Registration Dossier

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:
28 April 2009 to 16 July 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: USA EPA, TSCA and FIFRA guidelines
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese METI/MHLW guidelines for testing of new chemical substances
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
3-sec-[C15-18-(branched and linear)-alk-2-enyl]pyrrolidine-2,5-dione
EC Number:
701-350-3
Molecular formula:
Not possible to assign, UVCB
IUPAC Name:
3-sec-[C15-18-(branched and linear)-alk-2-enyl]pyrrolidine-2,5-dione
Test material form:
liquid
Details on test material:
- Appearance: brown liquid
- Storage: room temperature, in the dark

Test animals

Species:
mouse
Strain:
other: Hsd: ICR (CD-1)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately five to eight weeks old
- Weight at study initiation: 23 to 29 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: The animals were housed in groups of up to seven, by sex, in solid-floor polypropylene cages with wood-flake bedding.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: minimum of five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 – 25 °C
- Humidity: 30 – 70 %
- Air changes: approximately fifteen changes per hour
- Photoperiod: the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Amount of vehicle: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was freshly prepared as required as a solution at the appropriate concentration in arachis oil.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Once
Post exposure period:
24 or 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
800 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main test: 7 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- 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
- Justification for choice of positive control(s): Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test.
- Route of administration: oral
- Doses / concentrations: 50 mg/kg

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- 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 as follows: 2000 mg/kg (oral) and 100, 1600, 1200 and 800 mg/kg (intraperitoneal). All animals were dosed once only at the appropriate dose level by gavage using a metal cannula or with a hypodermic needle 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 within the time periods of 0.5, 1 and 2 hours after dosing and subsequently once daily for up to two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
- Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration; therefore, this was selected for use in the main test. The maximum tolerated dose (MTD) of the test material, 800 mg/kg, was selected for use in the main test, with 400 and 200 mg/kg as the lower dose levels.

TREATMENT AND SAMPLING TIMES:
- One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test material at 800 mg/kg was killed after 48 hours.
- In addition, three further groups of mice were included in the test; two groups (each of seven mice) were dosed via the intraperitoneal route with the vehicle alone (arachis oil) and a third group (five mice) was dosed orally with cyclophosphamide. Cyclophosphamide is 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.
- 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.

DETAILS OF SLIDE PREPARATION:
- Immediately following termination (i.e. 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 re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

METHOD OF ANALYSIS:
- Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micro nucleated 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 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.
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, dose­responsive, toxicologically relevant 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 fulfilled, 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 Ill (1989). The data was analysed following a √(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
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 800 – 2000 mg/kg
- Clinical signs of toxicity in test animals: No evidence of toxicity was observed in animals dosed with test material via the oral route and, therefore systemic absorption could not be confirmed using this dose route. In animals dosed with the test material via the intraperitoneal route premature deaths occurred at and above 1200 mg/kg, and clinical signs were observed at and above 800 mg/kg as follows: Hunched posture, ptosis, ataxia, pilo-erection, lethargy, pallor of the extremities, hypothermia, decreased respiratory rate, splayed gait and laboured respiration. 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 test. Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration; therefore, this was selected for use in the main test. The maximum tolerated dose (MTD) of the test material, 800 mg/kg, was selected for use in the main test, with 400 and 200 mg/kg as the lower dose levels.

RESULTS OF DEFINITIVE STUDY
- There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 400 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture, ataxia and ptosis.
- Evaluation of Bone Marrow Slides: Marked statistically significant decreases in the PCE/NCE ratio were observed in the 24 and 48-hour 800 mg/kg test material dose groups when compared to their concurrent control groups and, together with the observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved. There were no statistically significant increases in the frequency of micronucleated PCE in any of the test material dose groups when compared to their concurrent vehicle control groups. 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. The test material 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

 Table 1: Micronucleus Test - Summary of Group Mean Data

Treatment Group

Number of PCE With Micronuclei per 2000 PCE

PCE/NCE Ratio

Group Mean

SD

Group Mean

SD

Vehicle Control 10 mL/kg (48-hour Sampling Time)

2.0

2.1

0.81

0.18

Vehicle Control 10 mL/kg (24-hour Sampling Time)

1.7

1.4

0.80

0.16

Positive Control 50 mg/kg

(24 -hour Sampling Time)

29.0***

9.1

0.64

0.25

Test Material 800 mg/kg

(48 -hour Sampling Time)

1.0

1.2

0.49**

0.19

Test Material 800 mg/kg

(24 -hour Sampling Time)

0.7

0.8

0.52**

0.18

Test Material 400 mg/kg

(24 -hour Sampling Time)

1.6

1.5

0.71

0.21

Test Material 200 mg/kg

(24 -hour Sampling Time)

0.9

1.2

0.88

0.36

PCE = Polychromatic erythrocytes, NCE = Normochromatic erythrocytes, SD = Standard deviation, ** = P <0.01, *** = P <0.001

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test material was considered to be non-genotoxic.
Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 474, EU Method B12, USA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines, under GLP conditions.

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. A range-finding test was performed to find suitable dose levels of the test material, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test material between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) 800 mg/kg and with 400 and 200 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 (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups, each of 7 mice, were given a single intraperitoneal dose of arachis oil or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 400 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture, ataxia and ptosis.

Marked statistically significant decreases in the PCE/NCE ratio were observed in the 24 and 48-hour 800 mg/kg test material dose groups when compared to their concurrent control groups and, together with the observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

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 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.

Under the conditions of this study, the test material was considered to be non-genotoxic.

Categories Display