Reaction products of alkenyl dihydrofuran-2,5-dione and polyisoalkenyl dihydro-2,5-furandione with alkylene glycol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of the test material was assessed in accordance with OECD Guideline 471, Bacterial Reverse Mutation Assay, and OECD Guideline 473, in vitro mammalian chromosome abberation test. The test item was considered to be non-mutagenic and non-clastogenic under the conditions of these studies.

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:

between 26 July 1995 and 1 September 1995.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS harmonised guidelines

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA (TSCA) guidelines

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Not required

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Non-mammalian study

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Non-mammalian study

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1: Range-finding test
Salmonella strains: 0, 50, 150, 500, 1500, 5000 µg/plate
E.coli strain: 0, 50, 150, 500, 1500, 5000 µg/plate

Experiment 2: Main test
Salmonella strains : A0, 50, 150, 500, 1500, 5000 µg/plate
E.coli strain: 0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification: Analysis for concentration, homogeneity and stability of the test material formulations is not a requirement of the test guidelines and was, therefore, not determined.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent

True negative controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2, 3, 5 ug/plate respectively for WP2uvrA, TA100, TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 ug/plate for TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 ug/plate for TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

1 µg/plate for TA100, 2 µg/plate for TA1535 & TA1537, 10 µg/plate for WP2uvrA , 0.5 µg/plate for TA98

Details on test system and experimental conditions:

Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material
to the tester organisms. A mixture of 0.1 ml of bacterial suspension (TA100 or WP2uvrA-), 0.1 ml of test solution, 0.5 ml phosphate buffer and 2 ml of
molten, trace histidine/tryptophan supplemented media was overlaid onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Five doses of the
test material and a vehicle control (acetone) were tested in duplicate. After approximately 48 hours incubation at 37°C the plates were scored for
revertant colonies and examined for a thinning of the background lawn.

Mutation Study - Experiment 1 (Range-finding Study):
Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.
- Test Material and Vehicle Controls: Known aliquots (0.1 ml) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 ml of molten trace histidine/tryptophan supplemented top agar at 45°C, 0.1 ml of the appropriately diluted test material or vehicle control and either 0.5 ml of the S9 liver microsome mix or 0.5 ml of pH 7.4 buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material with and without S9-mix.
- Positive Controls Without Activation: A known aliquot (0.1 ml) of one of the positive control solutions (ENNG, 9AA or 4NQO) was added to a test tube containing 2.0 ml of molten, trace histidinel tryptophan supplemented top agar and 0.1 ml of the appropriate bacterial suspension. Finally, 0.5 ml of pH 7.4 buffer was added to the tube, the contents mixed and poured onto an agar plate. This procedure was then repeated, in triplicate, for each tester strain.
- The plates were incubated at 37'C for approximately 48 hours and the number of revertant colonies counted.

Mutation Study - Experiment 2 (Main Study): The second experiment was performed using methodology as described for experiment 1, using fresh bacterial cultures, test material and control solutions in triplicate.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at subtoxic dose levels. If the two experiments give conflicting results or equivocal results are obtained, then a third experiment may be used to confirm the correct response. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than two fold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 pg/plate. In this case the limiting factor was the maximum recommended dose.

Statistics:

MAHON, G.A.T., et al (1989). Analysis of data from microbial colony assays. In: KIRKLAND D.J., (eds.). Statistical Evaluation of Mutagenicity Test Data: UKEMS sub-committee on guidelines for mutagenicity testing. Cambridge University Press Report, pp. 26-65.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

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:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

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:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No toxicity to the bacterial background lawn was exhibited to any of the strains ofbacteria used, although small and inconsistent decreases in revertant colony frequency were observed.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material either with or without metabolic activation.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Conclusions:

The genetic toxicity of the test material was assessed in accordance with OECD Guideline 471, Bacterial Reverse Mutation Assay. The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (acetone) control plates produced counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation, and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. Decreases in revertant colony frequency were observed in several of the tester strains, but these responses were not accompanied by a weakening of the bacterial background lawn.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic: activation. The test material was found to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 28 November 1995 and 11 April 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Remarks:

Additional time-points were used.

Qualifier:

according to guideline

Guideline:

other: Safepharm Standard Method Number EEC 28B

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

N/A

Species / strain / cell type:

other: Chinese hamster lung (CHL)

Details on mammalian cell type (if applicable):

-Cells were grown in Eagle’s Minimal Essential medium with Earle’s Salts (MEM), supplemented with 10% foetal bovine serum and antibiotics, at 37°C
with 5% CO, in air.

- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Experiment 1
The dose levels selected for assessment of chromosome aberrations were 10, 20 and 40 µg/ml. without S9 - 12 hours continuous exposure to the test material prior to cell harvest
The dose levels selected for assessment of chromosome aberrations were 40, 80 and 160 µg/ml with S9. 4 hours exposure to the test material and S9-mix (0.5 ml per 4.5 ml culture medium, of 50% S9 in standard co-factors).

Experiment 2
The dose levels selected for assessment of chromosome aberrations were 9.75, 19.5 and 39 µg/mI without S9 - 24 hours continuous exposure to the test material prior to cell harvest.
The dose levels selected for assessment of chromosome aberrations were 4.88, 9.75 and 19.59 µg/mI without S9 - 48 hours continuous exposure to the test material prior to cell harvest.
The dose levels selected for assessment of chromosome aberrations were 19.5, 39 and 58.55 µg/mI without S9 - 6 hours exposure to the test material, a phosphate buffered saline wash and then a further 18 hours culture in treatment-free media prior to cell harvest.
The dose levels selected for assessment of chromosome aberrations were 10, 20 and 40 µg/mI without S9 - 12 hours continuous exposure to the test material prior to cell harvest.
The dose levels selected for assessment of chromosome aberrations were 39, 78.1 and 117.2 µg/ml with S9. 6 hours exposure to the test material and S9-mix (0.5 ml per 4.5 ml culture medium of 10% S9 in standard co-factors).
The dose levels selected for assessment of chromosome aberrations were 40, 80 and 160 µg/ml with S9. 4 hours exposure to the test material and S9-mix (0.5 ml per 4.5 ml culture medium of 10% S9 in standard co-factors).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
-The test material was accurately weighed and prepared in dimethyl sulphoxide (DMSO) and appropriate dilutions made. Analysis for concentration,
homogeneity and stability of the test material preparations was not a requirement of the test guidelines and therefore was not performed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

used in Experiment 1 at 0.075 µg/ml for the 12-hour cultures and at 0.05 µg/mI for the 24 and 48-hour cultures in Experiment 2.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

10 µg/ml for cultures treated for the 4(8)-hour culture with S9 in Experiment 1, the 6(18)-hour culture both with and without S9-mix, and the 4(8)-hour culture with S9-mix in Experiment 2.

Details on test system and experimental conditions:

The initial purity of the test material was 82%, therefore an allowance for test material purity was made when dosing solutions were prepared. There was no observable change in pH and when the test material was dosed into media.
The osmolality was outside the 50mOsm limiting range, at 5000 µg/ml however due to toxicity this concentration was not used as a treatment dose.

Cultures were established approximately 24 or 48 hours prior to treatment, 0.15 to 0.5 x 10^6 cells were seeded per flask for the 6, 12 and 24-hour
treatment cultures and 0.2 x 10^6 cells were seeded per flask for the 48-hour treatment cultures. The cells were exposed to doses of the test material, vehicle and positive controls, both with and without metabolic activation. All treatments were performed in duplicate (A + 6). Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2, in air.

Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mI) two hours before the required harvest time

Evaluation criteria:

Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 23 to 27 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing
Aberrations recorded by the slide scorer were checked by a senior cytogeneticist. Cells with 28 to 31 chromosomes were scored as aneuploid
cells. Cells with greater than 31 chromosomes were classified as polyploid cells with the o/o incidence of polyploid cells reported. The percentage of cells showing structural chromosome aberrations (gaps, breaks and exchanges) were calculated and reported as both including and excluding those with gaps.
Mitotic Index: A total of 1000 cells were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Vehicle and positive controls were used in parallel with the test material.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The results of the preliminary cytotoxicity test are presented in Table 1 (attached). It can be seen that the test material showed evidence of a dose-related increase in cell toxicity in every exposure case. The presence of a precipitate affected the electronic cell counter giving falsely elevated counts but only at dose levels which were toxic. Microscopic assessment of the slides prepared from the preliminary cytotoxicity test showed metaphases present up to 78.1 µg/ml in the 6 and 12-hour with S9-mix treatment cases and at up to 19.5 µg/ml in the 12-hour without S9-mix treatment and at up to 39 pg/ml in the 6-hour without- S9 treatment cases. The maximum dose with metaphases present was 39 µpg/ml in the 24 and 48-hour continuous exposure treatments.

Experiment 1.
The results of the cell counts from the cultures after their respective treatments are presented in Table 2 (attached) It can be seen that the test material showed the expected level of toxicity similar to that seen in the preliminary toxicity study except that in this case scorable metaphase cells were obtained at the maximum dose tested ie 40 µg/mI without49 and 160 pg/mI with-S9.
The results of the mitotic index are presented in Table 3. These data also show a clear dose-related increase in toxicity with increasing doses of the test material. The mitotic indices gave a measure of toxicity greater than that of the cell counts for the cultures in the absence of S9 in which 50% mitotic inhibition was achieved at 40 µg/mI. In the presence of S9, increases in the mitotic index were observed indicating toxicity-induced cell-cycle
synchronisation. The vehicle control cultures gave values of chromosome aberrations within the expected range (Table 6, Attached).
The positive control cultures gave significant increases in the frequency of aberrations (Table 6) indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected. It was considered that the poor response observed for cyclophosphamide was due to a toxicity-induced cell-cycle delay typical for early time-points.
The test material was seen to induce a statistically significant increase in the frequency of cells with aberrations at 160 µg/ml in the 12-hour treatment with metabolic activation only. There was an increase in the frequency of chromatid exchanges, this increase was only observed in one culture, but gave a statistical significance using Fisher’s Exact Test for the number of cells with aberrations excluding gap-type aberrations compared to the concurrent control. The response was not dose-related.
The test material induced a significant increase in the numbers of polyploid cells at 40 µg/mI without-% only. This increase was observed in one of the
duplicate cultures only and was therefore considered to be spurious.

Experiment 2.
The results of the cell counts from the cultures after their respective treatments are presented in Table 4 (attached). It can be seen that the test material showed the expected level of toxicity similar to that seen in the preliminary cytotoxicity study and Experiment 1.
The results of the mitotic index are presented in Table 5. As in Experiment 1 the mitotic indices generally indicate a higher level of toxicity than shown by the cell counts.
The vehicle control cultures gave values of chromosome aberrations within the expected range (Table 6).
The positive control cultures, except cyclophosphamide without S9 treatment, gave significant increases in the frequency of cells with aberrations (Table 6) indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected.
It was considered, as in Experiment 1, that toxicity-induced cell cycle delay was responsible for the relatively weak response seen in both the 12-hourwith and without S9 positive control groups.
The test material did not induce any statistically significant dose-related increases in the frequency of cells with aberrations at any dose level in any of
the six treatment cases. However, there was a slightly exceptional frequency of exchange-type aberrations at 11 7.2 µg/ml in the 6-hour treatment with metabolic activation, which, although it did not achieve statistical significance compared to the controls, was similar to, but lower than, that seen in
Experiment 1. The increase was not reproduced in the 4(8)-hour treatment with-S9 of Experiment 2 and was therefore considered to be of no toxicological significance.
The test material induced a small but significant increase in the numbers of polyploid cells at 39 µg/ml dose level in the 6-hour treatment with -S9 only which was not doserelated and therefore considered to be of no toxicological significance.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

See attached background material.

Conclusions:

The genetic toxicity of the test material was assessed in accordance with OECD Guideline 473, in vitro mammalian chromosome abberation test. The test material demonstrated no toxicologically significant dose-related increases in the frequency of cells with aberrations. The test material was shown to be toxic to CHL cells in vitro in all six treatment cases, with a very steep dose response curve. The test material was shown to be non-clastonenic to CHL cells in vitro.

Executive summary:

Chinese hamster lung (CHL) cells were treated with the test material at a minimum of four dose levels, in each treatment case, in duplicate, together with vehicle and positive controls, in each of two experiments. In Experiment 1 a single cell-harvest

time-point at 12 hours, both with and without metabolic activation, was used. In Experiment 2 six treatment regimes were used: 6 hours exposure both with and without the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 18 hours treatment-free incubation); 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 8 hours treatment-free incubation); 12 hours continuous exposure, 24 hours continuous exposure and 48 hours continuous exposure.

The dose range for metaphase analysis was selected from a series of at least four dose levels chosen on the basis of the results of a preliminary toxicity test. The test material was evaluated at dose levels between 4.88 and 160 µg/mI depending on the particular treatment regime used. The continuous exposures were more toxic than the pulse exposures.

The vehicle (solvent) controls gave frequencies of aberrations within the range expected for the CHL cell line.

The positive control treatments gave significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. A poor but typical response was seen in the 12-hour treatment case, particularly with cyclophosphamide, which was probably due to toxicity-induced cell cycle delay.

The test material induced no toxicologically significant dose-related increases in the frequency of cells with aberrations in Experiment 1 or Experiment 2. The test material was shown to be toxic to CHL cells in vitro in all six treatment cases, with a very steep dose response curve. The test material was shown to be non-clastogenic to CHL cells in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The genetic toxicity of the test material was assessed in accordance with OECD Guideline 474, Mammalian Erythrocyte Micronucleus Test.  The test item was considered to be non-genotoxic under the conditions of the test.

Link to relevant study records

Reference

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 1995 and 17 August 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Safepharm Standard Method Number EEC 29A

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

- Sufficient male and female albino CD-1 Strain mice were supplied by Charles River (UK) Limited, Manston, Kent.
- At the start of the main study the males weighed 23 to 30g and the females 20 to 24g, and were approximately 5 to 7 weeks old.
- Minimum acclimatisation period of 5 days.
- The animals were housed in groups of up to 5 in solid-floor polypropylene cages with woodflake bedding.
- Free access to mains drinking water and food.
- The animal room was maintained at a temperature of 19 to 25°C and relative humidity of 50 to 65%.
- The rate of air exchange was approximately 15 changes per hour.
- Lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.

Route of administration:

intraperitoneal

Vehicle:

Supplier's identification : Arachis oil
Supplier's batch number : 158
Saf-epharm serial number : CO/926
Date received : 7 June 1995
Description : straw coloured slightly viscous liquid
Storage conditions : room temperature

Details on exposure:

Groups, each of ten mice (five males and five females), were dosed once only via the intraperitoneal route with test material at 37.5, 75 or 150 mg/kg. One group of mice from each dose group was killed by cervical dislocation 24 hours following treatment and a second at 48 hours. In addition three further groups of ten mice (five males and five females) were included in the study; two groups were given a single intraperitoneal dose of the vehicle (arachis oil) and the third 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 and 48 hours following dosing and positive control group animals were killed 24 hours following dosing.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

once

Post exposure period:

No

Remarks:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Supplier's identification : Cyclophosphamide
Supplier's lot number : 73HO846
Safepharm serial number : CO/911
Date received : 17 May 1995
Description : white powder
Storage conditions : 4°C
The concentration, homogeneity and stability of the positive control material and its preparation were not determined by analysis.

Tissues and cell types examined:

Bone marrow polychromatic erythrocytes were examined for the presence or absence of micronuclei.

Details of tissue and slide preparation:

Immediately following sacrifice (ie. 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 and stained in May-Grünwald/Giemsa, dried and coverslipped using mounting medium.

Evaluation criteria:

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.

Statistics:

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.
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part lll (1989). The data was analysed following a*racine (x+1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs were observed in the RF study

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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 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 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.

There was no significant increase in the frequency of micronucleated PCEs in any of the test material dose groups (37.5, 75 and 150 mg/kg) when compared to their concurrent vehicle control groups.

There was no statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. However, the presence of clinical signs and premature deaths in the range-finding toxicity study indicated that systemic absorption did occur. 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.

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.

Conclusions:

The genetic toxicity of the test material was assessed in accordance with OECD Guideline 474, Mammalian Erythrocyte Micronucleus Test. The test item was considered to be non-genotoxic under the conditions of the test.

Executive summary:

A study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered via the intraperitoneal route to mice. The method followed was designed to comply with Method B12 of the EEC Commission Directive 92/69/EEC which constitutes Annex V of Council Directive 57/548/EEC.

Following a preliminary range-finding study to find a suitable dose level for the test material, the micronucleus study was conducted using the test material at the maximum tolerated dose level of 150 mg/kg with 75 and 37.5 mg/kg as the lower dose levels.

In the micronucleus study, groups of ten mice (five males and five females) were given a single intraperitoneal dose of the test material at 37.5, 75 or 150 mg/kg. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

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.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the concurrent vehicle control groups. No significant change in the PCE/NCE ratio was observed after dosing with the test material. Although, the presence of clinical signs and premature cleaths during the preliminary range-finding study would indicate systemic absorption had occurred.

The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes.

The test material was considered to be non-genotoxic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Key Bacterial Mutation Assay

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (acetone) control plates produced counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation, and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. Decreases in revertant colony frequency were observed in several of the tester strains, but these responses were not accompanied by a weakening of the bacterial background lawn.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic: activation. The test material was found to be non-mutagenic under the conditions of this test.

Key Mammalian Cell Chromosome Aberration Test

Chinese hamster lung (CHL) cells were treated with the test material at a minimum of four dose levels, in each treatment case, in duplicate, together with vehicle and positive controls, in each of two experiments. In Experiment 1 a single cell-harvest

time-point at 12 hours, both with and without metabolic activation, was used. In Experiment 2 six treatment regimes were used: 6 hours exposure both with and without the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 18 hours treatment-free incubation); 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 8 hours treatment-free incubation); 12 hours continuous exposure, 24 hours continuous exposure and 48 hours continuous exposure.

The dose range for metaphase analysis was selected from a series of at least four dose levels chosen on the basis of the results of a preliminary toxicity test. The test material was evaluated at dose levels between 4.88 and 160 µg/mI depending on the particular treatment regime used. The continuous exposures were more toxic than the pulse exposures.

The vehicle (solvent) controls gave frequencies of aberrations within the range expected for the CHL cell line.

The positive control treatments gave significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. A poor but typical response was seen in the 12-hour treatment case, particularly with cyclophosphamide, which was probably due to toxicity-induced cell cycle delay.

The test material induced no toxicologically significant dose-related increases in the frequency of cells with aberrations in Experiment 1 or Experiment 2. The test material was shown to be toxic to CHL cells in vitro in all six treatment cases, with a very steep dose response curve. The test material was shown to be nonclastoaenic to CHL cells in vitro.

In Vivo Mammalian Micronucleus Test

There was no significant increase in the frequency of micronucleated PCEs in any of the test material dose groups (37.5, 75 and 150 mg/kg) when compared to their concurrent vehicle control groups.

There was no statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. However, the presence of clinical signs and premature deaths in the range-finding toxicity study indicated that systemic absorption did occur. 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.

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.

Justification for selection of genetic toxicity endpoint
Three GLP studies compliant with the relevant OECD test guidelines, all providing non-genotoxic results.

Short description of key information:
GLP, OECD compliant bacterial mutagenicity, mammalian cell chromosome aberration and in vivo mouse micronucleus studies on a similar substance with a valid and robust read-across justification.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with the CLP Regulation, no 1272/2008, a substance should be classified as mutagenic if there is evidence that it may induce heritable mutations in the germ cells of humans. The test material was found to be non-mutagenic in an in vitro Ames test, non-clastogenic in an in vitro chromosome aberration study and was non-genotoxic in an in vivo mouse micronucleus study. Therefore, using read-across data on a surrogate test item, the substance does not meet the criteria for classification.