Dodecanedioic acid, 1,12-bis[2-[4-(4,6-diphenyl-1,3,5-triazin-2-yl)-3-hydroxyphenoxy]ethyl] ester

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:

experimental study

Adequacy of study:

key study

Study period:

15 May to 13 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in a GLP compliant laboratory according to EU regulatory Guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Official notice of MHLW, METI and MOE (31 March 2011) YAKUSHOKUHATSU 0331 No 7 SEIKYOKU No 5 KANPOKIHATSU No 110331009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

1,12-bis({2-[4-(4,6-diphenyl-1,3,5-triazin-2-yl)-3-hydroxyphenoxy]ethyl}) dodecanedioate

EC Number:

939-042-8

Cas Number:

1482217-03-7

Molecular formula:

C58H56N6O8

IUPAC Name:

1,12-bis({2-[4-(4,6-diphenyl-1,3,5-triazin-2-yl)-3-hydroxyphenoxy]ethyl}) dodecanedioate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): T-1620L
- Physical state: pale yellow powder
- Analytical purity: 99.9%
- Lot/batch No.: OF1211
- Expiration date of the lot/batch: 30 April 2014
- Storage condition of test material: RT in the dark
- Other:

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Reputable commercial supplier
- Age at study initiation: Preliminary test 40-47 days, males and females (Day 1); Micronucleus test 42 49 days males
- Weight at study initiation: Preliminary test 27.8g males and 23.6 to 23.8g females; Micronucleus test 28.6 to 33.4g males
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): free access to pelleted expanded rat and mouse No.1 maintenance diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12h light/ 12h dark

IN-LIFE DATES: From: To: 15 May to 13 June 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% methylcellulose
- Justification for choice of solvent/vehicle: at Sponsor request
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Type and concentration of dispersant aid (if powder): N/a
- Lot/batch no. (if required): N/a
- Purity:N/a

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Suspensions of the test substance were prepared in 1% methylcellulose in purified water.
Mitomycin C was used as the positive control compound. A solution was prepared using purified water at a concentration of
0.6 mg/mL just prior to administration.
All animals in the vehicle control and test substance dose groups were dosed orally by gavage using a dose volume of 10 mL/kg. The positive control group were dosed at 20 mL/kg via oral gavage.

Duration of treatment / exposure:

Animals were treated with T-1620L orally by gavage on two occasions approximately 24 hours apart.

Frequency of treatment:

Animals were treated with T-1620L orally by gavage on two occasions approximately 24 hours apart.

Post exposure period:

Following dosing, the animals were examined regularly during the working day for a period of 48 hours after the first dose and any mortalities or clinical signs of reaction during the experiment were recorded.

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

6 males per dose group for the main study - two male and two females used for preliminary toxicity test

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Justification for choice of positive control(s): standard reccommended choice for this test
- Route of administration: Positive control dosed once only approximately 24 hours prior to termination at a dose volume of 20 mL/kg.
- Doses / concentrations: 0.6 mg/mL

Examinations

Tissues and cell types examined:

The bone marrow from both femurs from each animal was examined and the polychromatic erythrocytes were examined for the presence of micronuclei

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Dosing as detailed previously. Animals from the vehicle control and test substance groups were sacrificed 24 hours after administration of the second dose. In addition animals in the positive control group were sacrificed 24 hours after a single dose.

DETAILS OF SLIDE PREPARATION:

1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2 Rinsed in purified water
3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4 Washed in purified water for 5 minutes
5 Rinsed in cold tap water for 2 minutes
6 Stored at room temperature until required
7 Immediately prior to scoring, slides are wet mounted with coverslips using purified water

METHOD OF ANALYSIS: Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei

OTHER:

Evaluation criteria:

The following criteria were applied for assessment of assay acceptability:
1. Each treated and control group should include at least 5 analysable animals.
2. Vehicle control values for micronucleated polychromatic erythrocytes must be consistent
with the laboratory historical vehicle control data.
3. Positive controls must show clear unequivocal positive responses.

Statistics:

For the proportion of polychromatic erythrocytes at 24 hours, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3, then on Groups 1 and 2. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
For incidences of micronucleated polychromatic erythrocytes at 24 hours, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an increase from control. If significant, then the analysis was carried out on Groups 1 to
3. Also, exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, were carried out on Group 1 (control) versus Groups 2, 3, 4 and 5.
Statistical significance was declared at the 5% level for all tests.
The data were received in an Excel document and analysed using SAS 9.1.3 (SAS Institute Inc., 2002) (Jonckheere's and Wilcoxon tests) and StatXact 3 (Cytel 1995) (Linear-by-Linear and Permutation tests).

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Micronucleated polychromatic erythrocyte counts (MPCE)

T-1620L did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes in male CD1 mice.

Mitomycin C caused a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (p<0.01) in male CD1 mice.

Micronucleated normochromatic erythrocytes (MNCE)

T-1620L did not cause any significant increases in the incidence of micronucleated normochromatic erythrocytes in male CD1 mice.

Proportion of polychromatic erythrocytes (%PCE)

T-1620L did not cause any statistically significant decreases in the proportion of polychromatic erythrocytes in male CD1 mice. Mitomycin C did not cause a statistically significant decrease in the proportion of polychromatic erythrocytes in male CD1 mice.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that T-1620L did not show any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male CD1 mice when administered orally by gavage in this in vivo test procedure.