Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material is not mutagenic in Salmonella typhymurium strains TA98,TA100, TA1535, TA1537, and TA1538 (method not stated but largely consistent with 1997 OECD TG 471) and mouse L5178Y cells (OECD Guideline 476).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 June 1995 - 02 February 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A bacterial strain with an AT base pair at the primary reversion site (e.g. S. typhimurium TA102 or E. coli WP2uvrA) was not included.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(see below)
Principles of method if other than guideline:
The study report does not state the study was conducted in accordance with a particular published test guideline, but the methodology used is largely consistent with the 1997 OECD TG 471 (plate incorporation method), except that a strain with an AT base pair at the primary reversion site (e.g. S. typhimurium TA102 or E. coli WP2uvrA) was not included.
2-aminoathracene was used as the sole indicator of the efficacy of the S9 mix in the assay. It is not clear whether prior to assay initiation, the S9 underwent any other characterisation with a mutagen that requires activation by microsomal enzymes.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
The target genes in the Salmonella strains control the synthesis of histidine.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
rat S9 (Arochlor 1254 pretreated Sprague Dawley rats)
Test concentrations with justification for top dose:
50, 100, 500, 1000 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone (Lot No. E41620) was used for the test substance.
DMSO (Lot No. 06935PZ) was used for the positive control substances.

- Justification for choice of solvent/vehicle:
A solubility test was performed to assess the solubility of the test substance in tetrahydrofuran (THF), methyl sulfoxide (DMSO), water, and acetone. Solubiity testing indicated the test substance was soluble in DMSO, and toxicity and initial assays were performed using DMSO as the vehicle. The results from the initial concentration verification were received after these assays were performed and indicated that the test substance was not completely soluble in DMSO. Therefore, the toxicity and initial assays were repeated using acetone as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
used with TA1537 without S9

Migrated to IUCLID6: Lot No. 96F05641
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (Lot No. 04507JV)
Remarks:
used with all strains with S9
Positive controls:
yes
Positive control substance:
other: N-methyl-N-nitro-N-nitrosoguanidine (Lot No. 08029JG)
Remarks:
used with TA100 and TA1535 without S9
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
used with TA98 and TA1538 without S9

Migrated to IUCLID6: Lot No. 01703EV
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


EXPOSURE DURATION: 2 days.


NUMBER OF REPLICATIONS: 3 (triplicate).


DETERMINATION OF CYTOTOXICITY
- Method: size of background lawn, and/or >50% reduction in the mean number of revertant colonies when compared to the vehicle control.
Evaluation criteria:
An individual dose was considered positive if the mean revertant count on the test plates was equal to or greater than three times the mean number of spontaneous revertants on the vehicle control plates. A positive result for the assay was defined as a dose-related increase in the mean number of revertant colonies over at least three concentrations of test substance including at least one positive dose. A lack of response in the positive controls or spontaneous revertant frequencies which were not in keeping with historical laboratory values would render that portion of the test invalid.

Toxicity was defined as a notable reduction in the background lawn and/or a greater than 50% reduction in the mean number of revertant colonies when compared to the vehicle control.

A positive result in this assay indicates that, under the test conditions, the test substance induced point mutations by base changes or frameshifts in the genome of Salmonella typhimurium. Negative results indicate that, under the test conditions, the test substance was not mutagenic in Salmonella typhimurium.
Statistics:
The mean revertant colony count and standard deviation were determined for each dose point.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest concentration of 5000 µg/plate in strains TA100 and TA1527
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest concentration of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test material was beaded on all plates at 5000 µg/plate in all tester strains with and without metabolic activation in both the initial and repeat assays.


RANGE-FINDING/SCREENING STUDIES: Prior to initiation of the initial assay, a rangefinding test was performed to determine the doses used for both assays. Toxicity, a notable reduction in the background lawn and/or a greater than 50% reduction in the mean number of revertant colonies when compared to the vehicle control, was observed at 5000 µg/plate without metabolic activation. At the 1000, 2000 and 5000 µg/plate dose levels (with and without metabolic activation) beading was observed increasing with increasing dose levels. Based on the results of the rangefinding test, the doses selected for the mutagenicity assay were 50, 100, 500, 1000 and 5000 µg/plate.


COMPARISON WITH HISTORICAL CONTROL DATA: The nontreated and vehicle controls responded in a manner consistent with data from previous assays.


ADDITIONAL INFORMATION ON CYTOTOXICITY: A greater than 50% reduction in the mean number of revertant colonies when compared with the vehicle (acetone) control was observed in the initial assay in TA100, TA1537, and TA1538 without metabolic activation at 5000 µg/plate. In the repeat assay, a greater than 50% reduction in the mean number of revertant colonies was observed in TA100, and TA1538 with and without metabolic activation at 5000 µg/plate. Pinpoint colony background was observed in several plates in TA1537 and TA1538 with and without metabolic activation at 5000 µg/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of this study, the test material was not mutagenic in any strain of Salmonella typhimurium, w hich included at least one dose that was toxic and above the solubility of the test substance (5000 ug/plate).
Executive summary:

The test material was assessed for mutagenic potential in a bacterial reverse mutation (Ames) assay conducted in accordance with GLP. The study was largely consistent with the 1997 OECD Test Guideline 471 (plate incorporation method), except that a bacterial strain with an AT base pair at the primary reversion site was not included. Mutagenic potential was assessed in five strains of Salmonella typhimurium - TA98, TA100, TA1535, TA1537 and TA1538 - in both the presence and absence of S9 metabolic activation. Five concentrations of test material were evaluated: 50, 100, 500, 1000 and 5000 ug/plate; the highest concentration being above the solubility of the test substance and also showing cytotoxicity. The assay positive and negative (vehicles and nontreated) control groups responded appropriately. The test substance did not induce a significant increase in revertant colonies (equal to or greater than three times the vehicle control) in any strain at any dose level, with or without metabolic activation, in either the initial or repeat assays. It is concluded that the test substance was not mutagenic under the conditions of this test.

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:
Between 16 March 2009 and 27 April 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed to a guideline and used GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection 19/08/2008. Date of signature 04/03/2009
Type of assay:
mammalian cell gene mutation assay
Target gene:
The thymidine kinase, TK +1-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ug/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ug/ml) and 10% donor horse serum (giving R10 media) at 37 oC with 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared in-house from the livers of male Wistar Han™ rats weighing -200g. These had each received, orally, three consecutive daily doses of phenobarbital/b-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day.
Test concentrations with justification for top dose:
Experiment 1 (ug/ml) without S9: 0, 0.63, 1.25, 2.5, 5, 10, 20, 30, 40
Experiment 1 (ug/ml) with S9: 0, 2.5, 5, 10, 20, 30, 40, 60, 80
Experiment 2 (ug/ml) without S9: 0, 1.25, 2.5, 5, 10, 15, 20, 30, 40
Experiment 2 (ug/ml) with S9: 0, 2.5, 5, 10, 20, 30, 40, 60, 80
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone.
- Justification for choice of solvent/vehicle:The test material was a complex mixture, insoluble in water, and acetone was selected as the solvent because the test material was soluble in it at the required concentration.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Cyclophosphamide (CP) at 2 ug/ml was used as the positive control in the presence of metabolic activation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Ethylmethanesulphonate (EMS) at 400 ug/ml and 150 ug/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ug/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ug/ml) and 10% donor horse serum (giving R10 media). 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 10^6 cells/ml in 10 ml aliquots in R10 medium in sterile plastic universals. The cells were exposed to doses of the test material, vehicle and positive control, both with and without metabolic activation. Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air.
The treatment regimes were as follows:
EXPERIMENT 1:
a). Without metabolic activation: 4-hour exposure, dose levels 0, 0.63, 1.25, 2.5, 5, 10, 20, 30, 40 ug/ml
b). With metabolic activation (2% S9): 4-hour exposure groups, dose levels 0, 2.5, 5, 10, 20, 30, 40, 60, 80 ug/ml
EXPERIMENT 2:
a). Without metabolic activation: 24-hour exposure, dose levels 0, 1.25, 2.5, 5, 10, 15, 20, 30, 40
ug/ml
b). With metabolic activation (1% S9):4-hour exposure, dose levels 0, 1.25, 2.5, 5, 10, 20, 30, 40, 50, 60 ug/ml.

DURATION
- Preincubation period: Not applicable.
- Exposure duration: 4 h (Experiment 1 and 2), or 24 h (Experiment 2).
- Expression time (cells in growth medium): 2 days (viability test)
- Selection time (if incubation with a selection agent): 10~14 days (plate scoring for colony formation)
- Fixation time (start of exposure up to fixation or harvest of cells): ~ 2 h


SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-200 x 10^-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should be within this range, experiments where the vehicle control values are markedly > 250 x 10^-6 mutant frequency per survivor will be repeated. Positive control chemicals should induce at least 3~5 fold increases in mutant frequency greater than the corresponding vehicle control.
The optimum toxicity is approximately 20% survival, but no less than 10% survival. Both %RSG and RTG values are used either individually or combined to designate the level of toxicity achieved by the test material for any individual dose level. Dose levels that have survival values less than 10% are excluded from any statistical analysis.
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The Global Evaluation Factor (GEF) value was set at 126 x 10^-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgment will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
The experimental data was analyzed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS statistical package. Dose levels that have survival values less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: mouse lymphoma L5178Y cells
Remarks:
Migrated from field 'Test system'.

Preliminary Toxicity Test

The dose range of the test material used in the preliminary toxicity test was 3.91 to 1000mg/ml. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (ug/ml)

for 4 h

% R8G (-S9) 4-Hour Exposure

% R8G (+S9) 4-Hour Exposure

 

Dose (ug/ml)

for 24 h

 % R8G (-S9) 24-Hour Exposure

0

100

100

0

100

19.53

76

94

0.31

40

39.06

3

57

0.63

 62

78.13

1

1

1.25

 70

156.25

0

0

2.5

 84

312.5

0

0

5

 58

625

0

0

10

 22

1250

0

0

20

12 

2500

0

0

40

 1

5000

0

0

60

 0

In all three of the exposure groups there was a marked reduction in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The toxicity curve was steep in all three of the exposure groups. A precipitate of the test material was observed at and above 156.25mg/ml. In the subsequent mutagenicity experiments the maximum dose was limited by test material-induced toxicity.

 

Mutagenicity Test

A summary of the results from the test is presented in Table 1.

Experiment 1

There was evidence of toxicity following exposure to the test material in both the absence (Table 3) and presence (Table 6) of metabolic activation, as indicated by the %RSG and RTG values. There was also evidence of a decrease in (%V) viabilities, therefore indicating that residual toxicity had occurred. However, it should be noted that significant decreases were only observed at dose levels that exceeded the usual acceptable upper limit of toxicity. The expected optimum levels of toxicity were not achieved in either the absence and presence of metabolic activation. However, it was considered that, due to the very steep toxicity curve of the test material, a higher dose level in each of the exposure groups would have resulted in excessive levels of toxicity. Therefore, with no evidence of any statistically significant increases in mutant frequency in this experiment or the second experiment, where optimum levels of toxicity were achieved in both the absence and presence of metabolic activation at similar dose levels, the test material was considered to have been adequately tested. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6)..

Neither of the vehicle control mutant frequency values were outside the range of 50 to 200 x 10-6viable cells that is acceptable for L5178Y cells at Harlan Laboratories Ltd,,. 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 material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). Precipitate of test material was not observed at any of the dose levels.

The numbers of small and large colonies also recorded and analyzed, no statistically significant differences were obtained as comparing vehicle control and treated groups.

 

Experiment 2

As was seen previously there was evidence of a dose-related reduction in % RSG and RTG values in cultures dosed with the test material in both the absence and presence of metabolic activation. On this occasion the expected optimum level of test material-induced toxicity was achieved in both the absence and presence of metabolic activation (Tables 9 and 12). There was no evidence of a reduction in (%V) viability, therefore indicating that no residual toxicity had occurred in either of the exposure groups. The (%V) viability values of the vehicle controls were very marginally lower than those usually observed at Harlan Laboratories Ltd,,. However, with no evidence of any responses, in either the first or second experiment, the data was considered acceptable for the purpose of this study. The excessive levels of toxicity observed at 40 μg/ml in the absence of metabolic activation, and at and above 80 μg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or TFT resistance. Both positive controls induced acceptable levels of toxicity (Tables 9 and 12).

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had no effect on the toxicity of the test material. The lowering of the S9 concentration to 1% S9 in this second experiment resulted in greater levels of toxicity than those observed in the presence of 2% S9 in the first experiment.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6viable cells. 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 9 and 12).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). Precipitate of test material was not observed at any of the dose levels.

The numbers of small and large colonies also recorded and analyzed, no statistically significant differences were obtained as comparing vehicle control and treated groups.

 

Key to Tables

$= Cell counts (x105cells/ml). Set up on previous day to 2 x 105cells/ml unless otherwise stated in parenthesis.

% RSG =Relative Suspension Growth

RTG = Relative Total Growth

%V = Viability Day 2

§ or # = Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis

A, B =  Replicate cultures

CP =   Cyclophosphamide

EMS= Ethylmethanesulphonate

MF§ = 5-TFT resistant mutants/106viable cells 2 days after treatment

NP =   Not plated due to toxicity or surplus to requirements

Ø= Not plated for viability or TFT resistance

X= Treatment excluded from test statistics due to toxicity

NS = Not significant

 

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.
Executive summary:

Introduction.The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK+/-,locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Methods.Two independent experiments were performed. In Experiment 1, L5178Y TK+/-3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 0.63 to 40 μg/ml in the absence of metabolic activation and 2.5 to 80 μg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 1.25 to 40 μg/ml in the absence of metabolic activation, and 2.5 to 100 μg/ml in the presence of metabolic activation.

Results.The maximum dose level used was limited by test material induced toxicity. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test material does not cause clastogenic effects in the in vivo mouse micronucleus assay based on U.S. EPA Health Effects Test Guideline OPPTS 870.5395.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 October 2001 - 05 December 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study consistent with OECD and EPA test guidelines
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
yes
Remarks:
Housing temperature was maintained at 64-79 deg F, a slightly wider range than the 19-25 deg C recommended by US EPA.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Housing temperature was maintained at 64-79 deg F, a slightly wider range than the 19-25 deg C recommended by OECD.
Principles of method if other than guideline:
Although only the US EPA OPPTS guideline is specifically referred to in the methods section of the study report, the study methodology used was consistent with the 1997 OECD TG 474, and this OECD guideline is included in the study report's list of references (and in the testing laboratory's assigned study number: 23254-0-455OECD).
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: Crl:CD-1(ICR) BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC.
- Age at study initiation: Animals were approximately 8 weeks old at the time of dosing.
- Weight at study initiation: 20-40 g (within ±20% of the mean weight of each sex).
- Assigned to test groups randomly: Yes.
- Housing: Group-housed in sanitary polycarbonate cages containing hardwood chip bedding.
- Diet (e.g. ad libitum): Ad libitum.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: At least five days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-79ºF (17.7-26.1ºC).
- Humidity (%): 30-70%.
- Air changes (per hr): At least 10.
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark.

IN-LIFE DATES: Animals in the main study were dosed on 13-15 November 2001 and observed for a further 24 hours.
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: White oil.
- Justification for choice of solvent/vehicle: Not stated.
- Concentration of test material in vehicle: Dose-range finding study: 25, 50, 100 and 200 mg/mL.
Main micronucleus study: 6.25, 12.5 and 25 mg/mL.
- Dosing volume: 10 mL/kg.
Details on exposure:
The test substance was administered by intraperitoneal injection in both the dose-range-finding assay and the main micronucleus assay.
Duration of treatment / exposure:
Dose-range-finding assay: Three days.
Main micronucleus assay: Three days.
Frequency of treatment:
Dose-range-finding assay: Daily (for three consecutive days).
Main micronucleus assay: Daily (for three consecutive days).
Post exposure period:
Dose-range-finding assay: Two days.
Main micronucleus assay: One day.
Remarks:
Doses / Concentrations:
250, 500, 1000 and 2000 mg/kg/day
Basis:
other: Injected dose levels in dose-range-finding assay
Remarks:
Doses / Concentrations:
62.5, 125 and 250 mg/kg/day
Basis:
other: Injected doses levels in main micronucleus assay
No. of animals per sex per dose:
Dose-range-finding assay: Three animals per sex per dose level.
Main micronucleus assay: Six males per dose level.
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control: Cyclophosphamide.
- Justification for choice of positive control(s):
- Route of administration: Oral gavage.
- Doses / concentrations: 80 mg/kg/day (8 mg/mL at 10 mL/kg).
Tissues and cell types examined:
Cytotoxicity was assesssed by scoring the number of polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal.
At least 2000 PCEs per animal were analysed for the frequency of micronuclei.
Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Approximately 24 hours after the last dose administration, the animals were euthanised by carbond dioxide inhalation followed by incision of the diaphragm. The hind limb bones (tibias) were removed for marrow extraction from five surviving animals in each treatment and control group. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL foetal bovine serum (one tube per animal). Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, stained in May-Grunwald solution followed by Giemsa, and protected by permanently mounted coverslips. For control of bias, all slides were coded prior to analysis.


METHOD OF ANALYSIS:
Slides prepared from the bone marrow collected from the first five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. the micronucleus frquency (expressed as percent micronucleated cells) was determined by analysing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least the first 500 erythrocytes per animal.
Evaluation criteria:
The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test article that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevance of the results in the final evaluation.
Statistics:
Assay data analysis was performed using an analysis of variance on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances. If the analysis of variance was statistically significant (p 0.05), a Dunnett’s t-test was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Signs of clinical signs toxicity and mortality were induced, but the test substance was not cytotoxic to the bone marrow
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250-2000 mg/kg/day.
- Clinical signs of toxicity in test animals: All animals in the 1000 and 2000 mg/kg/day dose groups, and one male in the 500 mg/kg/day group, were dead by the one-hour post-dose observation. A further male and two females in the 500 mg/kg/day dose group died before the third dose had been administered, and the remaining male and female in this group were sacrificed due to the excessive mortality. A female in the 250 mg/kg/day group died immediately after the third dose. Other animals in this group displayed various signs of clinical toxicity including hunched posture, rough haircoat, low activity, laboured breathing, and irregular breathing.
- Evidence of cytotoxicity in tissue analyzed: Not assessed.


RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: On Day 3, one 250 mg/kg/day animal died prior to dosing and one died following dosing. Clinical signs of toxicity seen in animals within the 250 mg/kg/day group included: urine stains, distended abdomen, hypoactive, rough haircoat, squinted eyes, eye sealed shut, hunched posture, loose faces and faecal stains.
- 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): There was no statistically significant decrease in the PCE:NCE ratio).
- Statistical evaluation: A statistical significant increase in micronucleated PCEs was not observed at any dose level.

The reported historical background frequency of micronucleated cells in the Crl:CD-1(ICR) BR strain at the test laboratory was about 0.0-0.4%, which was stated to be within the range reported in the published data.

The temperature range of the animal housing was reported as 64–79ºF, equivalent to 17.8–26.1ºC. Recommended animal housing conditions were not included in the June 1996 draft EPA guideline, but the final 1998 guideline (and OECD TG 474) recommends maintaining temperatures within the range of 19–25ºC.

Conclusions:
Interpretation of results (migrated information): negative
The test substance was negative in the mouse bone marrow micronucleus assay under the conditions of this test.
Executive summary:

The test substance was evaluated for clastogenic potential in a mouse micronucleus test. The GLP study was conducted in accordance with US EPA OPPTS and OECD guidelines. The test substance, in white oil vehicle, was administered by intraperitoneal injection. In the initial dose-range-finding study, doses of 250, 500, 1000 and 2000 mg/kg/day were adminstered to groups of male and female Crl:CD-1(ICR) BR mice causing extensive mortality in the higher dose groups. As there was no indication of sex-dependent effects, doses of 62.5, 125 and 250 mg/kg/day were administered to groups of six males in the main micronucleus study (with two additional males dosed at 250 mg/kg/day in case of mortality in this group). The animals were dosed for three days. 24 hours after the third dose, animals were sacrificed, bone marrow collected and prepared for microscopic analysis of polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs). Cytotoxicity to the bone marrow was not seen (i.e. there was no decrease in the PCE:NCE ratio) in any of the test substance treatment groups, but clinical signs of toxicity and two deaths occurred at 250 mg/kg/day. A statistically significant increase in micronuclated PCEs was not seen at any dose level. The positive control, cyclophosphamide administered by gavage, confirmed the sensitivity of the test. It is concluded that the test substance was not clastogenic under the conditions of this test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Based on the result of the available in vitro mutagenicity studies, this substance is not considered genotoxic. It is not mutagenic in Salmonellatyphymurium strains TA98,TA100, TA1535, TA1537, and TA1538 (method not stated by largely consistent with 1997 OECD TG 471) and mouse L5178Y cells (OECD Guideline 476). It also does not cause clastogenic effects in the in vivo mouse micronucleus assay based on U.S. EPA Health Effects Test Guideline OPPTS 870.5395.

Justification for classification or non-classification

In accordance with EU CLP (Regulation (EC) No.1272/2008), classification as a mutagen is not required.