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Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
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]
EC Number:
940-594-7
Molecular formula:
C52H86O4S3 , C37H58O3S2
IUPAC Name:
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]
Test material form:
liquid
Details on test material:
Intended use: Antioxidant for plastics
Appearance: Brown liquid
Storage conditions: Room temperature protected from light
Supplier: Sponsor
Lot number: 102Y1
Expiry date: 30 April 2014
Purity 98.8%
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

Test animals

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.

Administration / exposure

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
Doses / concentrations
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).

Examinations

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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.