[2-(Isopropoxycarbonyloxy)-benzoyl]-benzoylperoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In two bacterial reverse gene mutation tests conducted according to OECD 471, the substance did not induce mutagenicity. In a mammalian cell mutation assay (mouse lymphoma) conducted according to OECD 476, the test item CD08467 did not demonstrate biologically relevant increases in mutagenic activity at the tk locus of L5178Y mouse lymphoma cells. In conclusion, these in vitro studies are considered negative with respect to genotoxicity.

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

Histidine

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.

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

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

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)

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

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

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.

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

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-02-14 to 2011-08-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Specific details on test material used for the study:

- Identity: CD08467, also known as RN001351
- Appearance: white to almost white powder
- Purity (LC): 98.4%

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Batch No.: 10.01639 (CS10.038A.1001)
- Expiration date of the lot/batch: 2011-05-02

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated, 2-8 °C

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: All the tester strains, with the exception of strain TA102, were originally obtained from the UK NCTC. 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 (with the exception of strains TA98, TA100 and TA102 in Experiment 2 which were cultured for 6.25 hours, see minor deviation from protocol, Appendix 8) in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

A maximum concentration of 5000 µg/plate was selected for the Experiment l, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines. For Experiment 2 the maximum concentration tested was selected on the basis of toxicity seen in the assay.
- Concentrations:
Mutation Experiment 1 (with and without S9): 0.32, 1.6, 8, 40, 200, 1000, 5000 µg/plate
Mutation Experiment 2 for TA100 and TA1535 in the absence of S9 and strains TA98, TA100, TA1535 and TA1537 in the presence of S9 (with and without S9): 78.13, 156.3, 312.5, 625, 1250, 2500, 5000 µg/plate
Mutation Experiment 2 for TA98 and TA1537 in the absence of S9 and strain TA102 in the presence of S9 (with and without S9): 62.5, 125, 250, 500, 1000, 2000 µg/plate

Vehicle / solvent:

None- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was soluble in the vehicle to at least 100 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9, TA98, 5 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, TA100, TA1535, 2 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9, TA1537, 50 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9, TA102, 0.2 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9, TA98, 10 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (AAN)

Remarks:

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

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Plate incorporation (Experiment 1 with and without activation and Experiment 2 without activation)
- Pre-incubation (Experiment 2 with activation)

MUTATION EXPERIMENTS:
CD08467 was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S9. Negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate in both assays without and with S-9. 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
- 0.1 mL of test article solution or 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).
As the results of Experiment l were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step. Quantities of test article or control solution (reduced to 0.05 mL), bacteria and S9 mix 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 0.05 mL due to the vehicle (DMSO) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

COLONY COUNTING:
For each experiment, colonies were counted electronically using a Sorceror Colony Counter (Perceptive Instruments), or manually where confounding factors such as bubbles or splits in the agar affected the accuracy of the automated counter. The background lawn was inspected for signs of toxicity.

Evaluation criteria:

For valid data, the test article was considered to induce mutation if:
- Dunnett's test gave a significant response (p 5 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 ifnone of the above criteria were met.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Biological relevance will be taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments. Further experimental work may be deemed necessary to aid evaluation of the data.

Statistics:

For evaluation of test article and positive control data there are many statistical methods in use, and several are acceptable. The m statistic was calculated to check that the data are Poisson distributed, and Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate).

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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: Mean vehicle control counts fell within the normal historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: Evidence of toxicity ranging from a diminution of the background bacterial lawn and/or a reduction in the number of revertants to a complete killing of the test bacteria was observed in all strains at the highest one, two and/or three concentrations in the absence and presence of S9.
Experiment 2: Following the treatments, evidence of toxicity ranging from a diminution of the background bacterial lawn and/or a reduction in the number of revertants to a complete killing of the test bacteria was observed in all strains at the highest one, two or three concentrations in the absence and presence of S9.

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 January 2011 by CLEH Statistics, using data selected Without bias from studies# started during the

periods given below:

S. typhimuriums strains (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

# All studies had been audited prior to data collection.

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 (98.4% purity) in DMSO at concentrations of 0.32, 1.6, 8, 40, 200, 1000, 5000 µg/plate (Mutation Experiment 1) and 62.5, 125, 250, 500, 1000, 2000 µg/plate (Mutation Experiment 2 for TA98 and TA1537 in the absence of S9 and strain TA102 in the presence of S9) with and without metabolic activation. 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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-02-10 to 2011-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

- Identity: CD08467, also known as RN001351
- Appearance: white to almost white powder
- Purity (LC): 98.4%

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Batch No.: 10.01639 (CS10.038A.1001)
- Expiration date of the lot/batch: 2011-05-02

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated, 2-8 °C

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5+1% (v/v) CO2 in air. Growth media: RPMI 1640 media

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

A Range-Finder Experiment was performed to establish an appropriate concentration range for the Mutation Experiments. 5 concentrations were selected for the first Mutation Experiment, ranging from non-toxic to toxic (approximately 10-20% relative total growth). If a 24-hour treatment incubation period is required for Experiment 2, concentrations will be selected based on the results of a 24 hour treatment cytotoxicity Range-Finder.
Range-Finder: For freely soluble test articles, where the molecular weight is unknown, the highest concentration tested will be 5000 μg/mL.

Concentrations used in the Range-Finder: 19.53, 39.06, 78.13, 156.3, 312.5, 625.0, 1250, 2500, 5000 µg/mL
Experiment 1: 5, 10, 15, 20, 25, 30, 35, 40, 50 and 60 μg/mL in the absence of S9 and 20, 40, 50, 60, 70, 75, 80, 90, 105 and 120 μg/mL in the presence of S9 (3-hour treatment).
Experiment 2: 10, 20, 30, 40, 45, 50, 55, 60, 65, 70 and 80 μg/mL in the absence of S9 (24-hour treatment) and 20, 30, 40, 50, 55, 60, 65, 70, 80 and 100 μg/mL in the presence of S9 (3-hour treatment).
Experiment 3: 15, 30, 40, 50, 60, 65, 70, 80, 90 and 100 μg/mL in the presence of S9.

Vehicle / solvent:

Vehicle for test article: DMSO
Vehicle for positive control: DMSO- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:Preliminary solubility data indicated that CD08467 was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of approximately 107.8 mg active entity/mL.

Untreated negative controls:

yes

Remarks:

DMSO diluted 100-fold in the treatment medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9, 15.0 and 20.0 µg/mL (3 h treatment); 5 and 7.5 µg/mL (24 h treatment)

Untreated negative controls:

yes

Remarks:

DMSO diluted 100-fold in the treatment medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9, 2.0 and 3.0 µg/mL, 3 h treatment

Details on test system and experimental conditions:

CYTOTOXICITY RANGE-FINDER

Treatment and post-treatment dilution of cell cultures for the cytotoxicity Range-Finder
Experiment was as described below for the Mutation Experiments. However, single cultures only were used and positive controls were not included. The final treatment culture volume was 20 mL. In the absence of S-9, 3 and 24-hour treatment incubation periods were used, in the presence of S9 a 3-hour treatment incubation was used.
Following 3-hour treatment, cells were centrifuged (200 g) for 5 minutes, washed with tissue
culture medium and then resuspended in 50 mL RPMI 10. Following 24-hour treatment, cultures were centrifuged (200 g) for 5 minutes, washed and resuspended in 20 mL RPMI 10. Cell densities were determined using a Coulter counter and, where sufficient cells survived, adjusted to 2 x 10^5 cells/mL. All cultures were incubated at 37 ± 1 ºC for 1 day, recounted and where possible diluted to 2 x 10^5 cells/mL. Cultures were incubated for a further day, counted and adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtiter plate for determination of viability (plating efficiency). All 96 well plates were incubated at 37± 1 ºC in a humidified incubator gassed with 5+1% v/v CO2 in air for 7 days. Wells containing viable clones were identified by eye using background illumination and counted.

MUTATION ASSAYS

Treatment of cell cultures:
For Experiment 1 in the absence and presence of S9 and in Experiments 2 and 3 in the presence of S9 (3-hour treatments) at least 10^7 cells in a volume of 18.8 mL tissue culture medium (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were used. For Experiment 2 in the absence of S9 (24-hour treatment) at least 4 x 10^6 cells in a volume of 19.8 mL RPMI 10 were used. The cell suspensions were placed in a series of appropriate sterile disposable containers which were gassed with 5+1% v/v CO2 in air. For all treatments 0.2 mL vehicle, test article or positive control solution was added. For 3-hour treatments, S-9 mix or 150 mM KCl was added, as described. Each treatment, in the absence or presence of S9, was in duplicate (single cultures only used for positive control treatments) and the final treatment culture volume was 20 mL.
3-hour treatment: After 3 hours’ incubation at 37 ± 1 ºC with gentle agitation, cultures were
centrifuged (200 g) for 5 minutes, washed and resuspended in 50 mL fresh RPMI 10 medium.
24-hour treatment: After static incubation at 37 ± 1 °C for 24 hours, cultures were centrifuged
(200 g) for 5 minutes, washed and resuspended in fresh RPMI 10 medium (20 mL). Cell densities were determined using a Coulter counter and, where sufficient cells survived, adjusted to 2 x 10^5 cells/mL. Cells were transferred to tissue culture flasks for growth throughout the expression period. The solubility of the test article in culture was assessed, by eye, at the beginning and end of treatment. Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies. Osmolality and pH measurements on post-treatment incubation medium were taken in the cytotoxicity Range-Finder Experiment.

Expression period:
Cultures were maintained in flasks for a period of 2 days during which the tk-/- mutation would be expressed. During the expression period, subculturing was performed as required with the aim of not exceeding 1 x 10^6 cells/mL and, where possible, retaining at least 1 x 10^7 cells/flask.

Plating for viability:
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10^4 cells/mL in readiness for plating for TFT resistance. Samples from these were diluted to 8 cells/mL. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into
each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37 ± 1 ºC in a humidified incubator gassed with 5 ± 1% v/v CO2 in air until scoreable (7 to 8 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for TFT resistance:
At the end of the expression period, the cell densities in the selected cultures were adjusted to
1 x 10^4 cells/mL. TFT (300 μg/mL) was diluted 100-fold into these suspensions to give a final concentration of 3 μg/mL. Using an eight-channel pipette, 0.2 mL of each suspension was placed into each well of four 96-well microtiter plates (384 wells at 2 x 10^3 cells/well). Plates were incubated at 37 ± 1 ºC in a humidified incubator gassed with 5 ± 1% v/v CO2 in air until scoreable (12 days) and wells containing clones were identified as above and counted. In addition, the number of wells containing large colonies and the number containing small colonies were scored for the negative and positive controls and for concentrations of test article where a marked increase in mutant frequency (exceeding the GEF) was observed.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:

1. The mutant frequency of any test concentration exceeded the sum of the mean control mutant frequency plus global evaluation factor.

2. The linear trend test was positive.

The test article was considered as positive in this assay if both of the above criteria were met.

The test article was considered as negative in this assay if neither of the above criteria were met.

Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Test for linear trend: Chi-square test

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRECIPITATION
The test article was completely soluble in the aqueous assay system at all concentrations treated in each of the experiments performed.

ASSAY VALIDITY
Mutant frequencies in negative (vehicle) control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals Methyl methane sulphonate (without S-9) and Benzo[a]pyrene (with S9). The study was accepted as valid.

CYTOTOXIC EFFECTS
Experiment 1. Relative total growth (RTG) reduced to 17% at 30 μg/mL for 3 hour treatments without S-9 or 11% at 70 μg/mL for 3 hour treatments with S9 (limited by toxicity).
Experiment 2. RTG reduced to 11% at 60 μg/mL for 24 hour treatments without S-9 or 5% at 100 μg/mL for 3 hour treatments with S-9 (limited by toxicity).
Experiment 3: RTG reduced to 15% at 80 μg/mL for 3 hour treatments with S-9 (limited by toxicity).

For individual results see Tables 1 to 3 in box "Any other information on results incl. tables".


RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity Range-Finder Experiment, 3-hour treatment, six concentrations were tested in the absence and presence of S-9 ranging from 17.19 to 550 μg/mL (limited by solubility in culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations tested (137.5 to 550 μg/mL) in the absence and presence of S-9 but following the 3-hour treatment incubation period, no precipitate was observed at any concentration tested. The highest concentrations to provide >10% RTG were 34.38 μg/mL in the absence of S-9 and 68.75 μg/mL in the presence of S-9, which gave 19% and 26% RTG, respectively. In the cytotoxicity Range-Finder Experiment, 24-hour treatment, nine concentrations were tested
in the absence of S-9 ranging from 2.148 to 550 μg/mL. Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations tested (137.5 to 550 μg/mL) but following the 24-hour treatment incubation period, no precipitate was observed. The highest concentration to provide >10% RTG was 34.38 μg/mL, which gave 56% RTG.
No marked changes in osmolality or pH were observed in the 3- and 24-hour Range-Finder Experiment at the highest concentrations tested (550 μg/mL), compared to the concurrent vehicle controls.

Table 1: Results from Experiment I

Metabolic activation	Incubation conditions	Test Article	Experiment 2
			Concentration/Dose Level (µg/mL)	% Relative Total Growth	Mutant Frequency
+S9	3 h	DMSO	0	100	50.13
		Test item	10	71	52.03
		Test item	20	61	47.67
		Test item	30	47	54.59
		Test item	40	30	74.20
		Test item	45	38	63.53
		Test item	50	18	85.74
		Test item	55	16	59.07
		Test item	60	11	80.65
		Benzo[a]pyrene	5	10	498.74
		Benzo[a]pyrene	7.5	8	931.78

Table 2: Results from Experiment II

Metabolic activation	Incubation conditions	Test Article	Experiment 2
			Concentration/Dose Level (µg/mL)	% Relative Total Growth	Mutant Frequency
+S9	3 h	DMSO	0	100	58.89
		Test item	20	71	65.76
		Test item	30	83	54.51
		Test item	40	82	53.14
		Test item	50	70	45.37
		Test item	55	52	67.94
		Test item	60	46	73.79
		Test item	65	48	71.28
		Test item	70	36	67.32
		Test item	80	26	82.93
		Test item	100	5	112.51
		Benzo[a]pyrene	2	58	615.16
		Benzo[a]pyrene	3	27	1112.34

Table 3: Results from Experiment III

Metabolic activation	Incubation conditions	Test Article	Experiment 3
			Concentration/Dose Level (µg/mL)	% Relative Total Growth	Mutant Frequency
+S9	3 h	DMSO	0	100	54.66
		Test item	15	83	49.80
		Test item	30	68	50.26
		Test item	40	60	53.75
		Test item	50	63	66.37
		Test item	60	42	61.32
		Test item	65	32	75.39
		Test item	70	22	73.43
		Test item	80	15	90.65
		Benzo[a]pyrene	2	61	472.94
		Benzo[a]pyrene	3	25	948.71

Conclusions:

In this study, under the given conditions, the test item did not demonstrate biologically relevant increases in mutagenic activity at the tk locus of L5178Y mouse lymphoma cells. These conditions included treatments up to toxic concentrations in three independent experiments, in the absence and presence of a rat liver metabolic activation system.

Executive summary:

In a mammalian cell gene mutation assay conducted in accordance with OECD 476, mouse lymphoma L5178Y cells cultured in vitro were exposed to CD08467 (98.4% purity) in DMSO at concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 50 and 60 μg/mL in the absence of S9 and 20, 40, 50, 60, 70, 75, 80, 90, 105 and 120 μg/mL in the presence of S9 (3-hour treatment, Experiment 1), 10, 20, 30, 40, 45, 50, 55, 60, 65, 70 and 80 μg/mL in the absence of S9 (24-hour treatment, Experiment 2) and 20, 30, 40, 50, 55, 60, 65, 70, 80 and 100 μg/mL in the presence of S9 (3-hour treatment, Experiment 2) and 15, 30, 40, 50, 60, 65, 70, 80, 90 and 100 μg/mL in the presence of S9 (Experiment 3).

In the absence of S-9, the mutant frequency (MF) values of the concentrations plated in the absence of S-9 in Experiments 1 and 2 were all less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF, 126 mutants/106 viable cells), indicating a negative result. Statistically significant linear trends were observed in both experiments but in the absence of any marked increases in MF at any concentrations analysed in these experiments, the observations were not considered biologically relevant.

In the presence of S9 in Experiment 1, an increase in MF of 144 (thus greater than the GEF) was observed at the highest concentration analysed (70 μg/mL) and there was a statistically significantl inear trend. However, in Experiments 2 and 3, no increases in MF that exceeded the GEF were observed in any cultures analysed up to maximum (toxic) concentrations of 100 and 80 μg/mL in Experiments 2 and 3, respectively. Statistically significant linear trends were observed in Experiments 2 and 3 but no marked increases in MF were observed at any concentration analysed in either of these experiments. The positive result observed at one concentration (70 μg/mL) in the presence of S9 in Experiment 1 was therefore not reproduced in Experiments 2 and 3, where slightly higher maximum concentrations were analysed. These data did not provide evidence of mutagenic activity in the presence of S9 and the isolated observation in Experiment 1 may therefore be considered of little or no biological relevance.

Based on these results, the test item CD08467 is considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo micronucleus study conducted according to OECD 474, 6-10 weeks old male Han Wistar rats (6/dose) were exposed intraperitoneally to the test item CD08467 at a dose volume of 5 mL/kg at concentrations of 2.5, 5 and 10 mg/kg/day. In this study, CD08467 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 10 mg/kg/day. Based on these results, the test item is considered to be non-mutagenic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-02-16 to 2011-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

- Identity: CD08467, also known as RN001351
- Appearance: white to almost white powder
- Purity (LC): 98.4%

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Batch No.: 10.01639 (CS10.038A.1001)
- Expiration date of the lot/batch: 2011-05-02

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 °C in the dark

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd., Oxon UK or Charles River (UK) Ltd, Margate, UK
- Age at study initiation: 6-10 weeks old
- Assigned to test groups randomly: Yes
- Fasting period before study: No fasting
- Housing: Animals housed in groups of up to six
- Diet (e.g. ad libitum): ad libitum; SQC Rat and Mouse Maintenance Diet No 1, Expanded (Special Diets Services Ltd. Witham)
- Water (e.g. ad libitum): ad libitum; mains water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: PEG400/EtOH/NaCl 0.9%
- Justification for choice of solvent/vehicle: Previous trials confirmed that the maximum solubility of CD08467 in the vehicle, PEG400/EtOH/NaCl 0.9% (70/10/20 (w/w/w)), was 2 mg/mL.
- Concentration of test material in vehicle: PEG400/EtOH/NaCl 0.9% (70/10/20 (w/w/w))

Details on exposure:

All treatments were given via intraperitoneal injection in order to maximise exposure of the target organ to the test article. Animals were not fasted prior to dose administration.

Duration of treatment / exposure:

The test article was given as two administrations, 24 hours apart and animals were sampled 24 hours after the final administration, thus enabling examination of cells exposed to the test article over a period of 24 to 48 hours prior to sampling.

Frequency of treatment:

Two administrations, 24 hours apart

Post exposure period:

All animals were observed daily for signs of ill health or overt toxicity. An individual record was maintained of the clinical condition of all Range-Finder and Micronucleus animals dosed in the study.
In the Range-Finder Experiment, post-dosing observations were performed immediately after
each dose administration, at least four times in the 4 hours following each administration and prior to the second dose. Observations were also recorded at least once on each of the non-dose administration days.
The Range-Finder animals were killed and discarded without necropsy or sampling by an overdose of sodium pentobarbitone, given via intraperitoneal injection and subsequently ensured by cervical dislocation.
In the Micronucleus Experiment post-dosing observations were performed immediately after each dose administration, at least twice in the 4 hours following each dose and prior to the second administration. Observations were also recorded at least once on the day of bone marrow sampling.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

2.5 mg/kg bw/day

Dose / conc.:

5 mg/kg bw/day

Dose / conc.:

10 mg/kg bw/day

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control used: Cyclophosphamide (CPA) prepared in saline
- Route of administration: intraperitoneal
- Dose of CPA administered (mg/kg): 20
- Concentration of CPA solution (mg/mL): 4
- Dose volume (mL/kg): 5

Tissues and cell types examined:

Tissue: Bone marrow
Cell type examined: Erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on Range-Finder studies

DETAILS OF SLIDE PREPARATION: One femur from each animal was exposed, removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrows were flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes. An additional 4 mL of serum was added to each bone marrow sample prior to adding to prepared cellulose filtration columns.
Once filtered, the bone marrow cells were centrifuged at 200g for 5 minutes at room temperature and the majority of the supernatant removed. A further 3 mL of foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. A second centrifugation step at 200g for approximately five minutes followed with the serum aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette. From each tube one drop of suspension was placed on the end of each of three slides, labelled appropriately with details such as study number, assay type, sampling time, sex, date of preparation and animal number. A smear was made from the drop by drawing the end of a
clean slide along the labelled slide. Slides were allowed to air-dry and then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water. One slide from each set was taken and stained on the same day as slide preparation. Any remaining slides were kept in reserve at < -10 °C. Slides were stained for 5 minutes in 12.5 μg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then allowed to dry and stored in the dark at room temperature prior to analysis. Following analysis a reserve slide from vehicle animal 4 was stained for analysis. Prior to staining (as above), the slide was fixed again for 10 minutes in absolute methanol and rinsed several times in distilled water.

METHOD OF ANALYSIS: fluorescence microscopy

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic / aneugenic damage if:
- A statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (MN PCE) occurred at one or more dose levels
- The incidence and distribution of MN PCE in individual animals at such a point exceeded the laboratory’s historical vehicle control data
- The group mean MN PCE value at such a point exceeds the 95% calculated confidence interval for the mean historical vehicle control data
- A dose-response trend in the proportion of MN PCE was observed (where more than two dose levels were analysed).
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.

Statistics:

For each group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation. The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine chi-square. Probability values of p < 0.05 were accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: Based on the findings and the maximum test article solubility, 10 mg/kg/day was considered a suitable estimate of the maximum practicable dose. Two lower doses of 5 and 2.5 mg/kg/day were also selected for testing.
- Clinical signs of toxicity in test animals: On both days of dosing ataxia and aggressive behaviour were noted immediately after administration. On Day 1 female animals also displayed vocalization immediately after dosing. Decreased activity was seen in all animals up to 1hour post dose Day 1. The vehicle control was assessed for clinical toxicity at dose volumes of 10mL/kg and 5mL/kg. At 10mL/kg, clinical signs, including ataxia, decreased activity, lethargy, piloerection and hunched posture, persisted throughout post dose observations on Day 1. On Day 2 ataxia onlywas observed. With the 5mL/kg dose volume clinical signs were seen on Day 1 only, 2 & 4 hours post dose. These signs were limited to males and included decreased activity, hunched posture and aggressive behaviour. Due to the nature and severity of clinical signs observed in animals dosed with the vehicle control at 10mL/kg, the dose volume was limited to 5mL/kg for Range-Finder testing in this study.
As no substantial difference in toxicity was observed between males and females in the
Range-Finder Experiment, male animals only were used in the Micronucleus Experiment. - High dose with and without activation: 10 mg/kg/day

RESULTS OF DEFINITIVE STUDY
- Clinical signs: No clinical signs of toxicity were observed in the vehicle or positive control (CPA) animals. In test article treated animals (2.5, 5 and 10 mg/kg/day) clinical signs were limited to ataxia, which was noted in all animals 0.5 hour post the first administration. Following the first administration reductions in group mean bodyweight (ranging between 7-16 g) were noted (Day 1-2) in the vehicle and test article groups. There was no significant weight change following the second administration.
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): Groups of rats treated with CD08467 exhibited %PCE values that were acceptable compared to the vehicle control (Appendix 3 and Appendix 5) and which were within acceptable ranges. There was no evidence of any test article-induced toxicity to the bone marrow (as would usually be indicated by a notable decrease in %PCE values compared to the vehicle control group or dose dependent decrease).
- Statistical evaluation: For all dose groups, the group mean frequencies of MN PCE were comparable with, and not statistically (chi-square) different from those seen in concurrent vehicle control group. Individual frequencies of MN PCE for all treated animals were consistent with historical vehicle control distribution data and similar to frequencies observed in the concurrent controls.

For individual results see Table 1 in box "Any other information on results incl. tables".

Conclusions:

Under the reported experimental conditions, CD08467 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 10 mg/kg/day (the maximum practicable dose for this study).

Executive summary:

In an in vivo micronucleus study conducted according to OECD guideline 474, 6 -10 weeks old, male Han Wistar rats (6/dose) were exposed intraperitoneally to the test item CD08467 (purity: 98.4%) at a dose volume of 5 mL/kg at concentrations of 2.5, 5 and 10 mg/kg/day. The doses were given twice at an interval of 24 hours. The organisms were checked for clinical signs of toxicity and the bone marrow was sampled. Rats treated with CD08467 at all doses exhibited MN PCE frequencies that were similar to the values for the vehicle control group and which also fell within the laboratory's historical distribution data. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test article. The positive control chemical (CPA) induced a statistically significant (p < 0.001) increase in the frequency of micronucleated PCE, exhibiting a group mean of 30.17 MN/2000 PCE compared to the concurrent vehicle control which displayed a group mean of 3.00 MN/2000 PCE. It is concluded that CD08467 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 10 mg/kg/day (the maximum practicable dose for this study). Based on these results, the test item is considered to be non-mutagenic according to the results of the in vivo micronucleus test reported.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The genotoxic potential of CD08467 is assessed in a weight-of-evidence approach based on several in vitro studies (GLP studies, OECD 471 and OECD 476) and one in vivo study (GLP study, OECD 474).

In two bacterial reverse gene mutation tests conducted according to OECD 471, the test item CD08467 did not induce mutagenicity, and in a mammalian cell mutation assay conducted according to OECD 476, CD08467 did not demonstrate biologically relevant increases in mutagenic activity at the tk locus of L5178Y mouse lymphoma cells. In an in vivo micronucleus study conducted according to OECD 474, 6-10 weeks old male Han Wistar rats (6/dose) were exposed intraperitoneally to CD08467 at a dose volume of 5 mL/kg at concentrations of 2.5, 5 and 10 mg/kg/day. In this study, CD08467 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of male rats treated up to 10 mg/kg/day.

In conclusion, in all genotoxicity tests, CD08467 was tested negative. Based on the results, the substance can be considered as non-genotoxic and no classification is warranted in accordance with CLP Regulation 1272/2008.

Justification for classification or non-classification

Based on the lack of mutagenicity/genotoxicity in all of the conducted in vitro and in vivo assays, CD08467 is considered to be non-genotoxic and no classification is warranted in accordance with CLP Regulation 1272/2008.