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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-12-21 to 1995-02-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is classified reliable without restriction. The study was conducted according to OECD Guideline 474 and followed GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
1-dodecene dimer, hydrogenated
IUPAC Name:
1-dodecene dimer, hydrogenated
Details on test material:
- Substance type: 1-dodecene dimer, hydrogenated
- Physical state: liquid, clear colourless
- Lot/batch No.: C1527
- Storage condition of test material: Room temperature

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Manston, Kent
- Age at study initiation: 5-7 weeks
- Weight at study initiation: Male: 21-18 grams; Female: 20-24 grams
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: groups of up to 5 by sex in steel meshbottom polypropylene cages suspended above absorbant paper
- Diet (e.g. ad libitum): Rat and Mouse Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK ad libitum
- Water (e.g. ad libitum): Mains drinking water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21˚C
- Humidity (%): 50%-51%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

IN-LIFE DATES: From: 1994-12-21 To: 1995-02-16

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: No
- Concentration of test material in vehicle: 125, 250, and 500 mg/mL
- Lot/batch no. (if required): 012207
Duration of treatment / exposure:
Single intraperitoneal injection
Frequency of treatment:
Single injection
Post exposure period:
24, 48, and 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
1250, 2500, and 5000
Basis:
nominal conc.
mg/kg bw
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): No
- Route of administration: Orally
- Doses / concentrations: 50 mg/kg bw

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence o cytotoxicity up to a maximum recommended dose of 5000 mg/kg

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

Range-Finding Toxicity Study - All animals were dosed once only via the intraperitoneal route at the appropriate dose level using 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 1 hour after dosing and subsequently once daily for 3 days. Any deaths and evidence of overt toxicity were recorded at eac observation. No necropsies were performed.

Micronuclues Test - Groups, each of ten mice (five males and five females), were dosed once only via the intraperitoneal route with test material at 1250, 2500 or 5000 mg/kg. One group of mice from each dose group was killed by cervical dislocation 24 hours following treatment, a second at 48 hours and a third at 72 hours. In addition four further groups of ten mice (five males and five females) were included in the study: three groups were given a single intraperitoneal dose of the vehicle (arachis oil) and the fourth group was dosed orally with cyclophosphamide, a positive control material known to produce micronuclei under the conditions of the test. The vehicle control groups were killed 24, 48 and 72 hours followingdosing and positive control group animals were killed 24 hours following dosing.

DETAILS OF SLIDE PREPARATION: Immediately following sacrifice (ie. 24, 48 or 72 hours following dosing), one femur was dissected from each animal,
aspirated with foetal calf serum and bone marrow smears prepared following centrifugation for 30 seconds at approximately 6000 rpm, and re-suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, dried and coverslipped using mounting medium

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined 'blind' using light microscopy at x1000 magnification. The incidence of micronucleated cells per 1000 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 polychromatic 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 for males and females separately and combined.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the three test material groups and the number occurring in the corresponding vehicle control groups. A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24, 48, or 72-hour kill times when comparedto their corresponding control group. If these criteria are not demonstrated, then the test material is considered to be non-genotoxic under the conditions of the test. A positive response for bone marrow toxicity is demonstrated when the dose group mean normochromatic to polychromatic ratio is shown to be statistically significantly lower than that of the concurrent vehicle control group.
Statistics:
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
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1250, 2500, 5000 mg/kg bw
- Clinical signs of toxicity in test animals: No premature deaths or clinical observations were seen in animals dosed with the test material up to the maximum recommended dose level of 5000 mg/kg.
- Evidence of cytotoxicity in tissue analyzed: No
- Rationale for exposure: No

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There was evidence of a small but statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with test material at 2500 mg/kg after 48 hours when compared to the concurrent vehicle control group. The response was not part of a dose-related effect, was within current historical values for 48-hour group animals, and was, therefore, considered to be spurious and of no toxicological significance.
- Ratio of PCE/NCE (for Micronucleus assay): No significant change in the PCE/NCE ratio was observed after dosing with the test material.
- Appropriateness of dose levels and route: Appropriate per guideline
- Statistical evaluation: Approrpiate per guideline

Any other information on results incl. tables

Treatment Group

Number of PCE with Micronuclei per 1000 PCE

PCE/NCE Ration

 

Group Mean

Standard Deviation

Group Mean

Standard Deviation

Vehicle Control                    (72-hr Sampling)

0.7

1.3

1.26

0.30

Vehicle Control                    (48-hr Sampling)

0.6

0.5

1.30

0.55

Vehicle Control                    (24-hr Sampling)

0.4

0.5

1.40

0.48

Positive Control              (24-hr Sampling)

19.3***

5.0

1.33

0.31

TS 5000 mg/kg               (72 hr Sampling)

0.8

1.5

1.59

0.62

TS 2500 mg/kg               (72 hr Sampling)

0.6

0.5

1.29

0.51

TS 1250 mg/kg               (72 hr Sampling)

0.7

1.2

1.52

0.76

TS 5000 mg/kg               (48 hr Sampling)

0.5

1.0

1.66

0.84

TS 2500 mg/kg               (48 hr Sampling)

1.2*

0.6

1.47

0.33

TS 1250 mg/kg               (48 hr Sampling)

1.0

0.9

1.69

0.17

TS 5000 mg/kg               (24 hr Sampling)

0.8

0.7

1.71

0.88

TS 2500 mg/kg               (24 hr Sampling)

1.1

1.3

1.81

1.12

TS 1250 mg/kg               (24 hr Sampling)

0.7

0.5

1.30

0.42

PCE = polychromatic erythrocytes       

NCE = normochromatic erythrocytes

*p<0.05

***p<0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
The test material 1-dodecene dimer, hydrogenated, was considered to be non-genotoxic under the conditions of the test.
Executive summary:

In a micronucleus test conducted in the mouse (Duward, R., 1995; Klimisch score = 1) the potential of 1-dodecene dimer, hydrogenated to produce damage to chromosomes or aneuploidy post administration via the intraperitoneal route in CD-1 mice (5/sex/dose) was evaluated.

Following a preliminary range-finding study to confirm the toxicity of the test material, the micronucleus study was conducted using the test material at the maximum recommended dose level of 5000 mg/kg with 2500 and 1250 mg/kg as the lower two dose levels. Animals were administered a single intraperitoneal injection of the test material and killed 24, 48, or 72 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei, 1000 polychromatic erythrocytes were scored for each animal. Further groups of mice were dosed via the intraperitoneal route with arachis oil or orally with cyclophosphamide, to serve as vehicle and positive controls respectively.

 

A small but statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with test material at 2500 mg/kg after 48 hours was observed when compared to the concurrent vehicle control group. The response was not part of a dose-related effect, was within current historical values for 48-hour group animals, and was, therefore, considered to be spurious and of no toxicological significance. No significant change in the PCE/NCE ratio was observed after dosing with the test material. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes when compared to the concurrent vehicle control group.

 

Based on the results observed in this study, 1-dodecene dimer, hydrogenated is considered to be non-genotoxic in the micronucleus test in the mouse.