Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

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:

25 July to 17 August 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed to a guideline and used 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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

EC Number:

421-660-1

EC Name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

Molecular formula:

Hill formula: C50 H96 N2 O10 CAS formula: C42 H74 O8. 2(C4 H11 N O)

IUPAC Name:

bis((2-hydroxyethyl)dimethylazanium) (4E)-3-{[2-({3-carboxylato-3-[(2E)-hexadec-2-en-2-yl]propanoyl}oxy)ethoxy]carbonyl}-4-methyloctadec-4-enoate

Constituent 2

Reference substance name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

IUPAC Name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

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: Approximately 5 to 7 weeks old
- Weight at study initiation: Males: 23-30 g; Females: 20 to 24 g
- Assigned to test groups randomly: Yes
- Fasting period before study: NDA
- Housing: The animals were housed in groups of up to 5 in solid floor polypropylene cages with woodflake bedding.
- Diet: Ad libitum - rat and mouse expanded diet
- Water: Ad libitum - mains drinking water
- Acclimation period: Minimum acclimisation period of 5 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 50-65 %
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness


IN-LIFE DATES: NDA

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Arachis oil
- Justification for choice of solvent/vehicle: NDA
- Concentration of test material in vehicle: 0, 3.75, 7.5 and 15 mg test material per mL of vehicle
- Amount of vehicle (if gavage or dermal): N/A
- Type and concentration of dispersant aid: N/A
- Supplier's batch no.: 158
- Purity: NDA

Details on exposure:

The test material was administered by intraperitoneal injection uisng a hypodermic needle attached to a graduated syringe.

Duration of treatment / exposure:

One group of mice from each dose group was killed 24 hours following treatment and a second at 48 hours.

Frequency of treatment:

Animals were dosed only once.

Post exposure period:

Either 24 or 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 37.5, 75 and 150 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

Male: 37.5 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 75 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 150 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 37.5 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 75 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 150 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 37.5 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 75 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 150 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 37.5 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 75 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 150 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: Cyclophosphamide
- Justification for choice of positive control(s): It is known to produce micronuclei under the conditions of the test.
- Route of administration: Oral
- Doses / concentrations: Dose level: 50 mg/kg; Concentration: 5 mg/mL

Animals treated with the positive control were killed 24 hours after dosing.

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: With premature deaths at and above 300 mg/kg the maximum tolerated dose level selected for the main study was 150 mg/kg. These dose levels were selected with animal welfare issues in mind.


TREATMENT AND SAMPLING TIMES: All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable. Animals were sacrificed either 24 or 48 hours after administration of the test material.


DETAILS OF SLIDE PREPARATION: Immediately following sacrifice, both femurs were dissected from each animal, asprated with foetal calf serum and bone marrow smears prepared following centrifugation and resuspension. The smears were dried, fixed in absolute methanol and stained in May-Grünwald/Giemsa, dried and coverslipped.


METHOD OF ANALYSIS: Stained bone marrow smears were 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. 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.


OTHER: NDA

Evaluation criteria:

A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48 hour kill times when compared to 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 polychromatic to normochromatic ratio is shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

The data were analysed using Student's t-test (two tailed).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Remarks:

There was no statistically significant increase or change in the frequency of micronucleated PCEs or the PCE/NCE ratios in any of the test material dose groups when compared to their concurrent vehicle control groups.

Toxicity:

no effects

Remarks:

There were no premature deaths or clinical signs observed in any of the dose 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: 75-2000 mg/kg
- Solubility: NDA
- Clinical signs of toxicity in test animals: Premature deaths were seen in animals dosed with the test material at and above 300 mg/kg. Clinical signs were observed above 100 mg/kg and included hunched posture, lethargy, pilo-erecion, ptosis, decreased respiratory rate, laboured respiration, ataxia, pallour of the extremities, distended abdomen, dehydration and tiptoe gait. The presence of clinical signs and premature deaths indicated that systemic absorption did occur.
- Evidence of cytotoxicity in tissue analyzed: N/A - no necropsies were performed
- Rationale for exposure: N/A
- Harvest times: N/A
- High dose with and without activation: N/A
- Other: NDA


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): N/A
- Induction of micronuclei (for Micronucleus assay): There was no statistically significant increase 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 was no statistically significant change in the PCE/NCE ratios in any of the test material dose groups when compared to their concurrent vehicle control groups.
- Appropriateness of dose levels and route: The steep toxicity curve seen in the range-finding study resulted in the selection of a maximum tolerated dose level that was expected to produce clinical signs in the main study. The absence of clinical signs in the main study was considered not to affect its integrity or validity.
- Statistical evaluation: See Table 1

Any other information on results incl. tables

Table 1 Summary of group mean data

Treatment group	No. of PCE with micronuclei per 1000 PCE		PCE/NCE ratio
	Group mean	SD	Group mean	SD
VEHICLE CONTROL 48 h sampling time	0.5	0.7	0.95	0.27
VEHICLE CONTROL 24 h sampling time	1.0	0.7	0.97	0.36
POSITIVE CONTROL 24 h sampling time	28.0*	7.8	0.77	0.15
150 mg/kg 48 h sampling time	0.8	1.1	1.01	0.24
75 mg/kg 48 h sampling time	0.7	0.9	0.99	0.47
37.5 mg/kg 48 h sampling time	0.9	0.7	1.00	0.30
150 mg/kg 24 h sampling time	1.4	1.2	1.08	0.47
75 mg/kg 24 h sampling time	0.8	0.8	1.15	0.54
37.5 mg/kg 24 h sampling time	1.4	1.5	1.11	0.29

SD = standard deviation

* = p less than 0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
OS 114451A was considered to be non-genotoxic under the conditions of the test.

Executive summary:

In an albino CD-1 strain mouse bone marrow micronucleus assay, groups of 5 males and 5 females were treated via a single intraperitoneal dose with OS 114451A at doses of 0, 37.5, 75 and 100  mg/kg bw.  Bone marrow cells were harvested at 24 and 48 hours post-treatment.  The vehicle was arachis oil. 

 

There were no signs of toxicity during the study.  The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.