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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The work described was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1997 (SI 1997/654)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 87/18/EEC and 88/320/EEC.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Sponser's Identification: Ethyl Tetrahydrofurfuryl Ether
- Date received: 26 August 1998
- Description: colourless liquid
- Storage conditions: room temperature in the dark under nitrogen.

Data relating to the identity, purity and stability of the test material are the responsibilty of the Sponser.

Method

Target gene:
- designed to assess the potenital mutagenicity of the test material on the thymidin kinase, TK +/-, locus of the L5178Y mouse lymphoms cell line.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Obtained from Dr J Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- Cells were originally obtained from Dr D Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Vehicle / solvent:
Solvent (RO) treatment groups were used as the vehicle controls.
Controls
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
batch 87H0207 at 7.5 µg/ml.
Details on test system and experimental conditions:
CELL CULTURE: The stocks of cells are stored in liquid nitrogen at -196°C. Cells were routinely cultured at RPMI 1640 medium supplemented with 10% donor horse serum and 20mM Hepes buffer (R10) at 37°C with 5% CO2 in air. The cells have a generation time approximately 12 hours and were subcultured accoridngly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.

CELL CLEANSING: the TK +/- heterozygote cells are grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine 9 (µg/ml), Hypoxanthine (15 µg/ml), Methotrexate (0.3 µg/ml) and Glycine (22.5 µg/ml) For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

PREPARATION OF TEST ANd CONTROl MATERIAL: the test material was accurately weighed and dissolved in RO media before the appropriate dilutions were made. The molecular weight of the test material was calculated to be 130.2, thereofre the maximum dose level was initially limited to 1302 µg/ml which was equivalent to 10mM. However with no evidence of any marked toxicity with the test material and no measured increases in osmolality, the maximum dose level was increased to 5000 µg/ml. Analaysis for conc. homogeneity and stability of the test material preparations were not a requirement of the test method and were therefore not performed. Vehicle and positive control were used in parallel with the test material.

MICROSOMAL ENZYME FRACTIONS: Lot No. Aro. S9/22/08/98 used in experiemtn 1 was prepared in-house on 22 August 1998 from the livers of male Sprague-Dawley rats weighing ~250g. These had received a single ip injection of Aroclor 1254 at 500 mg/kg, five days before S9 preparation. The S9 was stored at -196°C in a liquid nitrogren freezer.

Lot No. PB/BNF S9/29/11/98 used in Experiment 2 was prepared in-house on 22 August 1998 from the livers of male Sprague-Dawley rats weighing ~250g. These had each received, orally, three consecutive daily doses of phenobarbitone/ß-naphthoflavone )80/100 mg per kg per day) prior to S9 preparation. The S9 was stored at -196°C.

10% S9-mix was prepared by mixing S9, NADP (5 mM), G6P (5 mM), KCl (33 mM) and MGCL2 (8 mM) in R0.

The final concentration was 1% throughout the course of the study.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
not specified
Additional information on results:
Interpretation of Results
The normal range for mutant frequency per survivor is 25-150 x 10"6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10"6 mutant frequency per survivor are not normally acceptable and will be repeated.
Positive control chemicals should give a significant increase in mutant frequency per survivor over the negative controls of at least a three to five-fold increase.
For a test material to give a 'significant' result then 2 or more of the following criteria should be met:
i) A statistically significant increase in mutant frequency.
ii) A greater than three-fold increase in the mutant frequency per survivor over the negative control value.
iii) A dose-related increase in the mutant frequency per survivor.
iv) An increase in the absolute number of mutants.
A test material may be reported as equivocal if only one of the above criteria is met.

Any other information on results incl. tables

Preliminary Toxicity Test

The dose levels given below were used in the preliminary toxicity test. The results of the Day 0 relative toxicity survivals (%RTS) were as follows:

Dose

%RTS of Control (-S9)

%RTS of Control ( + S9)

%RTS of Control (-S9)

(/yg/ml)

3-hour exposure

3-hour exposure

24-hour exposure

0

100

100

100

20.35

121

103

104

40.69

198

120

112

81.38

194

130

>240

162.75

105

127

>240

325.5

98

104

>240

651

89

116

202

1302

98

117

178

 

It can be seen, in both the absence and presence of S9, that no toxicity was observed with the test material. No precipitate was observed.

Mutagenicity Test Experiment 1

It can be seen that the test material did not induce a dose-related toxic response as indicated by the Day 0 viability (%S) values and relative survival values (%RS). There was no evidence of a reduction in Day 2 (%V) viability or relative total growth (RTG) with the test material indicating that there was no residual toxicity, either with or without S9. Both of the positive controls produced reductions in both Day 0 (%S) and Day 2 (%V) viabilities and RTG values.

The test material did not induce any statsically significant or dose related (linear trend increased in the mutant grequncy x10 -6 per viable cell either in the presence or absence of metabolic activation. Both the vehicle control mutant frequncies were within the range of 25 to 150 x 10-6 viable cells that is acceptable for L5178Y cells at Safepharm Laboratories. In the absence, and particularly in the presence of S9, some of the test material dose levels had mutant frequencies greater than the vehicle control value.

However, there was no clear dose-related response and they were considered to be artefactual and of no toxicological significance. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (see attached pdf. Report No 905/026 Tables 2 and 5).

Experiment 2

The 24-hour continuous exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had no marked effect on the toxicity of the test material up to the 10 mM dose level of 1302 /vg/ml. However, in the case of the 5000 µg/ml test material dose level, there was evidence of toxicity with a reduction in %S and %RS values. No reduction in %V was observed with the Day 2 viability value, but there was a reduction in the RTG value indicating some residual toxicity had occurred during the expression period.

As was seen in Experiment 1, there was no evidence of a dose-related reduction in Day 0 (%S) viability and relative survival (%RS) values in cultures dosed with the test material in the presence of metabolic activation. There was no evidence of a reduction in Day 2 (%V) viability or relative total growth (RTG), therefore indicating that no residual toxicity occurred in the presence of metabolic activation. Both positive controls produced reductions in both the Day 0 (%S) and Day 2 (%V) viabilities and RTG values.

Both the vehicle control mutant frequencies were within the acceptable range of 25 to 150 x 10-6viable cells. The test material did not induce any significant or dose-related increases in the mutant frequency per viable cell in the presence of metabolic activation and as was seen in Experiment 1, any increases in mutant frequency over the controls were small and considered to be artefactual and of no toxicological significance. However, in the absence of metabolic activation in the 24-hour continuous exposure cultures a modest, but statistically significant, dose-related (linear-trend) increase in mutant frequency was observed. The significant increase was only observed at the 5000 "g/ml test material dose level, however, it should be noted that the response was due to an increase in the absolute number of mutant colonies. It was decided to perform a confirmatory third experiment to see if this response was reproducible and therefore had toxicological significance. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The increase in mutant frequency observed in the absence of metabolic activation was partly due to small colony formation indicating clastogenic activity.

Experiment 3

As was seen in Experiment 2 there was evidence of test material toxicity as demonstrated by the Day 0 (%S) viabilities and relative survival (%RS) values. There was some residual toxicity with the test material after the two day expression period as indicated by the reduced Day2.(%V) viability values ar.d relative total growth (RTG) values for the 5000 µg/ml dose level. The positive control produced reductions in both the Day 0 (%S) and Day 2 (%V) viabilities and RTG values.

The vehicle control mutant frequency value was within the acceptable range of 25 to 150 x 10-6viable cells. The test material induced a significant dose- related increase in mutant frequency at 5000 µg/ml.

The positive control produced a marked increase in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

Applicant's summary and conclusion

Conclusions:
ETHYL TETRAHYDROFURFURYL ETHER induced small, toxicologically significant, dose-related increases in the mutant frequency at the TK +/- locus in L5178Y cells in the absence of metabolic activation following 24-hours continuous exposure at dose levels above 10 mM. ETHYL TETRAHYDROFURFURYL ETHER was therefore considered to be mutagenic under the conditions of the test.