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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 April 2002 (start of treatment) - 01 May 2002 (end of treatment); 06 June 2002 (end of in-life phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 84-2
Deviations:
no
GLP compliance:
yes
Type of assay:
transgenic rodent mutagenicity assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): furfural
- Analytical purity: 99.88%
- Lot/batch No.: 020220
- Expiration date: 30 September 2002
- Storage condition of test material: 2-10°C in the dark

Test animals

Species:
mouse
Strain:
other: λlacZ transgenic (CD2f1 (BALB/c x DBA/2)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan BV, the Netherlands
- Age at study initiation: approximately 7±2 weeks
- Weight at study initiation: 23.1-30.6 g (mean 26.7 g)
- Assigned to test groups randomly: [yes, under following basis: computer randomisation program]
- Fasting period before study: no
- Housing: individually in macrolon cages
- Diet: rat and mouse No 3 breeding diet, RM3 (Special diets services, Witham, England) ad libitum
- Water: community tap water (provided in polypropylene bottles) ad libitum
- Acclimation period: al least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 45-60%
- Air changes (per hr): approximately 10 per hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 04 April 2002 To: 06 June 2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Fresh solutions of the test substance in the vehicle were made once per week and stored at 2-10°C
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Post exposure period:
Mutation assessments were made on DNA from liver cells removed at necropsy from 8 mice in all groups on Days 62/63 (34/35 days after the end of the furfural dosing period).
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 37.5, 75, 150 and 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
13 males plus 2 reserves
Control animals:
yes, concurrent vehicle
Positive control(s):
- Ethylnitrosurea (ENU). The positive control group was dosed intraperitoneally with 50 mg/kg/day ethylnitrosourea for 5 days
- Solvent: dimethylsulfoxide (DMSO)
- Vehicle: phosphate buffered saline (PBS)

Examinations

Tissues and cell types examined:
Genomic DNA was isolated from the relevant tissue (liver). The IacZ genes were excised and single copies of the gene were packaged into infectious phage particles by use of commercially available lambda-packaging extracts.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Furfural dose levels (37.5, 75, 150 and 300 mg/kg/day) were selected on the basis of an existing 13 week toxicity study. The no-observed-adverse-effect-level (NOAEL) in the 13 week study was reported to be 75 mg/kg/day and it was anticipated that 300 mg/kg/day would elicit hepatotoxicity when administered orally once daily for 28 days.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
A subgroup of three mice in each of the Furfural treated groups and the negative control group was killed after 28 days of dosing to assess hepatotoxicity.
Mutation assessments were made on DNA from liver cells removed at necropsy from 8 mice in all groups on Days 62/63 (34/35 days after the end of the Furfural dosing period, to allow an untreated period for mutation fixation).
Finally, the potential mutagenicity of the test substance furfural was evaluated considering the biological relevance and statistical significance of th data by comparing the mean mutant frequencies of the groups.

METHOD OF ANALYSIS:
High molecular DNA was isolated from the liver samples by first isolating the nuclei, followed by proteinase K and SDS treatment, and salt precipitation. The DNA was packaged in phage particles by use of Transpack™ packaging extracts and the mutant phages were detected by use of the positive selection method.
Evaluation criteria:
The study was considered valid if the mean mutant frequency of the positive control was more than twice that of the negative (vehicle) control. However, if the mean mutant frequency of the positive control was not more than twice that of the negative control, but the test substance was considered positive based on the criteria below, the study was still considered valid.
The test substance was considered mutagenic if the mutant frequency in any of the dose groups was statistically significantly increased and/or dose-related increased compared to the mutant frequency observed in the negative control group.
The test substance was considered non-mutagenic if there was no statistically significant increase of the mutant frequency at any dose group, and if all dose groups fell within the range of the negative control group (±2 standard deviations).
If the test substance did not meet the criteria for either a mutagenic or non-mutagenic result, its mutagenicity was considered equivocal.
For the evaluation of the mutagenicity of the test substance both the biological relevance and the statistical significance were used.
Statistics:
The statistical procedures used in the evaluation of data were as follows: histopathological changes: Fisher's exact probability test. Body weight: one-way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests. Clinical chemistry values, food consumption and liver weights: one-way ANOVA followed by Dunnett's multiple comparison tests. Mutant frequency: Kruskal-Wallis ANOVA, Student's t-test and Jonckheere trend test for comparison of groups A to E. Mann-Whitney U-test and Student's t-test for comparison of groups A and F.
All statistical tests were two-sided. Probability values of p<0.05 were considered significant.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
three deaths at 300 mg/kg/day (the highest dose level) and 1 death at 75 mg/kg/day
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: confirmation that the test had been conducted using furfural dose levels close to the maximum tolerated dose was indicated by 3/15 early decedents at the highest dose level (300 mg/kg/day) and as observed from clinical chemistry (increased triglyceride levels), histopathological (centrilobular hypertrophy) evaluations and increased liver weights in a subgroup of mice killed after 28 days of dosing.

DOSE PREPARATION ANALYSIS
- Retrospective investigations confirmed that the method of dose formulation and the homogeneity and stability of the formulations was satisfactory for use on the study.

Any other information on results incl. tables

Table 2: Mutation analysis – mutant frequencies

Group

Treatment

Dose level (mg/kg/day)

Number of mice evaluated

Mean MF±SD (*10^6)

A

Vehicle control

0

8

61±23

B

Furfural

75

7

41±7

C

Furfural

150

8

54±21

D

Furfural

300

8

37±16

E

ENU (positive control)

50

8

246±95**

**statistically significant p<0.01 (Mann-Whitney U-test and Student’s t-test

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Oral administration of furfural in corn oil at levels up to and including 300 mg/kg/day was not associated with in vivo mutagenicity of liver cells using the λlacZ transgenic mouse model.
Executive summary:

Furfural was administered once daily by oral gavage for 28 consecutive days. It was dissolved in corn oil at concentrations of 3.75, 7.5, 15 and 30 mg/mL and administered at a constant dose volume of 10 mL/kg body weight to provide dose levels of 37.5, 75, 150 and 300 mg/kg bw/day. Vehicle controls were dosed with the vehicle only at a dose volume of 10 mL/kg body weight. Positive controls were dosed with 50 mg/kg ethylnitrosourea (ENU), administered intraperitoneally (i.p.) for 5 consecutive days (equivalent to Days 5-9 of the treatment period). ENU was dissolved in phosphate buffered saline (PBS) containing 5% dimethylsulfoxide (DMSO) at a concentration of 5 mg/mL and administered at a dose volume of 10 mL/kg.

Confirmation that the test had been conducted using furfural dose levels close to the maximum tolerated dose was indicated by 3/15 early decedents at the highest dose level (300 mg/kg/day) and as observed from clinical chemistry (increased triglyceride levels), histopathological (centrilobular hypertrophy) evaluations and increased liver weights in a subgroup of mice killed after 28 days of dosing. There was no increase in mutant frequency at this high dose level (300 mg/kg/day) or at the lower dose levels evaluated (75 and 150 mg/kg/day).

In conclusion, oral administration of furfural in corn oil at levels up to and including 300 mg/kg/day was not associated with in vivo mutagenicity of liver cells using the λlacZ transgenic mouse model.