Registration Dossier

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:
20 July - 3 Oct 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(adopted in 1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
No. 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
(August 1998)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: Japanese METI/MHLW/MAFF
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom, London, UK
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Triethylene Glycol Diacetate [2,2'-(ethane-1,2-diylbis(oxy))bisethyl Diacetate]
- Physical state/Appearance: clear, colourless liquid
- Analytical purity: 99.2%
- Lot/batch No.:
- Expiry date: 24 Feb 2015
- Storage conditions: room temperature, in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Details on strain: albino Hsd:ICR (CD-1)
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 6-10 weeks
- Weight at study initiation: 23-30 g
- Assigned to test groups randomly: yes, under following basis: no data
- Housing: groups of up to 7 in solid-floor polypropylene cages with wood-flake bedding
- Diet: Harlan Teklad 2014C Global Certified Rodent Diet (Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Phosphate buffered saline (PBS)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
Dosing solutions were prepared freshly by dissolving appropriate amounts of the test material in phosphate buffered saline (PBS) yielding a final concentration of 40, 80, 100, 150, 160, or 200 mg/mL in the pre-test and in the main test.

TEST MATERIAL
- Amounts applied: 1600, 800, and 400 mg/kg bw/day (Pretest: 1000, 1500, 1600, and 2000 mg/kg bw)
- Constant volume or concentration used: yes, 10 mL/kg bw
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
- Test groups: 24 or 48 h
- Control groups: 24 h
Doses / concentrations
Remarks:
Doses / Concentrations:
1600, 800, and 400 mg/kg bw
Basis:
other: intraperitoneal dose
No. of animals per sex per dose:
7 males per time point
(Pre-test: 2-4 animals per dose)
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: orally
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range-finding test was performed to find suitable dose levels of the test item, route of administration, and to investigate if there was a marked difference in toxic response between the sexes. 1600 mg/kg bw (i.p.) was selected as maximum tolerable dose (MTD) and thus, as the highest dose in the main test. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
7 males per group were dosed once via the intraperitoneal route with the test item at 1600, 800, or 400 mg/kg bw. One group of mice from each dose level was killed by cervical dislocation 24 h following treatment and a second group (1600 mg/kg bw) was killed after 48 h. Two additional groups of male mice were treated with PBS (vehicle) alone (7 mice, intraperitoneal) and cyclophosphamide (5 mice, orally), respectively.

DETAILS OF SLIDE PREPARATION:
Immediately following termination (24 or 48 h following dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears were 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.

METHOD OF ANALYSIS:
Stained bone marrow smears were examined blind using light microscopy at x 1000 magnification. Where possible, the incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE) per animal was scored. In addition, the number of normochromatic erythrocytes (NCE) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei. The ratio of PCE to NCE was calculated together with appropriate group mean values and standard deviations.
Evaluation criteria:
The number of micronucleated polychromatic erythrocytes occurring in each of the test item groups were compared to the number occurring in the vehicle control group. A positive mutagenic response would be demonstrated when a statistically significant dose-dependent, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48 hour killing time compared to the vehicle control.
If the ratio of polychromatic to normochromatic erythrocytes is found to be significantly lower than the control value, this is taken as being indicative of cytotoxicity.
Statistics:
Student's t-test (two-tailed), one way analysis of variance

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs were seen in the 1600 mg/kg bw groups
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 to 2000 mg/kg bw
- Clinical signs of toxicity in test animals: 2000 mg/kg bw, oral: hunched posture, ptosis, and some weight loss; 2000 mg/kg bw, i.p.: a premature death in one animal and hunched posture and ptosis in the other; 1500 and 1600 mg/kg bw, i.p.: hunched posture and ptosis; 1000 mg/kg bw: no clinical signs

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control.
- Ratio of PCE/NCE: No statistically significant decreases in the PCE/NCE ratio were noted in any of the test item dose groups when compared to the vehicle control group. The observation of clinical signs indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.
- Appropriateness of dose levels and route: dose levels and route were based on the result of a range-finding study
- Clinical signs of toxicity in test animals: at 1600 mg/kg bw, in both the 24 and 48 h dose groups, hunched posture and ptosis

Any other information on results incl. tables

Table 1: Micronucleus test – Summary of group mean data

Treatment group

Sampling time

No. PCE with micronuclei per 2000 PCE

PCE/NCE ratio

(hours after treatment)

Group mean

SD

Group mean

SD

Vehicle Control (PBS) 10 mL/kg bw

24

0.6

0.5

0.74

0.11

Positive control (Cyclophosphamide) 50 mg/kg bw

24

21.2***

9.7

0.97

0.23

Test item 1600 mg/kg bw

48

0.6

0.8

0.77

0.14

Test item 1600 mg/kg bw

24

1.4

1.3

0.70

0.13

Test item 800 mg/kg bw

24

0.7

1.1

0.88

0.35

Test item 400 mg/kg bw

24

0.3

0.5

1.06

0.43

*** = P < 0.001

SD = standard deviation

PCE = polychromatic erythrocytes

NCE = normochromatic erythrocytes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative