal animal colony room
during non-exposure hours. Food and water were withheld during each daily exposure period.
Prior to each exposure, the animals were placed into nose-only restraint tubes in the colony room
and transported to the exposure room. Animals were placed on the nose-only systems, exposed
for the requisite duration, and returned to their home cages in the animal colony room . After
being transported to the exposure rooms, the animals were held in restraint tu bes for
15-29 minutes before the initiation of exposure. Animals were rotated among the ports of the
CNOS on a daily basis.

All systems were operated under dynamic conditions. Exposure system temperature, relative
humidity, airflow rates and other generation parameters were recorded during each exposure.
Oxygen content was measured during the method development phase and was 20.9% for all
systems.

The control exposure system (0 ppm) was operated as follows. Humidified supply air was
delivered to the nose-only exposure system using a rotameter-type flowmeter connected in-line
with the WIL Research Inhalation Department supply air source.
Vapors of TBHP were generated from a solution of TBHP in water using a heated glass bead
column-type vaporization system. The column was filled with various sized glass bea ds and
heated using a heat tape. A syringe pump equipped was used to deliver liquid solution to near
the top of the bead column. Using a regulator and a rotameter-type flowmeter compressed air
was metered to the bottom of the bead column. Vaporization occurred as the solution flowed
over the surface of the heated beads, while the compressed air flowed up through the column.
The concentrated vapors were directed towards a ‘T’-fitting prior to the CNOS inlet, where the
concentration was reduced by mixing the concentrated vapors with dilution air prior to entering
the exposure system. Prior to mixing with dilution air, a portion of the concentrated vapor was
removed via a siphon, in order to achieve the target exposure concentrations. The amount of
concentrated TBHP vapor removed was controlled using a rotameter-type flowmeter connected
to the WIL Research Inhalation Department vacuum source.

Analyzed exposure concentrations of the test substance within each CNOS were determined at
approximately 45-minute intervals using a gas chromatograph (GC). Samples were collected
from the approximate animal breathing zone of the CNOS via Teflon® tubing. Test atmosphere
samples were collected automatically using an external multi-position valve. Gas sample
injection onto the chromatography column occurred via an internal gas-sampling valve with a
sample loop. The chromatograph was displayed, the area under the sample peak was calculated
and stored, and the concentration in parts per million (ppm) was calculated.

The positive control substance formulation (200 mg/kg) was prepared at a concentration of
20 mg/mL on day of dose administration (study day 2) as a weight/volume (EMS/0.9% sodium
chloride for injection) mixture. No correction factor was used for the preparation of positive
control formulation. The formulation was stirred continuously throughout the preparation
procedures and continuously throughout dose administration. The pH measurement of dosing
formulation was 2.95 using a pH meter.

Duration of treatment / exposure:

TBHP (Groups 2-4) or humidified, filtered air (Group 1) was administered to appropriately
restrained animals via nose-only inhalation exposures for 6 hours per day for 3 consecutive days.

Frequency of treatment:

6 hours per day for 3 consecutive days.

Post exposure period:

None

Dose / conc.:

0 ppm

Dose / conc.:

7.5 ppm

Dose / conc.:

15 ppm

Dose / conc.:

30 ppm

No. of animals per sex per dose:

12

Control animals:

yes

Positive control(s):

A single dose of EMS was administered to the positive control group

Tissues and cell types examined:

Nasal tissue

Details of tissue and slide preparation:

Animals were euthanized by carbon dioxide inhalation. Immediately following euthanasia, the nasal tissues from 5 animals in each group were collected and processed. The nasal tissue collected for the in vivo comet assay roughly corresponds to the area between Nasal Section II and III. The entire head of 5 or 6 additional animals in each group was removed and placed in 10% neutral-buffered formalin for
histopathology of the nasal cavity with turbinates (olfactory bulbs were severed from the brain and remained in the skull). The remaining up to 2 animals in each group were discarded without tissue collection.

Histopathology
After fixation, protocol-specified tissues were trimmed according to WIL Research SOPs. Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research SOPs, and mounted on glass microscope slides and the nasal cavity with turbinates for Groups 1-4 were stained with hematoxylin and eosin following receipt of the comet assay results. Six cross-sections of the nasal cavities were prepared for microscopic examination in accordance with the method described by Morgan (1991) and Mery et al. (1994). Olfactory bulbs were included in nasal level VI for examination. Microscopic examination was performed on the nasal cavity for all animals in Groups 1-4.

Comet Aassay
The rats were euthanized by CO2 inhalation on Study Day 2, between 2 and 4 hours after completion of the final exposure (Groups 1-4) or after the single dose of EMS (Group 5) for nasal tissue collection. Immediately following euthanasia, five (5) surviving rats in groups 1-5 had the nasal tissue collected.

A section of the nasal tissue (turbinates and septum lining from the nasal cavity) was placed in 3 mL of chilled mincing solution (Hanks’ balanced salt solution with EDTA and DMSO), then minced with fine scissors to release the cells. The cell suspension were strained into a pre-labeled conical polypropylene tube through a Cell Strainer were on wet ice during preparation of the slides

Preparation of Comet Slides
The following work was performed at the Test Facility by BioReliance personnel, except where noted.

Preparation of Slides
From each nasal cavity suspension, an aliquot of 2.5 μL was mixed with 75 μL (0.5%) of low melting agarose. The cell/agrose suspension was applied to microscope slides, commercially available pre-treated multi-well slides. The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. Trevigen, Inc 20-well slides were prepared. Slides were identified with a random code that reflects the study number, group, animal number, and organ/tissue. Three wells were used in scoring and the other wells were designated as a backup. Following solidification of agarose, the slides were placed in jars containing lysis solution.

Lysis
Following solidification of agarose, the slides were submerged in a commercially available lysis solution supplemented with 10% DMSO on the day of use. The slides were kept in this solution at least overnight at 2-8°C.
Unwinding After cell lysis, slides/wells were washed with neutralization buffer (0.4 M tris hydroxymethyl aminomethane in purified water, pH ~7.5) and placed in the electrophoresis chamber. The chamber reservoirs were slowly filled with alkaline buffer composed of 300 mM sodium hydroxide and 1 mM EDTA (disodium) in purified water. The pH was > 13. All slides remained in the buffer for 20 minutes at 2-10°C and protected from light, allowing DNA to unwind.

Electrophoresis
Using the same buffer, electrophoresis was conducted for 30 minutes at 0.7 V/cm, at 2-10°C and protected from light. The electrophoresis time was constant for all slides. Neutralization
After completion of electrophoresis, the slides (gels) were removed from the electrophoresis chamber and washed with neutralization buffer for at least 10 minutes. The slides were then dehydrated with 200-proof ethanol for at least 5 minutes then air dried for at least 2 hours and stored at room temperature with desiccant. These slides were shipped at ambient temperature to BioReliance and upon receipt were logged in by the Test Site repository.

Staining
Slides were stained at the Test Site with a DNA stain (i.e., Sybr-gold) prior to scoring at the Test Site (BioReliance). The stain solution was prepared by diluting 1 μL of Sybr-gold stain in 15 mL of 1xTBE (tris-boric acid EDTA buffer solution).

Evaluation of DNA Damage
Three wells were used for scoring and the remaining wells were stored as backups. Some wells did not have 50 scorable cells; therefore, additional cells were scored using backup wells. Fifty randomly selected, non-overlapping cells per slide/well were scored, resulting in a total of 150 cells evaluated per animal. The following endpoints of DNA damage were assessed and measured:
• Comet Tail Migration; defined as the distance from the perimeter of the comet head to the last visible point in the tail.
• % Tail DNA; (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
• Tail Moment (also known as Olive Tail moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/ 100; Olive et al. 1990)].

Each slide/well was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was determined by scanning 150 cells per animal, when possible (percentage of “clouds” was calculated by adding the total number of clouds for all slides/wells scored, dividing by the total number of cells scored and multiplying by 100). Every effort was made to score at least 150 cells; otherwise, the total number of scorable cells was used for calculations. The “clouds”, also known as “hedgehogs”, are a morphological indication of highly damaged cells however their etiology is uncertain. A “cloud” is produced when almost the entire cell DNA is in the tail of the comet and the head is reduced in size, almost nonexistent (Collins et. al., 2004). “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis. Slides were discarded prior to report finalization.

Evaluation criteria:

Vehicle Controls: The group mean for the % Tail DNA for each tissue analyzed should ideally be within the distribution of the historical negative control database for that tissue.
Positive Controls:The group mean for the % Tail DNA must be significantly greater than the concurrent vehicle control (p ≤ 0.05) and should be compatible with those observed in the historical positive control data base.
Test Conditions: Five animals/sex/group were available for analysis.
Cell Analysis: At least 150 cells/tissue/animal were scored for % Tail DNA whenever possible. In addition, at least 150 cells/tissue/animal were scored to determine the proportion of clouds as an indication of cytotoxicity.
Maximum Dose Evaluated: The maximum dose evaluated for Comets must (a) be the MTD or MFD, or( b) in the absence of cytotoxicity or MFD, a dose of 2000 mg/kg/day (limit dose) is used.

Statistics:

Comet
The median value of 150 counts of % Tail DNA, Tail moment and Tail migration were determined and presented for each animal in each treatment group. The mean and standard deviation of the median values only for % Tail DNA were presented for each treatment group. Statistical analysis was performed only for % Tail DNA. In order to quantify the test substance effects on DNA damage, the following statistical analysis was performed:
• The use of parametric or non-parametric statistical methods in evaluation of data
was based on the variation between groups. The group variances for % Tail DNA
generated for the vehicle and test substance groups were compared using
Levene’s test (significant level of p ≤ 0.05). The differences and variations
between groups were found not to be significant, therefore, a parametric one-way
ANOVA followed by a Dunnett’s post-hoc test was performed (significant level
of p < 0.05).
• A linear regression analysis was conducted to assess dose responsiveness in the
test substance treated groups (p ≤ 0.01).
• A pair-wise comparison (Student’s T-test, p ≤ 0.05) was used to compare the
positive control group to the concurrent vehicle/negative control group. If needed,
non-parametric statistical methods (Kruskal Wallis and/or Mann Whitney test)
may be used in evaluation of data.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Test substance-related microscopic findings were noted in nasal levels II, III, and IV in the 30 ppm group and in nasal level II in the 15 ppm group.

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Overall Mean Analyzed Exposure Concentrations:
Exposure System: 1 2 3 4
Target Concentration (ppm): 0 7.5 15 30
Mean Concentration (ppm): 0.0 7.4 15.6 30.5
Standard Deviation: 0.0 0.50 0.67 1.01
N: 3 3 3 3

Survival:
All animals survived to the scheduled necropsy; therefore, gross examinations were not performed.

Clinical Observations:
There were no test substance-related clinical observations. All clinical findings in the test substance-exposed groups were noted with similar incidence in the filtered air control group,
were limited to single animals, were not noted in an exposure-related manner, and/or were common findings for laboratory rats of this age and strain.

Body Weights:
Test substance-related statistically significantly lower mean body weight gains were noted throughout the exposure period in the 30 ppm group when compared to the control group. At the end of the exposure period (study day 2), mean body weights in the 30 ppm group were 4.7% lower than the filtered air control group. There were no test substance-related effects on body weight in the 7.5 and 15 ppm groups.

Food Consumption:
Test substance-related statistically significant lower mean food consumption was noted in the 30 ppm group when compared to the control group. There were no test substance-related effects
on food consumption in the 7.5 and 15 ppm groups.

Histopathology:
In nasal level II, there was test substance-related marked subacute inflammation noted in all animals in the 30 ppm group and minimal to mild subacute inflammation was noted in 2 of 5 animals in the 15 ppm group. Marked inflammation was locally extensive to diffuse, involving more than 75% of the respiratory and transitional epithelium in that particular section. The inflammation was characterized by an infiltration of neutrophils and fewer lymphocytes obscuring the respiratory epithelium lining the nasal septum and naso- and maxilla-turbinates, and also obscuring the transitional epithelium lining the lateral meatus. There was prominent inflammatory exudate containing proteinaceous material and neutrophils lining the inflamed mucosa, ulceration and/or erosion of epithelium, occasionally evidence of re-epithelialization of denuded mucosa with elongated squamous-like cells, and extension of the inflammation into the lamina propria. In 1 animal (no. 1326), the inflammation was accompanied by mild hyperplasia of respiratory epithelium along the septum. This change was characterized by crowded nuclei
and loss of mucus-containing cells. Subacute inflammation was limited to nasal level II, and did not include the respiratory epithelium lining the dorsal middle meatus or the vomeronasal organ. Minimal to mild subacute inflammation in the 15 ppm group was characterized by a multifocal erosion of respiratory epithelium lining the maxillary turbinates and of the transitional epithelium lining the lateral meatus. A small infiltration of mixed inflammatory cells were present in the submucosa associated with these erosions.

In nasal level III, there was test substance-related degeneration of olfactory epithelium lining the dorsal middle meatus of all 30 ppm group animals. The change was mild to marked, limited to the dorsal middle meatus, and characterized by loss of olfactory epithelial cells with replacement by undifferentiated low cuboidal to flattened epithelium.

In nasal level IV, there was mild multifocal degeneration of the olfactory epithelium lining the dorsal middle meatus of 1 animal in the 30 ppm group (no. 1329). This degeneration was an
extension of, and similar in nature to, that present in the nasal level III sections.

Comet Assay:
The test substance gave a negative response (non-DNA damaging) in this assay in nasal tissues of the male rats. None of the test substance-exposed animals had significant increases in the % tail DNA compared to the filtered air control group. The filtered air control group’s % tail DNA was within the BioReliance Corporation historical range and the positive control group had a statistically significant increase in % tail DNA compared to the filtered air control group. Thus, all criteria for a valid assay were met for nasal tissue. Under the conditions of this study, the nose-only exposure of rats to vapors of tert-Butyl hydroperoxide at concentrations up to 30 ppm did not cause a significant increase in % tail DNA in nasal tissue relative to the filtered air control group. Therefore, tert-Butyl hydroperoxide was concluded to be negative in the in vivo comet assay.



Conclusions:

Based on the results of this study, exposure of male Hsd:SD rats to tert-Butyl hydroperoxide via nose-only inhalation for 6 hours per day for 3 consecutive days at exposure concentrations of 7.5, 15, and 30 ppm resulted in a negative response (non-DNA damaging) for the in vivo comet assay in nasal tissues at exposure concentrations up to 30 ppm. In addition, exposure was associated with subacute inflammation of the respiratory and/or transitional epithelium in the 15 and 30 ppm groups in nasal section II and degeneration of olfactory epithelium in the 30 ppm group in nasal sections III and IV.

Executive summary:

The objective of this study was to assess the potential of the test substance to cause DNA damage in rat nasal tissue when administered via nose-only inhalation to Sprague Dawley rats for 6 hours per day for 3 consecutive days.

tert-Butyl hydroperoxide (TBHP; also known as Trigonox® A-W70) was administered via nose-only inhalation exposure for 6 hours per day for 3 consecutive days to 3 groups (Groups 2-4) of male Hsd:SD rats. Target exposure concentrations were 7.5, 15, and 30 ppm for Groups 2, 3, and 4, respectively. Overall mean analyzed exposure concentrations were 7.4, 15.6, and 30.5 ppm for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) was exposed to humidified, filtered air on a comparable regimen. A positive control group (Group 5) received a single oral gavage dose of 200 mg/kg ethyl methanesulfonate (EMS) on study day 2; the dose volume was 10 mL/kg. Each group (Groups 1-5) consisted of 12 males. On study day 2, between 2 and 4 hours after completion of the final exposure (Groups 1-4) or after the single dose of EMS (Group 5), animals were euthanized and subjected to collection of nasal tissue.

All animals were observed twice daily for mortality and moribundity. Clinical examinations were conducted prior to exposure, at approximately the mid-point of exposure, and at 0-1 hours (+ 0.25 hour) following exposure for Groups 1-4 and once daily on nondosing days and at 0.5-1 hour after dose administration for Group 5. Detailed physical examinations were conducted and body and food weights were recorded within 2 days of receipt, on the day of randomization, and on study days 0 (prior to exposure for Groups 1-4) and 2 (prior to dosing/exposure). All animals were euthanized on study day 2. The nasal tissues from 5 rats in each group were collected and processed for comet assay evaluation and the entire head from an additional 5 or 6 rats in each group was placed in 10% neutral-buffered formalin and the nasal cavity with turbinates were examined microscopically for Groups 1-4. The remaining up to 2 rats in each group were discarded without tissue collection.

There were no test substance-related effects on survival or clinical observations.

Test substance-related lower body weight gains were noted throughout the exposure period in the 30 ppm group. At the end of the exposure period (study day 2), mean body weights in the 30 ppm group were 4.7% lower than the filtered air control group. In addition, the lower body weight gains correlated with the lower mean food consumption throughout the study.

Test substance-related microscopic findings included minimal to mild subacute inflammation of the respiratory and/or transitional epithelium in the 15 ppm group in nasal section II; marked subacute inflammation of the respiratory and transitional epithelium in the 30 ppm group in nasal section II; and mild to marked degeneration of olfactory epithelium of the dorsal meatus in the 30 ppm group in nasal sections III and IV.

The test substance gave a negative response (non-DNA damaging) in the comet assay in the nasal tissues of the male rats. None of the test substance-exposed animals had significant increases in the % tail DNA compared to the filtered air control group. The filtered air control group’s % tail DNA was within the historical range and the positive control group had a statistically significant increase in % tail DNA compared to the filtered air control group. Additionally, it was concluded that the test system was exposed up to the maximum feasible dose, based on evidence of tissue cytotoxicity noted in the nasal cavity in the 30 ppm group. Thus, all criteria for a valid assay were met for nasal tissue.

Justification for classification or non-classification

DTBP is not genotoxic in vitro.

DTBP is not a germ cell mutagen and was negative in an in vivo micronucleus assay by the most relevant route of exposure, inhalation.

Di-tert-butyl has been officially classified (harmonized) as mutagenic, Cat 2. However, this is not in concordance with the currently available test data.