1,2-diethoxypropane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance 1,2-Diethoxypropane did not induce mutagenicity in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 in the presence or absence of Aroclor-1254 induced rat liver S9 (equivalent or similar to OECD 471, GLP).

In vitro cytogenicity (chromosome aberration) study in mammalian cells: the substance 1,2-Diethoxypropane was concluded to be negative for the induction of chromosome aberrations in the presence and absence of Aroclor-1254 induced rat liver S9 metabolic activation system in Chinese hamster ovary cells (equivalent or similar to OECD 473/GLP).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October 1991 - 13 January 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Only 4 guideline strains were used.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 relevant strains were used.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Synthetic Chemicals; 9059

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the dark at ambient temperature.

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test in S.typhimurium TA100: 33, 100, 333, 1000, 3333, 10000 µg/plate
Main tests: 33, 100, 333, 1000, 3333, 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: With S9: 2-Aminoanthracene (2-AAN), 2 µg per plate with TA 1535 and TA 1537 and 0.5 µg per plate with TA 1538, TA 98 and TA 100

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: background lawn activity

Evaluation criteria:

A test was considered acceptable if for each strain:

i) the bacteria demonstrated their typical responses to crystal violet, ampicillin and u.v. light.
ii) at least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and TA 1538, 5-35.
iii) on at least 2 of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5 the mean vehicle control mutant numbers per plate. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required on at least 2 of the positive control plates.
iv) no toxicity or contamination was observed in at least 4 dose levels.
v) in cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:
i) for S. typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98, at least a doubling of the mean concurrent vehicle control values at some concentration of the test substances and, for S. typhimurium strain TA 100, a 1.5-fold increase over the control value. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
ii) a dose related response, although at high dose levels this relationship could be inverted because of, for example, (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
iii) a reproducible effect in independent tests.

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

10,000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: There was no precipitation of the test material.

RANGE-FINDING/SCREENING STUDIES:
A toxicity test using strain TA 100 only was performed in the presence and absence of 59 mix to establish suitable dose levels for the mutation tests. One plate of each of the following concentrations
of 1,2-Di-ethoxy-propane was used: 33, 100, 333, 1000, 3333, 10000 µg/plate. A thin lawn of microcolonies was noted at 10000 µg/plate indicating toxicity to the bacteria (Table 1).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No
- Negative (solvent/vehicle) historical control data: The vehicle control values were within the normal ranges experienced in the laboratory (no dates provided) and reported in the literature with these strains of S. typhimurium.

Conclusions:

In a bacterial reverse mutation assay (Amest test), 1,2-Diethoxypropane was negative with and without Aroclor 1254-induced rat liver S9 metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (8341), strains of S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to 1,2-Diethoxypropane in DMSO at concentrations of 0, 33, 100, 333, 1000, 3333, 10000 µg/plate (pre-incubation, both experiments) in the presence and absence of mammalian metabolic activation (Aroclor-1254 induced rat liver S9).

1,2-Diethoxypropane was tested beyond the limit concentration of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 January 1992 - 28 April 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Shell International

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in a brown bottle with black plastic cap (containing cardboard wadding) in the dark, in a solvent store, at ambient temperature when not in use.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Darroudi, State University of Leiden, Netherlands

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F-10 medium with 5% C02:95% air

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test: 0, 20, 39, 78, 156, 313, 625, 1250, 2500 and 5000 µg/mL
Main test (6 hrs +S9): 0, 625, 1250, 2500, 5000 µg/mL
Main test (48 hrs -S9): 0, 625, 1250, 2500, 4000, 4500, 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 hrs +S9; 48 hrs -S9.
- Expression time (cells in growth medium): 18 hrs + S9; 48 hrs – S9.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (final concentration of 0.1 µg/mL)

STAIN (for cytogenetic assays): 5% Giemsa (Gurrs Improved R66).

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
-Centrifugation at 210-220 g for 5 min and removal of media.
-Resuspension in 1% tri-sodium citrate for 15 min.
-Centrifugation as before and removal of tri-sodium citrate.
-Resuspension in fixative (3:1, methanol:acetic acid) for ca 1 h at 0-4°C.
-Centrifugation as before and removal of fixative.
-Resuspension in fresh fixative for ca 10 min.
-Centrifugation as before and removal of excess fixative.
-Cells resuspended in the residual fixative.
-Slides made (one from each culture for toxicity test, 3 from each culture for main assay).
-Air dried (at least overnight).
-Stained in 5% Giemsa (Gurrs Improved R66).
-Rinsed in distilled water.
-Air dried.
-Mounted with coverslips and DPX mountant.


NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Whenever possible or practical, 100 cells from each culture were examined and scored for structural and numerical chromosomal abnormalities. From each culture 2 or 3 slides were examined.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and Relative increase in Cell Count (RICC)

OTHER EXAMINATIONS:
- Determination of polyploidy: Two types of polyploid cells were defined. Normal polyploid cells having multiple increased chromosome sets (4N, 8N etc) distributed in no obvious pattern in the metaphase.

- Determination of endoreplication: Endoreduplicating polyploid cells were recognised by the appearance of 'pairs' of chromosomes formed by 2 rounds of DNA replication without the normal intervening mitosis.

Evaluation criteria:

Structural Aberrations
A negative test substance response was recorded if the measured aberration parameter fell on or below the 95% confidence limits of mean historical values of vehicle control cultures. The number of aberrant cells (excluding gaps) was regarded as a more important parameter than either total lesion or aberrant cells (including gaps).

A positive response was recorded whenever the incidence of aberrations in treated cultures consistently equalled or rose above the upper 99% confidence limits of mean historical values of vehicle control cultures. Importance was also placed on the demonstration of dose related and reproducible increases in the assessed aberration parameters. Sporadic increases in structural aberrations in compound treated cultures whether over the 95 or 99% confidence levels were discussed individually. Where control values fell between the 95 and 99% limits, the frequency was deemed elevated. If the responses to the test and positive control substances gave a doubling of the elevated control frequencies these were then judged as positive. A test was rejected if vehicle or medium-only control values fell outside the upper 99% confidence limits for 2 of the 3 measured aberration parameters shown in the table above. Similarly, a test was rejected if positive control values (for at least one positive control) were not in excess of the upper 99% confidence limits for 2 of the 3 measured parameters shown in the table above.

Numerical Aberrations
As with structural aberrations, elevated numerical aberrations sometimes occur in negative controls. Such results were discussed individually and taken into consideration when assessing relevant responses in compound treated or positive control treated cultures, with a doubling over the elevated negative control required for a positive response. In negative control cultures, 2 Aneuploidy, 1 endoreduplication and 4 polyploidy cells were regarded as normal.

Statistics:

Statistical evaluation of in-house data from vehicle and untreated control cultures have enabled acceptable aberration frequency ranges for a negative response to be defined. These are based on 95% and 99% confidence limits of mean values.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

weakly positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: The osmolality of 1,2-Diethoxypropane was measured in the toxicity test, in the presence of S9 mix. The test compound had no effect on osmolality of the culture medium (Appendix 3). No further measurements were made in the main assay.
- Precipitation: 1,2-Diethoxypropane was tested to concentrations of 5000 µg/mL in both the presence and absence of S9 mix. The test compound gave a visible precipitate at 2500 µg/mL and higher.

RANGE-FINDING/SCREENING STUDIES:
1,2-Diethoxypropane was non toxic in the presence of S9 mix. In the absence of S9 mix, 5000 µg/mL reduced the cell count by about 50% with the lower concentration 2500 µg/mL being sub-toxic. In both the presence and absence of S9 mix, increases in mitotic indices were recorded. In the presence of S9 mix the effect was demonstrated over the range of 1250-5000 µg/mL. In the absence of S9 mix, this effect was less concentration dependent, suggestive effects being noted with 20 and 39 µg/mL and significant effects with 1250 and 2500 µg/mL. The relatively normal levels at 78-625 µg/mL may have been sporadic observations. The low value obtained with 5000 µg/mL probably reflected the toxic effects of this concentration (Tables 1,2).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No
- Negative (solvent/vehicle) historical control data: Historical Aberration Ranges for Vehicle and Untreated Control Cultures were presented (no timeframe).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main assay 1,2-Diethoxypropane was non-toxic in the presence of S9 mix. In the absence of S9 mix, intervening concentrations of 4000 and 4500 were included to provide cultures for analysis if 5000 µg/mL proved more toxic in this assay. In fact, the toxicity pattern achieved was very similar to the toxicity test. The extra concentrations gave some additional information, both 4000 and 4500 µg/mL had levels of toxicity very similar to 5000 µg/mL. This result reflected the precipitation pattern. That is, undissolved 1,2-Diethoxypropane (precipitate) had no additional effect on cytotoxicity (Table 3).

Conclusions:

In an in vitro mammalian chromosome aberration test in CHO celss, it was concluded that 1,2-Diethoxypropane was weakly clastogenic in the absence of S9 metabolic activation and negative in the presence of S9 metabolic activation.

Executive summary:

In an in vitro cytogenicity study (chromosome aberration; 8755), Chinese hamster ovary cells cultured in vitro were exposed to 1,2-Diethoxypropane in DMSO at 0, 625, 1250, 2500, 5000 µg/mL for 6 hours in the presence of Aroclor 1254-induced rat-liver S9 metabolic activation and 0, 625, 1250, 2500, 4000, 4500, 5000 µg/mL for 48 hrs in the absence of metabolic activation.

1,2-Diethoxypropane was tested up to cytotoxic concentrations.  In the absence of S9 metabolic activation, slightly elevated numbers of aberrant cells were found in single cultures treated with 2500 and 5000 µg/mL. A single culture treated with the lower concentration (1250 µg/mL) had a small increase in aberration frequencies. There was no evidence that 1,2-Diethoxypropane induced either structural or numerical aberrations in the presence of S9 metabolic activation. It was concluded that 1,2-Diethoxypropane was weakly clastogenic in the absence of S9 metabolic activation and negative in the presence of S9 metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo mammalian somatic cell study (cytogenicity/erythrocyte micronucleus): 1,2-Diethoxypropane did not induce micronuclei in bone marrow cells when tested to maximum tolerated doses in male and female CD-1 mice.

In vivo mammalian cell study: DNA damage and/or repair (unscheduled DNA synthesis): there was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear grain counts] was induced by 1,2-Diethoxypropane in male Wistar rats up to the limit dose.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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.

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

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

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

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.

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.

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.

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.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January 1993 - 13 July 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Shell International Chemical Company; 13412
- Purity: 99.91%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature under nitrogen and in the dark

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd, Margate, UK
- Age at study initiation: 43-53 days
- Weight at study initiation: 174-236 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: no more than 3 animals in polypropylene cages with wire mesh lids ard solid floors
- Diet: laboratory chow diet (Special Diet Services Ltd) ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 60-65%
- Air changes (per hr): 15 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs light/dark

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 1% (v/v) Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing preparations were made by suspending 1,2-DEP in 1% (v/v) Tween 80, aided by Silverson homogenization, to give 200 mg/ml. All test compound solutions were wrapped in foil to exclude light and administered within 1 hour of formulation.

Duration of treatment / exposure:

Experiment 1: 11 hours 59 minutes to 12 hours 45 minutes
Experiment 2: 2 hours 3 minutes to 2 hours 45 minutes

Frequency of treatment:

Once

Post exposure period:

For Experiment 1, animals were killed a nominal 12-14 hours after dosing, For Experiment 2, animals were killed approximately 2-4 hours post-treatment.

Dose / conc.:

632.5 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 males/group

Control animals:

yes, concurrent vehicle

Positive control(s):

2-Acetamidofluorene (2-AAF) (Sigma Chemical Co, Poole, UK) was freshly suspended in corn oil at 7.5 mg/ml to serve as the positive control for the 12-14 hour experiment.
Dimethylnitrosamine (DMN) (Sigma Chemical Co, Poole, UK) dissolved in reverse osmosis highly purified (ROHP) water at 1.0 mg/ml was used as the positive control for the 2-4 hour experiment.

Tissues and cell types examined:

Liver; hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
An initial range-finding study was conducted using groups of 5 male rats. Animals were treated with vehicle and 2000 mg/kg bw. There were no mortalities after 3 days. Animals dosed at 2000 mg/kg bw DEP showed signs of lethargy, prostration, unsteady gait and irregular breathing after dosing, but all recovered after approximately 3 hours. Thus, a dose of 2000 mgjkg was chosen as an appropriate upper dose level for the UDS study. A lower dose, 632.5 mg/kg bw, one half log dilution of the upper dose, was also used.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): In the main study, groups of 6 male rats were treated. To enable a manageable number of animals to be processed at any one time, dosing was carried out on 2 occasions approximately 2 hours apart, usually treating 3 animals from each dose group on each occasion.

DETAILS OF SLIDE PREPARATION:
Hepatocytes were prepared from 5 or 6 animals in each dose group. Three of the 6 slides from each animal (Experiment 2) or all 6 (Experiment 1) were coated in I1ford K2 liquid emulsion using a dipping technique. Each slide was dipped individually into the emulsion, ensuring that no air-bubbles were generated. After gelling over ice, for 10 minutes face upwards, the slides were incubated in a light-tight box at room temperature for approximately 90 minutes to let the emulsion dry. The slides were then packed in light-tight boxes containing desiccant, sealed with tape and refrigerated for 14 days. At the end of this time, the emulsion was developed in Kodak 019 developer and fixed using Ilford Hypam fixer. The cell nuclei and cytoplasm were then stained with Meyers haemalumfeosin Y. Slides were then dehydrated in alcohol, cleared in xylene and mounted with coverslips for microscopic examination. The spare, duplicate set of Experiment 2 slides was not required for autoradiography.

METHOD OF ANALYSIS:
Grain counting was performed using a microscope with a video camera connected to a Biotran (Experiment 1) or Artek (Experiment 2) colony counter and a computer programmed for automatic data capture. Each slide was examined to ensure that the culture was viable. A patch of cells was selected as a starting point and cells were scored in a regular fashion by bringing new cells into the field of view, moving only the x-axis. If the desired number of cells had not been scored before coming to the edge of the slide, the stage was moved 1 or 2 fields on the Y-axis and counting resumed. The circular field was centred over the nucleus of a suitable cell and the grains counted. The field of view was moved and counts obtained for 3 separate adjacent areas of cytoplasm. Nuclear and mean cytoplasmic grain counts were then recorded, and the net grains/nucleus (NG) determined. One hundred cells were analysed per animal, using 2 of the slides in each case.

Evaluation criteria:

A test chemical at a particular- dose level would be considered clearly positive in this assay if the chemical yielded at least 0 NG and 20% or more of cells responding. A dose-related increase in both NG and the percentage of cells in repair would also be required. Cytoplasmic and riuclear grain count values would be considered in relation to the overall net grain count.

Statistics:

Mean and Standard Deviation

Sex:

male

Genotoxicity:

negative

Remarks:

Experiment 1 (632.5 mg/kg bw)

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

Experiment 1 (2000 mg/kg bw)

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

Experiment 2 (632.5 mg/kg bw)

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

Experiment 2 (2000 mg/kg bw)

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

In an in vivo mammalian cell study: DNA damage and/or repair (unscheduled DNA synthesis), it is concluded that 1,2-DEP had no genotoxic activity in this test system.

Executive summary:

In an unscheduled DNA Synthesis test with mammalian liver cells in vivo (121/9), 6 male Wistar rats  were treated orally (gavage) with 1,2-Diethoxypropane (99.91%) in1% Tween 80 at doses of 0, 632.5, 2000 mg/kg bw. Approximately 12-14 hours (Experiment 1) or 2-4 hours (Experiment 2) after dosing animals were killed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from each of 5 animals in each dose group and were treated with [3H] thymidine.

1,2-Diethoxypropane was tested up to the limit concentration (2000 mg/kg bw). The liver is a target organ as effects were noted in the 28-day repeated dose toxicity study. The positive controls induced the appropriate response.  There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear grain counts] was induced.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Gene mutation (Bacterial Reverse Mutation Assay/Ames test)

There is one bacterial reverse mutation assay/Ames test available.

In a reverse gene mutation assay in bacteria (equivalent or similar to OECD 471/GLP), strains of S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to 1,2-Diethoxypropane in DMSO at concentrations of 0, 33, 100, 333, 1000, 3333, 10000 µg/plate (pre-incubation, both experiments) in the presence and absence of mammalian metabolic activation (Aroclor-1254 induced rat liver S9).1,2-Diethoxypropane was tested beyond the limit concentration of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In vitro cytogenicity (chromosome aberration) study in mammalian cells

There is one in vitro mammalian chromosome aberration test available.

In an in vitro cytogenicity study (chromosome aberration; equivalent or similar to OECD 473/GLP), Chinese hamster ovary cells cultured in vitro were exposed to 1,2-Diethoxypropane in DMSO at 0, 625, 1250, 2500, 5000 µg/mL for 6 hours in the presence of Aroclor 1254-induced rat-liver S9 metabolic activation and 0, 625, 1250, 2500, 4000, 4500, 5000 µg/mL for 48 hrs in the absence of metabolic activation. 1,2-Diethoxypropane was tested up to cytotoxic concentrations.  In the absence of S9 metabolic activation, slightly elevated numbers of aberrant cells were found in single cultures treated with 2500 and 5000 µg/mL. A single culture treated with the lower concentration (1250 µg/mL) had a small increase in aberration frequencies. There was no evidence that 1,2-Diethoxypropane induced either structural or numerical aberrations in the presence of S9 metabolic activation. It was concluded that 1,2-Diethoxypropane was weakly clastogenic in the absence of S9 metabolic activation and negative in the presence of S9 metabolic activation.

In vivo

In vivo mammalian somatic cell study (cytogenicity/erythrocyte micronucleus)

There is one mammalian erythrocyte micronucleus test available.

In a CD-1 mouse bone marrow micronucleus assay (OECD 474/GLP), 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.

In vivo mammalian cell study: DNA damage and/or repair (unscheduled DNA synthesis)

There is one unscheduled DNA Synthesis test with mammalian liver cells in vivo available.

In an unscheduled DNA Synthesis test with mammalian liver cells in vivo (121/9), 6 male Wistar rats  were treated orally (gavage) with 1,2-Diethoxypropane (99.91%) in1% Tween 80 at doses of 0, 632.5, 2000 mg/kg bw. Approximately 12-14 hours (Experiment 1) or 2-4 hours (Experiment 2) after dosing animals were killed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from each of 5 animals in each dose group and were treated with [3H] thymidine. 1,2-Diethoxypropane was tested up to the limit concentration (2000 mg/kg bw). The liver is a target organ as effects were noted in the 28-day repeated dose toxicity study. The positive controls induced the appropriate response.  There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear grain counts] was induced.

Justification for classification or non-classification

Based on the available information in the dossier, the substance 1,2 Diethoxypropane (CAS No. 10221-57-5) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.