[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-09-29 to 2011-02-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

EC Number:

814-835-0

Cas Number:

1310672-91-3

Molecular formula:

C18H16O7

IUPAC Name:

[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B
- Purity (HPLC): 96.95%area
- Expiration date of the lot/batch: 2015-03-02

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: The test item is unstable in dimethyl sulphoxide, but that in acetone stable solutions of at least 5% p/v could be attained. In this study test item solutions of at least 200 mg/mL in acetone were achieved.
- Storage: at 2 to 8 °C in the dark

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100, TA1535, TA1537 and TA102

Details on mammalian cell type (if applicable):

Not applicableCELLS USED
- Source of cells: Strains TA98, TA1OO, TA1535 and TA1537 were originally obtained from the UK NCTC. Strain TA97 was obtained from the Czech Collection of Microorganisms, Masaryk University, Czech Republic. Strain TA102 was derived from a culture obtained from Glaxo Group Research Limited

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: bacteria were cultured at 37 ± 1 °C for 10 hours in nutrient broth, containing ampicillin (TA97, TA98, TA1OO) or ampicillin and tetracycline (TA102) as appropriate.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

- Test concentrations:
Range-finder Experiment and Mutation Experiment 1 (with and without S9): 1.6, 8, 40, 200, 1000, 5000 µg/plate
Mutation Experiment 2 TA98, TA100, TA1535, TA97, TA102 (with and without S9): 78.125, 156.25, 312.5, 625, 1250, 2500 µg/plate
Mutation Experiment 2 TA1537 (with and without S9): 156.25, 312.5, 625, 1250, 2500, 5000 µg/plate
For concentrations see also Table 1 in box "Any other information on materials & methods incl. tables".

In this study CD08467 solutions of at least 200 mg/mL in acetone were achieved and a maximum recommended concentration of 5000 μg/plate (according to current regulatory guidelines was tested in the Range-finder Experiment and Experiment 1. In Experiment 2, the maximum concentration tested was selected based on solubility limitations seen in Experiment 1.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Information provided by the Sponsor confirmed that solutions of CD08467 in dimethyl sulphoxide were unstable, but that in acetone stable solutions of at least 5% p/v could be attained.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9, TA98, 5 µg/plate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, TA100, TA1535, 2 µg/plate

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9, TA1537 (50 µg/plate), TA97 (100 µg/plate)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9, TA102, 0.2 µg/plate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9, TA98, 10 µg/plate

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (AAN)

Remarks:

with S9, TA100, TA1535, TA1537, TA97 (5 µg/plate), TA102 (20 µg/plate)

Details on test system and experimental conditions:

EXPERIMENTAL PROCEDURE:

- Range - Finder experiment: CD08467 was tested for toxicity (and mutation) in strain TA100, at the concentrations detailed previously. Triplicate plates without and with S-9 mix were used. Negative (vehicle) and positive controls were included in quintuplicate and triplicate respectively, without and with S-9 mix.
These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46+1ºC :
- 0.1 mL of bacterial culture
- 0.05 mL of test article solution and vehicle control or 0.1 mL positive control
- 0.5 mL 10% S-9 mix or buffer solution.
Followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37+1ºC in the dark for 3 days. The plates were examined for signs of toxicity and revertant colonies counted (see Colony counting).

- Experiment I: CD08467 was tested for mutation (and toxicity) in six strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA97 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9. Experiment 1 mutagenicity data for strain TA100 were provided by the Range-Finder Experiment treatments. Negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate in both assays without and with S-9. Platings were achieved as described above.

Experiment II: Treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article and negative control (reduced to 0.025 mL) or positive control solution (reduced to 0.05 mL), bacteria and S-9 mix (as detailed above), were mixed together and incubated for 1 hour
at 37+1ºC, before the addition of 2.5 mL molten agar at 46+1ºC. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay. Volume additions for the Experiment 2 pre-incubation treatments were reduced to 0.025 mL due to the vehicle (acetone) employed in this study.

- Colony counting: For each experiment, colonies were counted electronically using a Sorceror Colony Counter (Perceptive Instruments), or manually where confounding factors such as precipitation or bubbles or splits in the agar affected the accuracy of the automated counter. The background lawn was
inspected for signs of toxicity.METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1); preincubation (Experiment 2)
- Cell density at seeding (if applicable):

EXPERIMENTAL PROCEDURE:
- Range - Finder experiment: CD08467 was tested for toxicity (and mutation) in strain TA100, at the concentrations detailed previously. Triplicate plates without and with S-9 mix were used. Negative (vehicle) and positive controls were included in quintuplicate and triplicate respectively, without and with S-9 mix.
These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46 ± 1 °C:
- 0.1 mL of bacterial culture
- 0.05 mL of test article solution and vehicle control or 0.1 mL positive control
- 0.5 mL 10% S9 mix or buffer solution.
Followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days. The plates were examined for signs of toxicity and revertant colonies counted (see Colony counting).
Experiment I: CD08467 was tested for mutation (and toxicity) in six strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, TA97 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S9. Experiment 1 mutagenicity data for strain TA100 were provided by the Range-Finder Experiment treatments. Negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate in both assays without and with S-9. Platings were achieved as described above.
Experiment II: Treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article and negative control (reduced to 0.025 mL) or positive control solution (reduced to 0.05 mL), bacteria and S-9 mix (as detailed above), were mixed together and incubated for 1 hour at 37 ± 1 °C, before the addition of 2.5 mL molten agar at 46 ± 1 °C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay. Volume additions for the Experiment 2 pre-incubation treatments were reduced to 0.025 mL due to the vehicle (acetone) employed in this study.
Colony counting: For each experiment, colonies were counted electronically using a Sorceror Colony Counter (Perceptive Instruments), or manually where confounding factors such as precipitation or bubbles or splits in the agar affected the accuracy of the automated counter. The background lawn was inspected for signs of toxicity.

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

For valid data, the test article was considered to induce mutation if:
- Dunnett's test gave a significant response (p < 0.01) which was concentration related
- the positive trend/effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis.
Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

Mean and standard deviation of revertant number per plate were determined.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA97 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only in TA102 at 1000 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: TA98, TA100, TA1535, TA1537, TA97, TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only in TA102 at 2500 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY:
Following treatments of all the test strains in the absence and presence of S9, only Experiment 1 treatments of strain TA1537 in the absence of S-9 at 5000 µg/plate resulted in an increase in revertant numbers that was statistically significant when the data were analysed at the 1% level using Dunnett’s test. Although the increase in revertants was large and appeared to be concentration-related (occurring solely at 5000 µg/plate) it was not reproduced in Experiment 2. It was considered likely that the precipitation observed at 5000 µg/plate may have confounded manual scoring of this concentration, and therefore observed increase in revertant colonies was considered to be an artefact and not evidence of mutagenic activity. No other increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no clear evidence of any CD08467 mutagenic activity in this assay system.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Experiment 1: Test article precipitate was observed on all plates treated at 1000 μg/plate and above.
Experiment 2: Test article precipitate was observed on all plates treated at 625 μg/plate and above.

RANGE-FINDING/SCREENING STUDIES: No evidence of toxicity was observed. Test article precipitate was observed on all plates treated at 1000 μg/plate and above. These data were considered to be acceptable for mutation assessment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control chemicals all induced large increases in revertant numbers in the appropriate strains, which fell within the normal historical ranges
- Negative (solvent/vehicle) historical control data: The mean vehicle control counts were comparable with the normal historical ranges
See Tables 2 and 3 in box "Any other information on results incl. tables".

Remarks on result:

other: Experiment 1

Any other information on results incl. tables

Table 2: Historical negative (vehicle) control values for S. typhimurium strains

Strain	S9	No. of studies	No. of plates	Mean	99% reference range(1)	99% confidence interval for group mean of
						4 values(2)	5 values(2)	6 values(2)
TA98	-	51	525	25	9.0-44.0	16.0-34.3	16.8-33.1	17.4-32.3
TA98	+	51	538	35	16.0-58.0	25.0-46.6	26.0-45.3	26.7-44.4
TA100	-	51	600	111	73.0-156.5	88.7-133.5	90.8-130.9	92.4-129.0
TA100	+	51	604	117	71.0-168.0	91.8-144.3	94.3-141.2	96.2-139.0
TA1535	-	50	520	17	5.0-30.0	10.8-24.8	11.4-23.9	11.8-23.3
TA1535	+	50	525	17	6.0-32.0	10.8-24.5	11.4-23.6	11.8-23.0
TA1537	-	51	530	13	3.0-30.0	6.3-20.1	6.9-19.2	7.3-18.5
TA1537	+	51	527	18	4.0-33.0	10.4-25.8	11.0-24.8	11.5-24.1
TA102	-	50	520	270	184.0-350.0	227.7-313.3	231.9-308.5	235.0-304.9
TA102	+	50	528	233	153.0-328.0	191.5-275.2	195.6-270.4	198.6-266.6

(1) Reference ranges are calculated from percentiles of the Observed distributions.

(2) Calculated from square-root transformed data.

Ranges calculated in July 2010 by CLEH Statistics, using data selected Without bias from studies# started during the

periods given below:

S. typhimurium strains (TA102) Feb 08 to Jul 09

S. typhimurium strain TA102 Feb 08 to Jul 09

# All Studies had been audited prior to data collection.

Table 3: Historical positive control values for S. typhimurium strains

Strain	S9	No. of studies	No. of plates	Mean (Induced numbers for individual plates)	Reference ranges(1)
					95%	99%
TA98	-	51	315	824	386.8-1760.4	275.2-1923.4
TA98	+	51	324	321	164.4-532.6	99.6-647.2
TA100	-	51	360	660	339.2-1094.6	263.8-1171.6
TA100	+	51	363	1172	542.0-2068.4	407.4-2384.8
TA1535	-	50	312	601	325.4-876.2	271.4-989.4
TA1535	+	50	314	212	107.6-326.8	84.4-364.6
TA1537	-	51	316	108	40.6-281.4	32.2-576.2
TA1537	+	51	317	119	36.2-257.0	19.4-327.6
TA102	-	50	312	450	235.6-672.8	140.2-931.6
TA102	+	50	318	1370	450.2-2742.6	313.4-3045.4

(1) Reference ranges are calculated from percentiles of the Observed distributions.

Ranges calculated in July 2010 by CLEH Statistics, using data selected Without bias from studies# started during the periods given below:

S. typhimurium strains (TA102) Feb 08 to Jul 09

S. typhimurium strain TA102 Feb 08 to Jul 09

# All studies had been audited prior to data collection.

Applicant's summary and conclusion

Conclusions:

In conclusion, the test item is not genotoxic in the bacterial reverse gene mutation assay in the presence and absence of mammalian metabolic activation..

Executive summary:

In a reverse mutation assay in bacteria conducted according to OECD 471, strains of S. typhimurium (TA97, TA98, TA100, TA1535, TA1537 and TA102) were exposed to the test item CD08467 (97% purity) in acetone at concentrations of 1.6, 8, 40, 200, 1000, 5000 µg/plate (Mutation Experiment 1 (with and without S9)), 78.125, 156.25, 312.5, 625, 1250, 2500 µg/plate (Mutation Experiment 2 TA98, TA100, TA1535, TA97, TA102 (with and without S9)) and 156.25, 312.5, 625, 1250, 2500, 5000 µg/plate (Mutation Experiment 2 TA1537 (with and without S9)). The positive controls did induce the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.