N-ethyl-N-(3-methylphenyl)propionamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial Mutation test (OECD 471): not mutagenicwith and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 & TA100.

- Chromosome aberration test (OECD 473): not clastogenic in either the absence or presence of metabolic activation.

Based on the available results, especially the lack of mutagenic effect in the in vivo erythrocyte micronucleus test, the missing result on E.coli in the AMES test have no impact on the overall consensus that the substance is not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 September to 29 October

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human lymphocytes from whole blood culture

Details on mammalian cell type (if applicable):

Human blood samples were obtained by venepuncture in lithium heparin tubes on the days of culture initiation from healthy, non-smoking male donors

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The metabolic activation system (S9-mix) used in this study was prepared as required (on each day of culture treatment) as a 1:1 mixture of S9 fraction and cofactor solution.
S9 was prepared from male Sprague Dawley rats, dosed once daily (by oral gavage) for 3 days with a combined phenobarbital (80mg/kg bodyweight) and beta-naphthoflavone (100mg/kg bodyweight) corn oil preparation. The treated animals were sacrificed on the day following the third dose. A 25% w/v homogenate (the S9 fraction) was prepared according to the method given in Callander et al (1995).
The cofactor solution was prepared as a single stock solution of Na2HPO., KCl, glucose-6- phosphate, NADP (Na salt) and MgCl2 (150: 49.5 : 7.5 : 6: 12mM) in sterile double deionised water and adjusted to a final pH of 7.4

Test concentrations with justification for top dose:

Donor 1: 500 µg/ml, 250 µg/ml, 50 µg/ml (with S9 mix), 68 hours harvest
Donor 1: 250 µg/ml, 150 µg/ml, 25 µg/ml (without S9 mix), 68 hours harvest
Donor 2: 500 µg/ml, 250 µg/ml, 50 µg/ml (with S9 mix), 68 hours harvest
Donor 2: 250 µg/ml, 150 µg/ml, 25 µg/ml (without S9 mixture), 68 hours harvest
Donor 2: 500 µg/ml (with S9 mix) 92 hours harvest
Donor 2: 100 µg/ml (without S9 mix) 92 hours harvest

Vehicle / solvent:

dimethylsulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-

Details on test system and experimental conditions:

EXPERIMENTAL DESIGN
Duplicate human peripheral blood cultures were exposed to the solvent, test substance or positive control substances at appropriate concentrations in the following experiments:
a) A cytogenetic test using blood from Donor I in the presence and absence of S9-mix with a standard sampling time of 68 hours after culture initiation. Solvent and positive control cultures were included.
b) A second independent cytogenetic test using blood from Donor 2 in the presence and absence of S9-mix with a standard sampling time of 68 hours after culture initiation and a later sampling time of92 hours after culture initiation. Solvent control cultures were included at both sampling times whereas the positive control cultures were only included at the 68 hour sampling time

In both experiments a range of concentrations of the test item was used in order to define suitable concentrations for chromosomal aberration analysis.
The standard sampling time of 68 hours after culture initiation used in this study was based on a measured mean cell cycle time for cultured human peripheral blood lymphocytes of 13.5 hours in this Laboratory (September 1992 and November 1993). The later sampling time was selected to be 24 hours after the standard sampling time.

CULTURE HARVESTING
Approximately 2 hours prior to harvesting, the cultures were treated with colcemid at a final concentration of 0.4 µg/ml. Sixty-eight hours or 92 hours after culture establishment the cultures were centrifuged, the supernatant was removed and the cells were re-suspended in approximately 10ml of 0.075M KC! at room temperature for approximately 10 minutes. The cultures were centrifuged, the supernatant was removed and the remaining cells were fixed in freshly prepared methanol/glacial acetic acid fixative (3:1 v/v) added dropwise and made up to a volume of approximately 10ml. The fixative was removed following centrifugation and replaced with freshly prepared fixative. This fixation process was repeated at least twice prior to slide preparation on clean, moist labelled microscope slides. The slides were air dried, stained in filtered Giemsa stain (10% Gurr's R66 in buffered [pH 6.8] double deionised water) for 7 minutes, rinsed in water, air-dried and mounted with coverslips in DPX

SLIDE ANALYSIS
Where appropriate, the mitotic index was determined by examining 1000 lymphocytes per culture and calculating the percentage of cells in metaphase. The number of polyploid metaphases, in 1000 cells, was determined during scoring of the mitotic index.
For each donor, both in the presence and absence of S9-mix, duplicate cultures treated with Agarbois at three concentrations were selected for chromosomal aberration analysis at the 68 hour sampling time along with the appropriate solvent and positive control cultures. In each case the highest concentration was selected on the basis of a significant reduction in mean mitotic activity (approximately 50%) and the suitability of the metaphase preparations for chromosomal aberration analysis. In addition, duplicate cultures from the Donor 2 treated with Agarbois at the highest concentration suitable for chromosomal aberration analysis (but not exceeding that selected at the 68 hour sampling time) in the presence and absence of S9- mix were selected for chromosomal aberration analysis at the 92 hour sampling time along with the appropriate solvent control cultures.

The slides were coded prior to analysis and one hundred cells in metaphase, where possible, were analysed blind using light microscopy from each selected culture for the incidence of structural chromosomal damage, according to the principles of the criteria recommended by Scott et al (1990).

Rationale for test conditions:

The human peripheral blood lymphocyte was used as it is a sensitive target cell for the induction of in vitro chromosomal damage when stimulated to provide large numbers of rapidly dividing cells in culture (Scott et al, 1990).

Evaluation criteria:

a) No statistically significant increase in the percentage of aberrant cells (at any concentration) above concurrent solvent control values - NEGATIVE.
b) A statistically significant increase in the percentage of aberrant cells above concurrent solvent control values, which falls within the laboratory solvent control range - NEGATIVE.
c) An increase in the percentage of aberrant cells, at least at one concentration, which is substantially greater than the laboratory historical solvent control values - POSITIVE.
d) A statistically significant increase in the percentage of aberrant cells which is above concurrent solvent values and which is above the historical solvent control range upper value but below that described in (c) may require further evaluation.

Key result

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Small increases in the percentage of aberrant cells (up to 6%) were observed in cultures treated with the test item in the absence of S9-mix. Although these increases were small, they were statistically significant and were outside the laboratory historical control range of 5.5% aberrant cells. It is concluded that the test item has shown weak evidence of clastogenic activity in this test system.

Conclusions:

Under the conditions of this assay, small increases in the percentage of aberrant cells (up to 6%) were observed in cultures treated with the test item in the absence of S9-mix at only the top concentration tested. Although these increases were small, they were statistically significant and were outside the laboratory historical control range. Therefore, under the conditions of this study, the test item showed evidence of weak mutagenic potential

Executive summary:

The test item was evaluated for its clastogenic potential in anin vitrocytogenetic assay using human lymphocytes from two male donors treated in the presence and absence of a rat liver­ derived metabolicactivationsystem(S9 -mix).Cultures from both donors were harvested at the standard time of 68 hours after culture initiation and additional cultures from Donor 2 were harvested at the later time of 92 hours after culture initiation. Donors I and 2 were assayedonseparateoccasions.

Cultures treated with the test item at the following concentrations were selected for chromosomal aberration analysis along with the appropriate solvent and positive control cultures.

Donor 1 68 Hours		Donor268 Hours			Donor 2 92 Hours
+S9-mix	-S9-mix	+S9-mix	-S9-mix		+S9-mix	-S9-mix
500µg/ml	250µg/ml	500µg/ml	250µg/ml		500µg/ml	100 µg/ml
250µg/ml	150µg/ml	250µg/ml	150µg/ml		-	-
50µg/ml	25µg/ml	50µg/ml	25µg/ml		-	-

Concentration related reductions in mitotic activity were observed in cultures from both donors, thus demonstrating that test item is biologically active in this test system.

 

No statistically or biologically significant increases in the percentage of aberrant cells, compared to the solvent control values, were observed at the 68 hour sampling time in either Donor I or Donor 2 cultures treated with test item in the presence of S9-mix, or at the 92 hour sampling time in Donor 2 cultures treated with test item in the presence of S9-mix.

Small, but statistically significant increases in the percentage of aberrant cells, compared to the solvent control values, were observed at the 68 hour sampling time in both Donor I and Donor 2 cultures treated with Agarbois in the absence of S9-rnix. A small increase was also observed at the 92 hour sampling time in Donor 2 cultures treated with Agarbois in the absence of   S9-mix.

The sensitivity of the test system, and the metabolic activity of the S9 -mix employed, were clearly demonstrated by the increases in the percentage of aberrant cells induced by the positive control agents, mitomycin C and cyclophosphamide.

Under the conditions of this assay, small increases in the percentage of aberrant cells (up to 6%) were observed in cultures treated with the test item in the absence of S9-mix at only the top concentration tested. Although these increases were small, they were statistically significant and were outside the laboratory historical control range. Therefore, under the conditions of this study, test item showed evidence of weak mutagenic potential.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 June to 30 July 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

PREPARATION OF S-9 FRACTION
Species Rat
Sex Male
Strain Sprague-Dawley derived Harlan Olac Ltd
Source Harlan Olac Ltd
Age 7-8 weeks
Weight <300g
Diet Biosure Rodent Diet LAD 1

Mixed function oxidase systems in the livers of a group of rats were stimulated by Aroclor 1254, administered as a single intra-peritoneal injection in Arachis oil at a dosage of 500 mg/kg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed, and their livers aseptically removed.

The following steps were carried out at 0-4°C under aseptic conditions. The livers were placed in 0.15 M KCl (3 ml KCl : 1 g liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation, the homogenates were centrifuged at 9000 g for 10 minutes. The supernatant fraction (S-9 fraction) was dispensed into aliquots and stored at -80°C until required. The efficacy of each batch of S-9 fraction was tested with the carcinogens 7, 12-dimethylbenzanthracene and 2-aminoanthracene before use.


PREPARATION OF S-9 MIX

S-9 mix contained: S-9 fraction (10% or 30% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter­ sterilised before use.

Test concentrations with justification for top dose:

5 000, 1500, 500, 150, 50 and 15 µg/plate
The test item was toxic towards all the tester strains at the highest concentration. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests, followed by a further five concentrations to ensure that a sufficient number of non toxic dose levels would be obtained.

Vehicle / solvent:

dimethyl sulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S-9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S-9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 37 °C for 72 h

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test substance can be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance is assessedby the following criteria:
(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis performed.

(b)If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis performed.

(c)If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.

(i)Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.

(ii)If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al. (1989) and will usually be analysis of variance followed by Dunnett's test.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2

Remarks:

Missing result on E.coli in the AMES test have no impact on the overall consensus that the substance is not mutagenic.

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with Agarbois at any dose level, in the presence or absence of S-9 mix, in either mutation test.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations

Conclusions:

It is concluded that, when tested in dimethyl sulphoxide, the test item shows no evidence of mutagenic activity in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of the test item, histidine dependent auxotrophic mutants of Salmonella typhimurium(strains TA 1535, TA 1537, TA 98 and TA 100) were exposed to the test substance, diluted in dimethyl sulphoxide which was also used as a  negative control.

 

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. Only the first test included a 60 minute preincubation step at 37°C. The second test was a standard plate incorporation assay.

 

In the preliminary toxicity test, which included a preincubation step, with dose levels of up to 5000 µg/plate toxicity was observed towards all the tester trains at the highest concentration. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150, 50 and 15µg/plate.

 

No evidence of mutagenic activity was seen at any dose level of test item in either mutation test.

 

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

 

It is concluded that, when tested in dimethyl sulphoxide, the test item shows no evidence of mutagenic activity in this bacterial system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- Mammalian erythrocyte micronucleus test (OECD 474 - in vivo): not clastogenic in the mouse bone marrow micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Male and female CD-1 mice from Charles River Breeding Laboratories, Margate, UK

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Margate, UK
- Age at study initiation:
Phase I: 4-12 weeks
Phase II: 6-13 weeks
Phase III: 6-7 weeks
- Fasting period before study: No
- Housing: mobile mouse cage racks
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): l 9-25°C
- Humidity (%): 40-70%.
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light followed by 12 hours darkness

Route of administration:

oral: unspecified

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An individual stock solution of the test substance was prepared in com oil for each group of animals.
The positive control substance was prepared as a solution in sterile double deionised water (CTL test substance reference number Y045 l 7/0 IO).
All test and positive control substance dosing preparations were prepared as close to the time of dosing as possible. The test substance, vehicle and positive control substance were dosed at a volume of 10 ml/kg bodyweight.

Duration of treatment / exposure:

Bone marrow was collected 24 and 48 hours after dose administration.

Frequency of treatment:

Single oral dose

Post exposure period:

24, 48 h

Dose / conc.:

125 mg/kg bw (total dose)

Dose / conc.:

250 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose:

2 (two males and two females)
Phase II: 4 animals at 500 mg/kg (four males and four females)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide at 65 mg/kg

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

SLIDE PREPARATION:
The animals were killed by asphyxiation in halothane Ph. Eur. (FLUOTHANE, Zeneca Pharmaceuticals) followed by cervical dislocation 24 and 48 hours after dosing. All animals treated with Agarbois were examined internally for signs of colouration. or abnormalities to organs/tissues.
Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush was rinsed in saline, wiped to remove the excess and wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and two smears were painted on an appropriately labelled clean, dry microscope slide. This procedure was repeated to give four smears of marrow per slide.
The slides were allowed to air dry and were stained with polychrome methylene blue and eosin using an automatic staining machine.

SLIDE ANALYSIS:
Slides were coded and scored blind. Two thousand (2 x 1000) polychromatic erythrocytes were examined for the presence of micronuclei for each animal. The slides were also examined for evidence of cytotoxicity, which may be manifest by alterations in the ratio of different cell types in the bone marrow. This was assessed by counting the ratio of polychromatic to normochromatic erythrocytes in a sample of 1000 erythrocytes.

Evaluation criteria:

a) No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above concurrent vehicle control incidences - NEGATIVE.
b) A statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above the concurrent vehicle control incidences but which falls within the laboratory historical vehicle control range - NEGATIVE.
c) A statistically and biological significant increase in the incidence of micronucleated polychromatic erythrocytes which is in excess of a three-fold increase when compared with both historical and concurrent vehicle control incidences - POSITIVE.
d) An incidence of micronucleated polychromatic erythrocytes which is statistically significantly different from the concurrent vehicle control incidences, but less than 3-fold in excess of both historical and concurrent vehicle control incidences may require further evaluation.

Statistics:

Analyses were carried out using the GLM procedure in SAS (1989). Each treatment group mean was compared with the control group mean at the corresponding sampling time using a one-sided Student's t-test, based on the error mean square in the analysis.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No statistically significant increases in the incidence of micronucleated polychromatic erythrocytes were observed in the repeat experiment. When the data from the original test and the repeat test were combined, again no statistically significant increases in micronucleated polychromatic erythrocytes were observed.

No statistically significant differences in the percentage of polychromatic erythrocytes, between the vehicle control and test item treated animals, were observed in either males or females at any dose level or either sampling time investigated.

The test system positive control, cyclophosphamide, induced statistically and biologically significant increases in the frequency of micronucleated polychromatic erythrocytes in both male and female mice at the 24 hour sampling time.

MEAN INCIDENCE OF MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES/1000 POLYCHROMATIC ERYTHROCYTES ± STANDARD DEVIATION (SD) AT ONE SAMPLING TIME

GROUP MEAN ANIMAL DATA - MALES (PHASEIll)

Group	Treatment	Dose	Mean incidence of MPE/1000 PE± SD
			24 hours
16	Vehicle Control	10 ml/kg	0.8 ± 0.8 .
17	Cyclophosphamide	65mg/kg	22.4 ± 1.5**
18	Agarbois	125mg/kg	0.3 ± 0.5
19	Agarbois	250mg/kg	0.4 ± 0.4
20	Agarbois	500mg/kg	0.6 ± 0.7

 

PE = polychromatic erythrocytes

MPE    = micronucleated polychromatic erythrocytes.

SD      = Standard deviation

**       Statistically significant increase in micronucleated polychromatic erythrocytes at p<0.01 in the Student's t-test (one-sided) on transformed data.

 

All means based on 5 counts of 2000 PE per animal.

MEAN INCIDENCE OF MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES/1000 POLYCHROMATIC ERYTHROCYTES±

STANDARD DEVIATION (SD) AT ONE SAMPLING TIME

GROUP MEAN ANIMAL DATA- FEMALES (PHASEIll)

Group	Treatment	Dose	Mean incidence of MPE/1000 PE±SD
			24	hours
16	Vehicle Control	lOml/kg	0.4±0.4
17	Cyclophosphamide	65mg/kg	25.1±7.2**
18	Agarbois	125mg/kg	0.5±0.6
19	Agarbois	250mg/kg	0.1±0.2
20	Agarbois	500mg/kg	0.2±0.3

PE = polychromatic erythrocytes.

MPE    = micronucleated polychromatic erythrocytes.

SD = standard deviation.

**       Statistically significant increase in micronucleated polychromatic erythrocytesatp<0.01 in the Student's t-test (one-sided) on transformed data.

 

All means based 5 counts of 2000 PE per animal.

Conclusions:

Under the conditions of test, the test item is not clastogenic in the mouse bone marrow micronucleus test.

Executive summary:

Study design

Agarbois has been evaluated for its ability to induce micronucleated polychromatic erythrocytes in the bone marrow of CD-1 mice. A single oral dose was given to groups of male and female mice at dose levels of 125, 250 and 500 mg/kg.The highest dose level used represents the maximum tolerated dose (MTD) based on patterns of clinical signs and lethalities over a four day observation. Bone marrow samples were taken 24 hours after dosing for the vehicle control, positive control and each of the Agarbois dose levels. Bone marrow samples were taken at the 48 hour sampling time for the vehicle control and the highest dose level of the test item.

A repeat experiment was conducted in which groups of male and female mice were given a single oral dose of test item at doselevels of 125,250 and 500mg/kg.Bone marrow samples were taken 24 hours after dosing.

Results

In the first experiment, small statistically significant increases in the incidence of micronucleated polychromatic erythrocytes were observed in males treated with test item at dose levels of 250 and 500mg/kg at the 24 hour sampling time.

A small statistically significant increase was also observed in females treated with Agarbois at the lowest dose level of  l 25mg/kg.

 

Further testing was conducted with both males and females at dose levels of 125, 250 and 500mg/kg. Bone marrow smears were taken 24 hours after dosing.

In the repeat experiment, no statistically significant increases in the incidence of micronucleated polychromatic erythrocytes were observed in either males or females at any of the dose levels tested. When the data from the original test and the repeat test were combined, again no statistically significant increases in micronucleated polychromatic erythrocytes were observed.

Comparison of the percentage of polychromatic erythrocytes showed no statistically or biologically significant differences at any of the dose levels at either of tlie sampling times between the vehicle control animals and those treated with the test item.

The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes,compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Conclusion:

Under the conditions of test, Agarbois is not clastogenic in the mouse bone marrow micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)