O,O-tert-butyl isopropyl monoperoxycarbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the Ames test, it was concluded that TRIGONOX BPIC-C75 is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. In the Mouse Lymphoma Assay, the test item, O,O-tert-butyl isopropyl monoperoxycarbonate (CAS # 2372 -21 -6) induced marked toxicologically and statistically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells in the absence and presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 19 October 1999 , Experimental completion date 25 October 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: TRIGONOX BPIC·C75
Description: Colourless liquid
Batch: 0419908130562
Purity: 75.8%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Not indicated
Expiry date: 04 October 2000 (allocated at NOTOX, 1 year after receipt of the test substance)

Target gene:

The histidine locus in Salmonella typhimurium.
The tryptophan locus in Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 extracted from the livers of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Range-finding test:
with and without S9-mix - 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate

Based on results from the range-finding study, the following concentrations were used in the mutagenicity test:
with and without S9-mix - 100, 333, 1000, 3330 and 5000 μg/plate

Vehicle / solvent:

dimethylsulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for strain TA1535

Positive control substance:

sodium azide

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Remarks:

60 µg/plate for strain TA1537

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxid

True negative controls:

no

Positive controls:

yes

Remarks:

4 µg/plate for strain TA98

Positive control substance:

other: daunomycine

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Remarks:

650 µg/plate for strain TA100

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for strain WP2uvrA

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethylsulphoxide

True negative controls:

no

Positive controls:

yes

Remarks:

2.5 µg/plate for strain TA1537, 1 µg/plate for strains TA1535, TA98 and TA100 and 5 µg/plate for strain WP2uvrA

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 0.4 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.

DURATION
- Exposure duration: 37ºC for 48 h. (Inadvertently, the plates of the tester strains TA 1535, TA 1537 and T A98 were incubated for 72 hours)
- Selection time (if incubation with a selection agent): 37ºC for 48 h. (Inadvertently, the plates of the tester strains TA 1535, TA 1537 and T A98 were incubated for 72 hours.)
- Environmental conditions: All incubations were carried out in the dark at 37°C. The temperature was monitored during the experiment.

SELECTION AGENT (mutation assays): histidine or tryptophan deficient media

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
To determine the toxicity of TRIGONOX BPIC-C75, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING TEST
TRIGONOX BPIC·C75 was tested in the tester strains TA100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of S9-mix.
This dose range finding test is reported as a part of the mutation test.

Precipitate
The test substance did not precipitate in the top agar. Precipitation of TRIGONOX BPIC-C75 on the plates was not observed at the start or at the end of the incubation period in tester strain TA100 and WP2uvrA.

Toxicity
No reduction of the bacterial background lawn and no decrease in the number of revertants was observed.

Mutagenicity
In the absence of S9-mix, TRIGONOX BPIC-C75 did not induce a dose·related, two·fold, increase in the number of revertant (His+) colonies in tester strain TA 100 and in the number of revertant (Trp+) colonies in tester strain WP2uvrA.
In the presence of S9-mix in tester strain TA 100, TRIGONOX BPIC-C75 induced an up to 5.0·fold, dose related, increase in the number of revertant colonies compared to the solvent control.
In tester strain WP2uvrA, TRIGONOX BPIC-C75 induced an up to 8.7-fold, dose related, increase in the number of revertant colonies compared to the solvent control.

MUTATION ASSAY
Based on the results of the dose range finding study, TRIGONOX BPIC-C75 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the mutation experiment. The mutation experiment was performed with the strains TA 1535, TA 1537 and TA98.

Precipitate
TRIGONOX BPIC-C75 did not precipitate in the top agar. Precipitation of TRIGONOX BPIC-C75 on the plates was not observed at the start or at the end of the incubation period in all tester strains.

Toxicity of the test substance
The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed.

Mutagenicity
In the absence of S9-mix, TRIGONOX BPIC-C75 did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the three tester strains (TA 1535, TA1 537 and TA98).
In the presence of S9-mix in tester strain TA 1537, TAIGONOX BPIC-C75 induced an up to 3.0-fold, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, TRIGONOX BPIC-C75 induced an up to 1.5-fofd, dose related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA 1535, TRIGONOX BPIC-C75 did not induce a dose-related increase in the number of revertant colonies.

MUTAGENIC RESPONSE OF TRIGONOX BPIC-C75 IN THE SALMONELLA TYPHIMURIUM REVERSE MUTATION ASSAY AND IN THE ESCHERICHIA COLI REVERSE MUTATION ASSAY

Dose µg/plate	Mean number of revertant colonies/3 replicate plates (±S.D) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	382 ± 49	226 ± 21	706 ± 78	686 ± 81	908 ± 133
Solvent control	16 ± 3	11 ± 3	35 ± 9	76 ± 6	12 ± 3
3				75 ± 16	9 ± 4
10				73 ± 6	15 ± 4
33				57 ± 5	10 ± 5
100	16 ± 8	8 ± 3	32 ± 4	81 ± 10	12 ± 2
333	18 ± 6	6 ± 2	31 ± 5	74 ± 16	9 ± 3
1000	22 ± 1	9 ± 2	37 ± 7	74 ± 17	8 ± 1
3330	19 ± 7	11 ± 5	40 ± 9	83 ± 13	9 ± 2
5000	21 ± 5	14 ± 1	37 ± 5	96 ± 5	12 ± 2
With S9-mix^1
Positive control	237 ± 18	699 ± 34	1154 ± 55	1141 ± 99	71 ± 4
Solvent control	21 ± 2	10 ± 2	48 ± 2	85 ± 5	11 ± 2
3				80 ± 18	18 ± 2
10				78 ± 17	13 ± 4
33				81 ± 18	11 ± 1
100	15 ± 2	10 ± 3	48 ± 4	73 ± 13	12 ± 1
333	17 ± 5	11 ± 2	57 ± 9	101 ± 8	13 ± 3
1000	24 ± 13	15 ± 5	57 ± 8	102 ± 10	18 ± 6
3330	16 ± 6	24 ± 2	71 ± 8	386 ± 30	87 ± 9
5000	17 ± 4	30 ± 3	64 ± 6	429 ± 9	96 ± 14

Solvent control: 0.1 ml dimethylsulphoxide

¹ The S9-mix contained 5% (v/v) S9 fraction

Conclusions:

Based on the results of this study it is concluded that TRIGONOX BPIC-C75 is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

TRIGONOX BPIC-C75 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and T A98)

and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvr A.

TRIGONOX BPIC-C75 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix. TRIGONOX BPIC-C75 did-not precipitate on the plates at this dose level.

The bacterial background lawn was not reduced at all concentrations tested and no decrease in the n umber of revertants was observed.

In the absence of S9-mix, TRIGONOX BPIC-C75 did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA 1535, TA 1537, TA98 and TA 100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA. ·

In the presence of S9-mix in tester strain TA 1537, TRIGONOX BPIC-C75 induced an up to 3.0-fold, dose-related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA98, TAIGONOX BPIC-C75 induced an up to 1.5-fold, dose-related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA 100, TRIGONOX BPIC-C75 induced an up to 5.0-fold, dose-related, increase in the number of revertant colonies compared to the solvent control. In tester strain WP2uvrA, TRIGONOX BPIC-C75 induced an up to 8.7-fold, dose-related, increase in the number of revertant colonies compared to the solvent control. In tester strain TA 1535, TRIGONOX BPIC-C75 did not induce a dose-related increase in the number of revertant colonies.

Based on the results of this study it is concluded that TRIGONOX BPIC-C75 is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

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

Study period:

Experimental starting date: 27th February 2015 Experimental completion date: 23rd March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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 relevant results.

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: Method designed to be in alignment with the following Japanese Guidelines •Kanpoan No. 287 - Environment Protection Agency •Eisei No. 127 - Ministry of Health and Welfare •Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade and Industry

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/- locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1: 0, 2.5, 5, 10, 20, 30, 40, 50 and 60 µg/mL

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent treatment groups were used as the vehicle controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

-S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

Remarks:

Solvent treatment groups were used as the vehicle controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+S9

Details on test system and experimental conditions:

Test Item Preparation
Following solubility checks performed in-house, the test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared. The test item had a molecular weight of 176.2 therefore the maximum proposed dose level in the solubility test was set at 1762 µg/mL, the maximum recommended 10mM dose level, and the purity of the test item was 74.6% which was not accounted for at the request of the sponsor. However the maximum dose level used was 881 µg/mL, as acetone is dosed at 0.5% in this assay, due to the toxicity of acetone at higher concentrations. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991). The pH and osmolality readings are in the following table:

Dose level
µg/mL 0 3.44 6.88 13.77 27.53 55.06 110.13 220.25 440.5 881
pH
7.86 7.84 8.00 7.99 8.00 7.98 7.94 7.97 7.98 7.94
Osmolality
mOsm 370 377 - - 379 - 377 - 374 378
- Not recorded

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Control Preparation
Vehicle and positive controls were used in parallel with the test item. Solvent (Acetone) (CAS No. 67-64-1) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (CAS No.62-50-0) Sigma batch BCBK5968V 400 µg/mL. Cyclophosphamide (CP) (Cas No. 6055-19-2) Acros batch MKBS0021V at 1.5 µg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO

Microsomal Enzyme Fraction
PB/BNF S9 was prepared in-house on 23 November 2014 from the livers of male Sprague-Dawley rats weighing approximately 250g. These had each received, orally, three consecutive daily doses of phenobarbital/β-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day. This procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012. The conduct of the procedure may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process. The S9 was stored at approximately 196 °C in a liquid nitrogen freezer.

S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.

20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 3.44 to 881 µg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.

The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:

i) Maximum recommended dose level, 5000 µg/mL or 10 mM.

ii) The presence of excessive precipitate where no test item-induced toxicity was observed.

iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002).


Mutagenicity Test
Experiment 1
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (2.5 to 60 µg/mL in the absence of metabolic activation and 5 to 100 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.1 mL of the treatment dilutions, (0.2 or 0.15 mL for the positive controls) and sufficient R0 medium to bring the total volume to 20 mL.

The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.
Experiment 2 was not performed as the result of Experiment 1 was clearly positive. This meets the requirements of the OECD 476 guideline.

Evaluation criteria:

Please see "any other information on materials and methods"

Statistics:

Please see "any other information on materials and methods"

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was evidence of marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls in all three of the exposure groups. The onset of test item-induced toxicity was sharp in all three of the exposure groups. A precipitate of the test item was observed at and above 220.25 µg/mL in all three exposure groups increasing with dose. A greasy oily precipitate was observed at 881 µg/mL in all three exposure groups. Based on the %RSG values observed, the maximum dose level in the subsequent mutagenicity experiment was limited by test item induced toxicity.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

 Experiment 1

There was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and %RSG values (Tables 3 and 6). There was evidence of moderate reductions in viability (%V) in both exposure groups, therefore indicating that residual toxicity had occurred (Tables 3 and 6). Based on the RTG and %RSG values observed, optimum levels of toxicity were achieved in both exposure groups (Tables 3 and 6). The excessive toxicity observed at 60 µg/mL in the absence of metabolic activation and at 100 µg/mL in the presence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

 

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

 

The test item induced both statistically significant and dose related (linear-trend) increases in the mutant frequency x 10^-6per viable cell, in both the absence and presence of metabolic activation (Tables 3 and 6). The GEF value is exceeded at several dose levels both in the absence and presence of metabolic activation. The highest increases are observed with optimum toxicity. Precipitate of the test item was not observed at any of the dose levels.

 

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7. The colony formation was predominantly small, indicative of clastogenic activity, in the both the absence and presence of metabolic activation.

 

Experiment 2 was not performed as the result of Experiment 1 was clearly positive. This meets the requirement of the OECD 476 guideline.

Conclusions:

Interpretation of results (migrated information):
positive

The test item, O,O-tert-butyl isopropyl monoperoxycarbonate (CAS # 2372-21-6) induced marked toxicologically and statistically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells in the absence and presence of metabolic activation.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In VitroMammalian Cell Gene Mutation Tests" adopted 21 July 1997, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

Methods…….

One experiment was performed. L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle, acetone, and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). 

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows:

 

Experiment 1

Group	Concentration ofO,O-tert-butyl isopropyl monoperoxycarbonate (CAS # 2372-21-6)(µg/mL) plated for mutant frequency
4-hour without S9	5, 10, 20, 30, 40, 50
4-hour with S9 (2%)	10, 20, 30, 40, 50, 60

 

Results……..

The maximum dose levels used in thein vitroMutagenicity Test were limited by test item-induced toxicity. No precipitate of the test item was observed. The vehicle controls (acetone) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

 

The test item induced both statistically significant and dose-related (linear-trend) increases in the mutant frequency both with and without metabolic activation. In addition, the GEF (Global Evaluation Factor) value was exceeded at several dose levels, and the colony formation was predominantly small, indicative of clastogenic activity.

 

Conclusion

The test item,O,O-tert-butyl isopropyl monoperoxycarbonate (CAS # 2372-21-6)induced marked toxicologically and statistically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells in the absence and presence of metabolic activation.

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

TRIGONOX BPIC-C75 was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. It is concluded that TRIGONOX BPIC-C75 is NOT mutagenic in the micronucleus test under the in vivo experimental conditions described in the report.

A read across document using an analogue CAS 34443 -12 -4, which showed the lack of genotoxicity in vivo in several tissues. QSAR predictions on CAS# 2372 -21 -6 are in line with the above results.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start: 05 March 2001 Completed: 12 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: Micronucleus Test in mice

Specific details on test material used for the study:

Identification: Trigonox BPIC-C75
Chemical name: Tert-Butylperoxy isopropyl carbonate
CAS-Number: 2372-21-6
Description: Colourless liquid
Batch: 0007130467
Test substance storage: At room temperature in the dark
Stability under storage conditions: Not indicated
Expiry date: 11 January 2002

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Recommended test system in international guidelines (e.g. EPA, FDA, OECD, EEC). The NMRl BR mouse is used as test system because it is a readily available rodent species, which is commonly used for this purpose, with documented susceptibility to a wide range of toxic substances.

Sex:

male

Details on test animals or test system and environmental conditions:

Source: Charles River, Sulzfeld, Germany.
Age at Start of Treatment: Young adult animals were selected (6-8 weeks old}.
Identification: By unique number on the tail.
Allocation: Allocated to treatment groups as they came to hand from delivery boxes.
On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.
Mean body weights immediately prior to dosing (g) (mean ± S.D.):
A 32.6 ± 2.3
B 32.8 ± 1.3
C 33.0 ± 1.0
D 31.8 ± 0.4

ANIMAL HUSBANDRY
Conditions
The animals were housed in an air-conditioned room with approximately 15 air changes per hour and a controlled environment with a temperature of a temperature of 21 ± 3°C and a relative humidity of 30-70%. Due to cleaning procedures or performance of functional observations in the room, temporary deviations from the maximum level for humidity (with max. 20) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity. The room was illuminated with 12
hours artificial fluorescent light and 12 hours dark per day.

Accommodation
Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material (Sawi, Jelu Werk, Rosenberg, Germany). Certificates of analysis were examined and then retained in the NOTOX archives. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

Diet
Free access to standard pelleted laboratory animal diet (Altromin (code VRF 1), Lage, Germany). Certificates of analysis were examined and then retained in the NOTOX archives.

Water
Free access to tap-water. Certificates of analysis (performed quarterly) were examined and then retained in the NOTOX archives.

Route of administration:

other: oral intubation

Vehicle:

TRIGONOX BPIC-C75 was dissolved in corn oil (OPG, Utrecht, The Netherlands). TRIGONOX BPIC-C75 concentrations were treated with ultra-sonic waves until the test substance had completely dissolved.

Details on exposure:

TRIGONOX BPIC-C75 concentrations were dosed within 4 hours after preparation.
The route of administration was chosen to maximize the chance of the test article reaching the target tissue.
Feed was withheld 3- 4 h prior to dosing until administration of TRIGONOX BPIC-C75. The dosing volume was 10 ml/kg body weight. The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test article.

Duration of treatment / exposure:

Single dose

Post exposure period:

Dose range finding study: 3 days
Main study: 24 or 48 hours

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

recommended maximum dose

No. of animals per sex per dose:

In the dose range findng study 2 male and 2 female mice were treated at a single dose
In the main study 5 male mice per sampling time in each treatment group.

Control animals:

yes, concurrent vehicle

other: yes, concurrent positive

Positive control(s):

The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, F.R.G.) dissolved in physiological saline (Fresenius B.V., 's-Hertogenbosch, The Netherlands) dosed at a single oral intubation of 50 mg salt/kg body weight.

Tissues and cell types examined:

erythrocytes in the bone marrow of the femora

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation 24 or 48 h after dosing of TRIGONOX BPIC-C75, 24 h after dosing of the vehicle and 48 h after dosing of the positive control. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1 :1 mixture of 9 6 % (v/v) ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with roundwhetted sides at an angle of approximately 45 ' over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.

Staining of the bone marrow smears
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

Analysis of the bone marrow smears for micronuclei
All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. Al first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same lime. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range finding study
In a dose range finding study 4 animals (2 males and 2 females per group) were dosed orally with 2000 mg/kg body weight.
All treated animals showed no abnormalities during an observation period of 3 days.

Micronucleus Test
Since no substantial differences between the sexes in toxicity or any observable toxic effects were observed, the micronuclues test was performed with male animals only and one dose level: 2000 mg/kg body weight. Five male animals were used in each treatment group.

Mortalitv and systemic toxic signs
All animals of the groups treated with TRIGONOX BPIC-C75 and the animals of the vehicle treated group and the positive control group showed no abnormalities.

Micronucleated polychromatic ervthrocytes
The mean number of micronucleated polychromatic erythrocytes scored in TRIGONOX BPIC-C75 treated groups were compared with the corresponding solvent control group. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of TRIGONOX BPIC-C75 treated animals compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical solvent control data range.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

Ratio polychromatic to normochromatic erythrocytes
The animals of the groups which were treated with TRIGONOX BPIC-C75 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups which were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes.

Conclusions:

II is concluded that this test is valid and that TRIGONOX BPIC-C75 is not mutagenic in the micronucleus test under the in vivo experimental conditions described in this report.

Executive summary:

TRIGONOX BPIC-C75 was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Two groups each comprising 5 males received a single oral intubation of 2000 mg/kg body weight. After dosing all animals of the treatment group showed no abnormalities.

A vehicle treated group (corn oil) served as negative control, a group treated with a single oral intubation of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow of the groups treated with TRIGONOX BPIC-C75 was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone

marrow from the positive control group was harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. The dose range finding study at 2000 mg/kg/d had clinical findings suggesting that the substance was absorbed.

No increase in the frequency of micronuclealed polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with TRIGONOX BPIC-C75.

The groups that were treated with TRIGONOX BPIC-C75 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle

controls.

It is concluded that TRIGONOX BPIC-C75 is not mutagenic in the micronucleus test under the in vivo experimental conditions described in this report.

Reference 2

Endpoint:

genetic toxicity in vivo, other

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification

Justification for type of information:

1. SOFTWARE Six softwares used. Please see "Any other information on methods and materials" Section

2. MODEL (incl. version number): Please see "Any other information on methods and materials" Section

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: CC(C)OC(=O)OOC(C)(C)C

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL : These are standard models, as recognized by ECHA in its guidance document.
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]
- Defined endpoint:
- Unambiguous algorithm:
- Defined domain of applicability:
- Appropriate measures of goodness-of-fit and robustness and predictivity:
- Mechanistic interpretation:

5. APPLICABILITY DOMAIN : These are standard models, as recognized by ECHA in its guidance document.
[Explain how the substance falls within the applicability domain of the model]
- Descriptor domain:
- Structural and mechanistic domains:
- Similarity with analogues in the training set:
- Other considerations (as appropriate):

6. ADEQUACY OF THE RESULT : These are standard models, as recognized by ECHA in its guidance document.
[Explain how the prediction fits the purpose of classification and labelling and/or risk assessment]

Guideline:

other: REACH guidance on QSARs R.6

Principles of method if other than guideline:

- Software tool(s) used including version:
- Model(s) used:
- Model description: see field 'Justification for non-standard information', 'Attached justification' and/or 'Cross-reference'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification' and/or 'Cross-reference'

Specific details on test material used for the study:

CC(C)OC(=O)OOC(C)(C)C

Sex:

not specified

Genotoxicity:

negative

Remarks:

by concensus opinion

Toxicity:

no effects

Remarks:

Lack of genotoxicity by weight of evidence

Vehicle controls validity:

not applicable

Negative controls validity:

other:

Positive controls validity:

other:

Remarks on result:

other: See discussion

See "Any other information -Materials and Methods incl. tables.

Conclusions:

Conclusions
Overall, the QSAR predictions, by Weigh of Evidence, do not support a genotoxicity potential of CAS# 2372 -21 -6. This substance was negative for in vivo micronucleus test in mice. It is concluded that the requirement for an in vivo genotoxicity testing of this substance can be justifiably waived based on:
(1) Negative in vivo GLP genotoxicity test results (OECD 489: Comet assay; OECD 474) as well as other in vivo tests on an analogue substance, CAS # 34443 -12 -4, presented as a supportive evidence by read across;
(2) Weight of Evidence conclusions from QSAR predictions based on several models (mentioned in Appendix 1 of ECHA’s Practical Guide, “How to use and report (Q)SARs”, v.3.1, July 2016); and
(3) Within REACH, the obligation to conduct tests with vertebrate animals should be considered as a last resort. From the animal welfare perspective, the above approaches help to replace animal experiments.

Executive summary:

Introduction

The purpose of this QSAR analysis is to provide a weight of evidence support to the lack of genotoxic potential of BPIC (CAS# 2372-21-6), and thus waive the need for an in vivo genotoxic testing in animals. Within REACH, the obligation to conduct tests with vertebrate animals should be considered as a last resort, only after exhausting all potential sources of information on the physical and toxicological properties of chemicals (EU 2006). Potential sources of information recommended include those based on (Quantitative) Structure-Activity Relationship [(Q)SAR)], referred as one of the non-testing approaches. The advantages of the non-testing approaches include ease of use, speed and low costs. On an animal welfare perspective, they can assist to reduce, refine, and/or replace animal experiments (Russell and Birch, 1959).

 

Approach used for QSAR Predictions:

Chemical structures were drawn using SMILES codes as input parameter. CAS numbers were used in some cases, depending on the QSAR model input requirement. The following QSAR software was used:

 

ACD/ToxSuite, v.2.95 (Build 417)

OECD QSAR Toolbox, v. 4.3; 2019

Biovia Discovery Studio (TOPKAT); 2018

VEGA-QSAR v.1.1.4; 2016

DEREK Nexus v.6.0.1; 2017

Danish QSAR database (which includes Leadscope, CASE Ultra and SciQSAR models), 2016.

Conclusions

Overall, the QSAR predictions, by Weigh of Evidence, do not support a genotoxicity potential of CAS# 2372 -21 -6. This substance was negative for in vivo micronucleus test in mice. It is concluded that the requirement for an in vivo genotoxicity testing of this substance can be justifiably waived based on:

(1)    Negative in vivo GLP genotoxicity test results (OECD 489: Comet assay; OECD 474) as well as other in vivo tests on an analogue substance, CAS # 34443 -12 -4, presented as a supportive evidence by read across;

(2)    Weight of Evidence conclusions from QSAR predictions based on several models (mentioned in Appendix 1 of ECHA’s Practical Guide, “How to use and report (Q)SARs”, v.3.1, July 2016); and

(3)    Within REACH, the obligation to conduct tests with vertebrate animals should be considered as a last resort. From the animal welfare perspective, the above approaches help to replace animal experiments.

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

A read across study of an in vivo rat Comet assay (OECD 489) on an analogue (read across substance, CAS 34443-12-4), was negative for genotoxicity. There is also an in vivo micronucleus on the test substance (CAS 2372-21-6) available, and was negative for genotoxicity. A QSAR for genotoxicty prediction is also presented. Finally, in view of the 3R principle of saving animals, an in vivo gene mutation study is deemed not necessary.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A QSAR analysis for (negative) genotoxicity was done and attached as WofE.

Justification for classification or non-classification

The data are complete and not sufficient for classification.