Reaction mass of 2-(1,1-dimethylethyl)-4-{[5-(1,1-dimethylethyl)-4-hydroxy-2-methylphenyl]thio}-5-methylphenyl 3-(dodecylthio)propionate and thiobis[2-(1,1-dimethylethyl)-5-methyl-4,1-phenylene] bis[3-(dodecylthio)propionate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

It was concluded that AO-26 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

It is concluded that the test substance AO-26 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

The highest concentration of AO-26 tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. The highest concentration in each test was diluted with acetone to produce a series of lower concentrations, separated by approximately half-log10 intervals.

5 μg
15 μg
50 μg
150 μg
500 μg
1500 μg
5000 μg

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone;
- Justification for choice of solvent/vehicle: The Sponsor indicated that AO-26 was poorly soluble in water and in dimethyl sulphoxide but was soluble in acetone. Acetone (analytical reagent grade) was, therefore, used as the vehicle for this study.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

In the absence of S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9 mix

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of S9 mix

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

In the absence of S9 mix

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

In the presence of S9 mix

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

First test
Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 μg/plate), positive control or vehicle control were placed in glass tubes. The vehicle control was acetone. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer). Any toxic effects of the test substance may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both. In the absence of any toxic effects, the maximum concentration selected for use in the second test is the same as that used in the first. If toxic effects are observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at this maximum
concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate is observed on the plates at the end of the incubation period, at least one precipitating concentration should be included in the second test, unless otherwise justified by the Study Director.

Rationale for test conditions:

Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The pre-incubation procedure is not suitable for acetone, which is toxic under such conditions. The variation used was, therefore, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system. If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.

Statistics:

No statistical analysis is performed.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Conclusions:

It was concluded that AO-26 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

In this in vitro assessment of the mutagenic potential of AO-26, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to AO-26 diluted in acetone. Acetone was also used as a vehicle control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. Both tests were standard plate incorporation assays.

Concentrations of AO-26 up to 5000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

No signs of toxicity towards the tester strains were observed in either mutation test following exposure to AO-26.

No evidence of mutagenic activity was seen at any concentration of AO-26 in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

It was concluded that AO-26 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes:

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, the osmolality and pH of AO-26 in medium was tested at 5000 μg/mL. However, as a fluctuation in osmolality of more than
50 mOsm/kg was observed when compared with the vehicle control (with no evident change in pH), the osmolality and pH of AO-26 was performed at the lower concentration of 3000 μg/mL. No fluctuation in osmolality of more than 50 mOsm/kg, and no change in pH of more than 1.0 unit were observed when compared with the vehicle control. In this case, the highest final concentration used for subsequent testing was 3000 μg/mL, as osmolality and pH values were within acceptable parameters. In addition, this maintained precipitate (visible by eye) in culture medium.

Vehicle / solvent:

AO-26 was miscible in acetone at 500 mg/mL. On dosing a 500 mg/mL solution at 1% v/v into aqueous tissue culture medium, giving a final concentration of 5000 μg/mL, precipitate (visible by eye) was observed, when compared to the vehicle control. On serial dilution to the lower concentration of 3000 μg/mL, precipitate was also observed.

Negative solvent / vehicle controls:

yes

Remarks:

Sterile purified water

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 mix

Negative solvent / vehicle controls:

yes

Remarks:

Sterile purified water

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

Preliminary toxicity test
Cultures were treated approximately 48 hours after commencement of incubation of lymphocyte cultures. All cultures were identified using unique number/colour codes. Cultures were prepared for each treatment (3-hour treatment in the absence and presence of S9 mix, and 21-hour continuous treatment in the absence of S9 mix).
Duplicate cultures were used for treatment with the vehicle, and single cultures for treatment with the test substance for each test condition. No positive control cultures were prepared.
All cultures were centrifuged and resuspended in fresh medium before treatment, and S9 homogenate was present in appropriate cultures at a final concentration of 5% v/v.
AO-26 was added to each culture in 50 μL aliquots to give final concentrations of 30.2, 50.4, 84, 140, 233.3, 388.8, 648, 1080, 1800 and 3000 μg/mL. Acetone was used as the vehicle control.
At the end of the 3-hour treatment period, cultures were examined for the presence of precipitate and any observations noted in the study file. Cultures were then washed in saline (5 mL), and resuspended in fresh medium. The cultures were then incubated (for approximately 18 hours) until the scheduled harvest time. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope. At the end of the 21-hour treatment period, cultures were examined for the presence of precipitate and any observations noted in the study file. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope.

Harvesting and fixation
Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 μg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g. The cell pellets were treated with a hypotonic solution (0.075M KCl), pre-warmed at 37°C. After a 10 minute period of incubation at 37°C, the suspensions were centrifuged at 500g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear.

Slide preparation
The pellets were resuspended, then centrifuged at 500g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative were stored at approximately 4°C until slide analysis was completed.

Main test - Treatment of cells with test substance
Vehicle control, treatment, and positive control cultures were treated approximately 48 hours after commencement of incubation of lymphocyte cultures. Duplicate cultures were prepared throughout for each treatment (3-hour treatment in the absence and presence of S9 mix, and 21-hour continuous treatment in the absence of S9 mix). All cultures were centrifuged and resuspended in the required volume of fresh medium before treatment, taking into account the treatment volume and S9 mix volume, where required.

n the absence of S9 mix, 3-hour treatment
AO-26 was added to each culture in 50 μL aliquots to give final concentrations of 648, 1080, 1800 and 3000 μg/mL. Acetone was used as the vehicle control, and Mitomycin C at a final concentration of 0.2 μg/mL was the positive control. After 3-hour treatment, precipitate (visible by eye) was present at 3000 μg/mL only, when compared to the vehicle control. Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline (5 mL) and centrifuged at 500g for 5 minutes. The saline was then removed and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope.

In the presence of S9 mix, 3-hour treatment
For treatments in the presence of S9 mix, 1 mL of S9 mix was added to give a concentration of 5% v/v in the final test medium. AO-26 was added to each culture in 50 μL aliquots to give final concentrations of 648, 1080, 1800 and 3000 μg/mL. Acetone was used as the vehicle control, and Cyclophosphamide at a final concentration of 5 μg/mL was the positive control. After 3-hour treatment, precipitate (visible by eye) was present at 3000 μg/mL only, when compared to the vehicle control. Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline (5 mL) and centrifuged at 500g for 5 minutes. The saline was then removed and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope.

In the absence of S9 mix, 21-hour continuous treatment
AO-26 was added to each culture in 50 μL aliquots to give final concentrations of 648, 1080, 1800 and 3000 μg/mL. Acetone was used as the vehicle control, and Mitomycin C at a final concentration of 0.1 μg/mL was the positive control. After 21-hour treatment, precipitate (visible by eye) was present at 1800 and 3000 μg/mL, when compared to the vehicle control. The cultures were then harvested, slides prepared, and microscopically examined for cytotoxicity using a light microscope.

Rationale for test conditions:

The prepared slides were examined by light microscopy and the incidence of mitotic cells per 1000 cells assessed. Slides were assessed for a depression in mitotic index. In the absence of S9 mix following 3-hour treatment, AO-26 caused no reduction in the mitotic index at any treatment concentration, when compared to the vehicle control value. Precipitate (visible by eye) was observed at the highest tested concentration of 3000 μg/mL only. Therefore, the maximum final concentration selected for testing in the main test was 3000 μg/mL.

In the presence of S9 mix following 3-hour treatment, AO-26 caused no reduction in the mitotic index at any treatment concentration, when compared to the vehicle control value. Precipitate (visible by eye) was observed at the highest tested concentration of 3000 μg/mL only. The maximum final concentration selected for testing in the main test was 3000 μg/mL.
In the absence of S9 mix following 21-hour continuous treatment, AO-26 caused no reduction in the mitotic index at any treatment concentration, when compared to the vehicle control value. Precipitate (visible by eye) was observed at the highest tested
concentrations of 1800 and 3000 μg/mL. The maximum final concentration selected for testing in the main test was 3000 μg/mL.

The concentrations used in the main test were based upon these data.

Evaluation criteria:

An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range. The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.

Key result

Species / strain:

other: Human Lymphocytes

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

It is concluded that the test substance AO-26 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Executive summary:

A study was performed to assess the ability of AO-26 to induce structural chromosome aberrations in human lymphocytes cultured in vitro.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Vehicle and positive control cultures were also included. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

A preliminary toxicity test was performed to determine the toxicity of AO-26 to cultured human lymphocytes. A 3-hour treatment in the absence and presence of S9 mix, and a 21-hour continuous treatment in the absence of S9 mix were used to determine toxicity.

In the main test, the mitotic index was assessed for all cultures treated with AO-26 and the vehicle control, acetone. Justification for the highest analysed concentration was based on precipitate (visible by eye) in the final culture medium at the end of treatment.

On the basis of these data, the following concentrations were selected for metaphase analysis:

In the absence of S9 mix, 3-hour treatment: 1080, 1800 and 3000 μg/mL.

In the presence of S9 mix, 3-hour treatment: 1080, 1800 and 3000 μg/mL.

In the absence of S9 mix, 21-hour continuous treatment: 648, 1080 and 1800 μg/mL.

In the absence and presence of S9 mix following 3-hour treatment, AO-26 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the vehicle control.

In the absence of S9 mix following 21-hour continuous treatment, AO-26 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the vehicle control.

No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared to the vehicle control.

Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It is concluded that AO-26 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

It was concluded that the test item, AO-26, was considered to be non-genotoxic under the conditions of the 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:

The study was conducted between 2 March 2016 and 5 May 2016

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian germ cell cytogenetic assay

Specific details on test material used for the study:

Identification: AO-26
Physical state/Appearance: Dark brown viscous liquid
Batch: 10176
Purity: 98.8%
Expiry Date: 30 July 2017
Storage Conditions: Room temperature, in the dark

Species:

mouse

Strain:

ICR

Details on species / strain selection:

albino Hsd: ICR (CD-1)

Sex:

male

Details on test animals or test system and environmental conditions:

At the start of the main test the mice weighed 23.6 to 31.5g and were approximately six to ten weeks old. The bodyweights of the individual mice were within 20% of the dose group mean. After a minimum acclimatization period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.

The animals were housed in groups of five in solid-floor polypropylene cages with wood-flake bedding. Free access to mains drinking water and food (Envigo Teklad 2014C Global Certified Rodent Diet supplied by Envigo Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. Representative analyses of food and water quality are retained in the laboratory archive.

The temperature and relative humidity were set to achieve limits of 19 to 25ºC and 30 to 70%, respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: unspecified

Vehicle:

Identification: Arachis oil BP
Envigo Serial Number: V-6498
Expiry Date: 12 May 2018
Storage Conditions: Room temperature
Purity: Treated as 100%
Expiry: 12 May 2018

Details on exposure:

Test and Control Item Preparation
For the purpose of this study the test item was freshly prepared as required as a solution at the appropriate concentration in arachis oil.

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

Procedure
Range-finding Toxicity Test
A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the micronucleus test. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. The range-finding toxicity test was also used to determine if the main test was to be performed using both sexes or males only.

Groups of mice were dosed orally as follows:
Level (mg/kg) Concentration (mg/mL) Dose Volume (mL/kg) Number of Mice
Male Female
2000 200 10 4 2

All animals were dosed once only at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
Animals were observed one hour after dosing and subsequently once daily for two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed. In addition, bone marrow smears were prepared at the end of the exposure period to confirm that any bone marrow toxicity was within acceptable levels.

Micronucleus Test
Two groups, each of five mice, were dosed once only via the oral route with the test item at 2000 mg/kg. One group of mice was euthanized by cervical dislocation 24 hours following treatment and the second group was euthanized after 48 hours. In addition, two further groups of five mice were included in the study; one group was dosed via the oral route with the vehicle alone (arachis oil) and the second group was dosed orally with cyclophosphamide.
The vehicle and positive controls were euthanized 24 hours following dosing. The experimental design is summarized as follows:

Dose Group Dose Level (mg/kg) Concentration (mg/mL) Dose Volume (mL/kg) Kill Time (Hours After Dosing) Animal Numbers
Vehicle Control (Arachis oil) 0 0 10 24 1 - 5
Positive Control (Cyclophosphamide) 50 5 10 24 6 - 10
AO-26 2000 200 10 48 11 - 15
AO-26 2000 200 10 24 16 - 20

All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.

Duration of treatment / exposure:

24 or 48 hours

Frequency of treatment:

Once

Post exposure period:

24 or 48 hours

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

4 males, 2 females at 2000 mg/kg for the preliminary test
10 males dosed at 2000 mg/kg for the micronucleus test

Control animals:

yes, concurrent vehicle

other: Positive control (cyclophosphamide)

Positive control(s):

Positive Control Item Preparation
For the purpose of this study the positive control item (cyclophosphamide) was freshly prepared as required as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant 3012436).

Tissues and cell types examined:

Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.

The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Details of tissue and slide preparation:

Slide Preparation
Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald / Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

Evaluation criteria:

Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.
A positive mutagenic response is demonstrated when a statistically significant and toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to the vehicle control group and the number of micronucleated polychromatic erythrocytes in the animals exceed the historical control data range.

If these criteria were not fulfilled, then the test item was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

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:

Range-finding Toxicity Test
The mortality data are summarized as follows:

Dose Level (mg/kg) Sex Number of Animals Treated Route Deaths on Day Total Deaths
0 1 2
2000 Male 4 oral 0 0 0 0/6
Female 2 0 0 0

No evidence of toxicity was observed in animals dosed with test item via the oral route at the maximum recommended dose level of 2000 mg/kg. Therefore, the maximum recommended dose (MRD) of the test item, 2000 mg/kg, was selected for use in the main test using the treatment schedule for non-toxic test items.

Micronucleus Test
Mortality Data and Clinical Observations
There were no premature deaths or clinical signs observed in either of the test item dose groups.

Evaluation of Bone Marrow Slides
There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test item groups when compared to the vehicle control group.
There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group, and the group mean values were within the range of the group mean values of the historical vehicle control data.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all vehicle control animals were within the historical vehicle control data range.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusions:

The test item, AO-26, was considered to be non-genotoxic under the conditions of the test.

Executive summary:

Introduction

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 474 “Mammalian Erythrocyte Micronucleus Test” (adopted 29 July 2016), Method B12 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

Methods

A range-finding test was performed to find suitable dose levels of the test item and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in the toxicity of the test item between the sexes; therefore, the main test was performed using only male mice using the treatment schedule for non-toxic test items. The micronucleus test was conducted using the oral route in groups of five mice (males) at the maximum recommended dose (MRD) of 2000 mg/kg using arachis oil as the vehicle. Animals were euthanized 24 or 48 hours after dosing, the bone marrow was extracted and smear preparations were made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei and PCE/NCE ratio was calculated as an indicator for toxicity.

Two additional groups of five mice were given a single oral dose of arachis oil, or dosed orally with cyclophosphamide, to serve as vehicle and positive controls respectively. Vehicle and positive control animals were euthanized 24 hours after dosing.

Results

There were no premature deaths or clinical signs in either of the test item dose groups. There were also no marked decreases in the PCE/NCE ratio observed in the 24 or 48-hour test item dose groups when compared to the vehicle control group.

There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group, and the group mean values were within the range of the group mean values of the historical vehicle control data.

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

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusion

The test item, AO-26, was considered to be non-genotoxic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

According to the CLP regulation a substance will only be considered a mutagen based on a positive response.

The Ames and study and Chromosone abberation study both provided negative results therefore the test substance AO-26 can be considered as a non mutagenic substance.