reaction mass of isomers of: C7-9-alkyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

The experimental phases of the study were performed between 10 February 2000 and 14 March 2000.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of International Trade and Industry guidelines for testing of new chemical substances.

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): OS 144267
- Molecular formula (if other than submission substance): C21H34O3
- Molecular weight (if other than submission substance): 334.491
- Smiles notation (if other than submission substance): Oc1c(C(C)(C)C)cc(cc1C(C)(C)C)CCC(=O)OCCCC
- InChl (if other than submission substance): 1S/C21H34O3/c1-8-9-12-24-18(22)11-10-15-13-16(20(2,3)4)19(23)17(14-15)21(5,6)7/h13-14,23H,8-12H2,1-7H3

- Structural formula attached as image file (if other than submission substance): see Fig.1
- Substance type: organic
- Physical state: yellow crystalline solid block
- Storage condition of test material: room temperature in the dark
- Other: homogeneity and stability of the test material preparations were not determined by analysis (not required by the test method).

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent.
- Age at study initiation: five to eight weeks old.
- Weight at study initiation: 26 to 30g
- Assigned to test groups randomly: [yes ]
- Fasting period before study:
- Housing: in groups of up to seven in solid-floor polypropylene cages with woodflakes bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: [arachis oil]
- Justification for choice of solvent/vehicle: test material is well soluble in oil.
- Concentration of test material in vehicle: 100, 150 and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): T36 (Supplier's batch number), VEH/1868 (Safepharm serial number)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test material was freshly prepared as required as a solution at the appropriate concentration in arachis oil.

DIET PREPARATION
- Rate of preparation of diet (frequency): mice were dosed once only
- Mixing appropriate amounts with (Type of food): not applicable, all animals were dosed by gavage using a metal cannula.

Duration of treatment / exposure:

single dosage.

Frequency of treatment:

single dosage

Post exposure period:

Range-finding study: Animals were observed one hour after dosing and subsequently once daily for two days.
Mutagenicity study: 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 sacrifice

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
375 mg/kg bw
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
750 mg/kg bw
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1500 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Range finding study: 2 (m/f)
Main study: 7 (only males) in test material dose groups and in vehicle control groups; 5 (only males) in the positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): cyclophosphamide is known to produce micronuclei under the conditions of the test.
- Route of administration: oral/gavage
- Doses / concentrations: 50 mg/kg bw, 5 mg/mL
For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water.

Examinations

Tissues and cell types examined:

Bone marrow; erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A range-finding toxicity study was performed to determine a suitable dose level and route of administration for the micronucleus study. 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 study was also used to determine if the main study was to be performed using both sexes or males only. Using existing toxicology data it was considered to be unnecessary to investigate the intraperitoneal route of administration.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Groups, each of seven mice, were dosed once only via the oral route with the test material at 1500, 750 or 375 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed at 1500 mg/kg at 48 hours. In addition, three further groups of mice were included in the study; two groups (seven mice) were dosed via the oral route with the vehicle alone (arachis oil) and a third group (five mice) was dosed orally with cyclophosphamide, a positive control material known to produce micronuclei under the conditions of the test. The vehicle controls were killed 24 or 48 hours following dosing and positive control group animals were killed 24 hours following dosing.

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice (ie. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum
and bone marrow smears prepared following centrifugation and resuspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, allowed to airdry and coverslipped.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind using light microscopy at xl000 magnification. The incidence of micronucleated cells
per 2000 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 were 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.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group. A positive mutagenic response was demonstrated when a statistically significant dose-responsive increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group. If these criteria were not demonstrated, then the test material 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 concurrent 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 I l l (1989).The data was analysed following a square root of (x+1) 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

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: One death and severe signs of toxicity were obtained at 1500 mg/kg.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1500 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: In animals dosed with the test material via the oral route a premature death occurred at 2000 mg/kg, and clinical signs were observed at and above 1000 mgkg as follows: hunched posture, lethargy, ataxia, decreased respiratory rate, laboured respiration, splayed gait, hypothermia, increased lacrimation and ptosis.
- Evidence of cytotoxicity in tissue analyzed:
- Rationale for exposure: 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.
- Harvest times: 2 days after dosage
- Other:
The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main study. Adequate evidence of test material toxicity was demonstrated via the oral route of administration, therefore, this was selected for use in the main study. The maximum tolerated dose (MTD) of the test material, 1500 mg/kg, was selected for use in the main study, with 750 and 375 mg/kg as the lower dose levels.

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): no structural aberrations were found
- Induction of micronuclei (for Micronucleus assay): There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.
- Ratio of PCE/NCE (for Micronucleus assay): There were no statistically significant decreases in the PCVNCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups. However, the presence of a premature death and clinical observations indicated that systemic absorption had occurred.
- Appropriateness of dose levels and route: There was a premature death seen in the 48-hour 1500 mg/kg test material dose group. Clinical signs were observed in animals dosed with the test material at 1500 mg/kg in both the 24 and 48-hour groups, these included as follows: hunched posture, lethargy, prostration, decreased respiratory rate, laboured respiration, ptosis, increased lacrimation, dehydration and hypothermia. It was considered that the loss of an animal due to premature death did not affect the integrity of the study, because at least five analysable animals were available per group as recommended in the OECD test guidelines.
- Statistical evaluation: The test material, OS 144267, was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Any other information on results incl. tables

MICRONUCLEUS STUDY - SUMMARY OF GROUP MEAN DATA
TREATMENT GROUP	NUMBER OF PCE WITH MICRONUCLEI PER 2000 PCE		PCE/NCE RATIO
	GROUP MEAN	SD	GROUP MEAN	SD
1. VEHICLE CONTROL 48-hour sampling time	1.6	1.3	1.32	0.59
2. VEHICLE CONTROL 24-hour sampling time	1.7	2.6	1.02	0.24
3. POSITIVE CONTROL 24-hour sampling time	44.6***	12.5	1.30	0.40
4. OS 144267 1500 mg/kg 48-hour sampling time	2.3	1.4	1.28	0.45
5. OS 144267 1500 mg/kg 24-hour sampling time	1.4	1.5	1.43	0.38
6. OS 144267 750 mg/kg 24-hour sampling time	1.9	1.8	1.30	0.96
7. OS 144267 375 mg/kg 24-hour sampling time	1.9	2.0	1.36	0.60
Key:
PCE = polychromatic erythrocytes
NCE = normochromatic erythrocytes
SD = standard deviation
*** = p < 0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The study was conducted according to the OECD Guideline 474 without deviations and therefore considered to be of the highest quality (reliability Klimisch 1). The vehicle and the positive control substances fulfilled validity criteria of the test system. The test material did not produce statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups. Hence, test material was considered to be non-genotoxic under the conditions of the test.

Executive summary:

A study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method used has been designed to comply with the OECD Guideline 474, EU Method B12 and the USA EPA, TSCA and FIFRA guidelines.

A range-finding study was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes, therefore the main study was performed using only male mice. The micronucleus study was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) 1500 mg/kg with 750 and 375 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single oral dose of arachis oil (7 mice) or dosed orally with cyclophosphamide(5mice), to serve as vehicle and positive controls respectively.

No statistically significant decreases in the PCE/NCE (polychromatic erythrocytes/normochromatic erythrocytes) ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. However, the presence of a premature death and clinical signs indicated that systemic absorption had occurred.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes. The test material, OS 144267, was considered to be non-genotoxic under the conditions of the test.