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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 June 1992 - 4 February 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-diethoxypropane
EC Number:
412-180-1
EC Name:
1,2-diethoxypropane
Cas Number:
10221-57-5
Molecular formula:
C7H16O2
IUPAC Name:
1,2-diethoxypropane
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The material, a colourless liquid, was stored at ambient temperature in the dark when not in use.

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac Limited, Shaw's Farm, Blackthorn, Bicester, Oxfordshire
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 25-37g (male) 20-29g (female)
- Assigned to test groups randomly: Yes (main test)
- Housing: Animals were kept individually in polypropylene and stainless steel cages measuring 48 cm x 15 cm x 13 cm. White wood shavings provided the bedding in the cages
- Diet (e.g. ad libitum): SDS Rat and Mouse Maintenance Diet No. 1 obtained from Special Diet Services.Limited, Witham, Essex, England ad libutum (Appendix 10).
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22 °C
- Humidity (%): 37-65%
- Photoperiod (hrs dark / hrs light): 12 h light-dark cycle


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Immediately prior to dosing, the test compound was dissolved in Tween 80 to give the required test concentration. The dose volume used for both the control and test compound treated animals was a constant 10 ml/kg body weight. Cyclophosphamide was prepared fresh as an 8 mg/ml solution in distilled water and administered to the positive control animals in dose volumes of 10 ml/kg to give a target dose of 80 mg/kg. The mice were weighed immediately before each exposure event and the dose volume adjusted using a graded, gavage-fitted 1 ml syringe.
Duration of treatment / exposure:
24 and 48 hrs
Frequency of treatment:
Once
Doses / concentrationsopen allclose all
Dose / conc.:
1 600 mg/kg bw/day (nominal)
Remarks:
Males
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Females
No. of animals per sex per dose:
10 males and 10 females - test item and vehicle control
5 males and 5 females - positive control
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide;
- Route of administration: oral gavage
- Doses / concentrations: 80 mg/kg sampled at 24 hrs

Examinations

Tissues and cell types examined:
Femora, bone marrow, erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A toxicity study consisting of 2 parts, dose range finding and main toxicity test, was. undertaken prior to the micronucleus test. In the dose range finding, 6 groups of one male and one female CD-1 mice were dosed orally at 0 h with 1,2-Diethoxypropane as follows: 50, 125, 350, 800, 2000, 5000 mg/kg. The mice were observed for clinical signs or mortality at frequent intervals (1 min, 0.5 h, 1 h, 2 h and 4 h) post dosing, then twice daily until the end of the observation period. Surviving animals were killed 5 days after dosing by CO2 asphyxiation. A gross post mortem examination was performed on all animals, following compound induced death or scheduled kill. Animals deaths were induced at compound doses of 2000 and 5000 mg/kg in the males and at 5000 mg/kg in the females. The deaths were preceded by clinical signs consisting of piloerection, increased activity, subdued behaviour, ataxia, hunched appearance, laboured breathing, pale, tremors, increased breathing and prostration. The gross post mortem examination showed no abnormal findings (Appendices 1-3).

Based on the results of the dose range finding test, the exposure levels for the main toxicity test were set at 800, 1200 and 1600 mg/kg in the case of males, and 1200, 1600 and 2000 mg/kg in the case of females. No animal deaths were induced by these treatments. Clinical observations included piloerection, hunched appearance, increased activity, subdued behaviour, prostration, tremors, agitated, leaning to one side, ataxia, pale and laboured breathing. The gross post mortem examinations revealed no unusual findings. Taken together, these toxicity data indicated that the oral, single, dose MTD for I, 2-Diethoxypropane in CD-1 mice was around 1600 mg/kg for the males and 2000 mg/kg for the females (Appendices 4-8).

DETAILS OF SLIDE PREPARATION:
A drop of the suspension prepared from erythrocytes from the bone marrow was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube and animal. The smear was left to air dry, fixed in methanol for ca 10 min and stained with 1% May-Grunwald in methanol and Sorenson's buffer for 5 min, then counterstained in 15% Giemsa (Gurr) in Sorenson's buffer for 15 min. The stained smears were rinsed in 3 changes of distilled water and air dried. Finally, the smears were cleared in Histo-clear and permanent slide preparations obtained by sealing glass coverslips onto the microscopic slides using DPX mountant.

METHOD OF ANALYSIS:
The better of the 2 prepared slides was selected for examination and the coded slides, assessed blind by the same operator. One thousand (1000) polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micronucleated cells (MN-PCE) determined. As a control against inclusion of artefacts, or action of a mutagen on the G2 and/or mitotic phase of the cell cycle, the number of micronucleated normochromatic erythrocytes (MN-NCE). In addition, scored micronuclei were assigned on the basis of size into small or large categories, historically defined as micronuclei occupying less or more than 25% of the visible cellular area. This classification provided a non-specific measure of compound induced spindle disfunction, as large micronuclei appear to derive from lagging chromosomes caused by damage to the mitotic apparatus during bone marrow erythropoiesis. The PCE/NCE ratio, a measure of compound induced systemic toxicity, was calculated by counting a minimum total of 500 erythrocytes (PCE + NCE) per bone marrow preparation. Leitz Dialux 20 binocular microscopes were used for this purpose. Magnification was nominally x 1000 using x 10 magnification eye pieces and a x 100 oil immersion objective.
Evaluation criteria:
Assuming a mean frequency of MN-PCE of 0.128% per mouse and 0.122% in CD-1 mice for both sexes combined, a positive response was suspected if the total numbers of micronuclei within any one sample group of a given number of mice exceeded the corresponding value shown in table A. The cumulative historical micronucleus incidence in vehicle control dosed CD-l mice, treated with distilled water, corn oil or 0.5% carboxymethyl cellulose, has in this 1aboratory been determined as 0.121%. This figure represents 629 observations of MN-PCE in a total sample of 520 mice with 1000 PCEs evaluated per animal. Average spontaneous micronucleus incidences equal to or in slight excess of 0.20% were noted in 11 out of 26 conducted experiments using 5 mice per sample and sex as a reference point. The mean frequency of MN-PCE in these high range groups was 0.24 ± 0.03%. Taken together these in-house findings support the validity of the overleaf tabulated criteria for a negative or positive test response.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
The frequency of micronucleated bone marrow polychromatic erythrocytes (MN-PCE) in mice dosed with the vehicle, 10 ml/kg Tween 80 was 0. 06% for the 24 h samp1e and 0. 04% for the 48 h sample. These values conformed to the in-house historical frequency range for vehicle control dosed mice (=0.00-0.24%/10 mice). Exposure of the mice to the positive control agent, 80 mg/kg Cyclophosphamide induced large increases of bone marrow micronuclei. The combined MN-PCE frequency for both sexes was 2.51%. An increase in the numbers of MN-NCE was similarly noted. Systemic toxicity was also in evidence, as shown by a prominent suppression of the PCE/NCE ratios in exposed mice.

The incidence of MN-PCE in bone marrow erythrocytes of the 1,2- Diethoxypropane dosed mice was within established negative control frequencies for all test groups. The highest MN-PCE frequency recorded for the test material (0.16%, males 48 h) was fully consistent with a negative micronucleus response, as assessed by concurrent and cumulative control criteria. An apparent suppression of the PCE/NCE ratio was observed in the 24 h bone marrow samples taken from males, indicating minor systemic toxicity at the lethal dose used. No evidence of toxicity was observed in female mice.

Any other information on results incl. tables

Two unscheduled deaths occurred following treatment with 1,2-Diethoxypropane. The deaths were in the group of females scheduled for sampling at 48 h. To balance the sample sizes, 1 animal was therefore, sampled at 48 h instead of the scheduled 24 h. Thus, 4 females were killed for each of the 2 sampling times. Clinical signs observed in the test compound group were piloerection, increased activity, ataxia, subdued behaviour, prostration, tremors, hunched appearance, laboured breathing, pale and hypothermic.

Applicant's summary and conclusion

Conclusions:
In a mammalian erythrocyte micronucleus test, 1,2-Diethoxypropane did not induce micronuclei in bone marrow cells when tested to maximum tolerated doses in male and female CD-1 mice.
Executive summary:

In a CD-1 mouse bone marrow micronucleus assay (9003), groups of 10 male and female mice were treated by oral gavage with 1,2-Diethoxypropane at doses of 1600 mg/kg bw (males) and 2000 mg/kg bw (females). The animals were dosed once and bone marrow cells were harvested at 24 and 48 hrs post-treatment. The vehicle was Tween 80.

There were clinical signs of toxicity during the study.  1,2-Diethoxypropane was tested at an adequate dose (MTD; based on the dose range finding 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.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.