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in vivo insect germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from a publication

Data source

Reference Type:
Genetic toxicity study of the test chemical
Valencia et al
Bibliographic source:
Environmental Mutagenesis

Materials and methods

Test guideline
equivalent or similar to guideline
EU Method B.20 (Sex-linked Recessive Lethal Test in Drosophila melanogaster)
Principles of method if other than guideline:
Equivalent or similar to EU Method B.20 (Sex-linked Recessive Lethal Test in Drosophila melanogaster)
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 2-(3-oxo-6-oxidoxanthen-9-yl)benzoate
EC Number:
EC Name:
Disodium 2-(3-oxo-6-oxidoxanthen-9-yl)benzoate
Cas Number:
Molecular formula:
disodium 2-(3-oxo-6-oxidoxanthen-9-yl)benzoate
Test material form:
Details on test material:
Name of test material (as cited in study report): Fluorescein disodium salt
Molecular formula: C20H10Na2O5
Molecular weight (if other than submission substance): 376.274g/mol
Smiles notation: c1(c2c(oc3c1ccc(c3)[O-])cc(=O)cc2)c1c(cccc1)C(=O)[O-].[Na+].[Na+]
InChl (if other than submission substance): NJDNXYGOVLYJHP-UHFFFAOYSA-L
Substance type: Organic
Physical state: Solid
Purity: The test material was of 79.8% purity

Test animals

Drosophila melanogaster
not specified
Details on test animals or test system and environmental conditions:
The males to be treated were collected from a Canton-S wild-type stock within 8 hr of emergence and kept on regular culture medium until exposure.

Administration / exposure

Route of administration:
other: The chemical was first tested by feed. If non-mutagenic by this route, the chemical was injected.
Dietary exposure: 5% aqueous sucrose
Injection: 0.7% aqueous NaCl
Details on exposure:
Males to be fed were shaken into vials containing glass fiber filter material that was soaked with the feeding solution or mixture. At 24 hr and again at 48 hr, the flies were transferred to vials with freshly prepared feeding mixture and were mated at 72 hr. Males to be injected were held on regular food for 1-3 days. They were then injected with 0.7% NaCl solution containing the test chemical. They were held for 24 hr to recover, then mated.
Duration of treatment / exposure:
1-2 days
Frequency of treatment:
Feeding: 1-2 days of exposure
Injection: one injection
Post exposure period:
Injection: 24 hr recovery period
Doses / concentrations
0 and 50000 ppm (feed)
0 and 10000 ppm (injection)
No. of animals per sex per dose:
no data
Control animals:
yes, concurrent vehicle
yes, plain diet
Positive control(s):


Tissues and cell types examined:
Germ cells
Evaluation criteria:
Generally, a test was considered positive if the frequency of lethals in the treated series exceeded 0.2% over the control frequency.
Data were then tested for significance using the normal test as described by Margolin et al (Environ Mutagen 1983; 5: 705-16). A SLRL result was called negative if P > 06, equivocal if P = 0.04-0.06, either questionable or positive if P = 0.01-0.04 (depending on the control frequency), and positive if P < 0.01.

Results and discussion

Test results
Key result
19% mortality bu injection, 17% mortality by feeding
Vehicle controls validity:
Negative controls validity:
not specified
Positive controls validity:
Remarks on result:
other: Negative
Additional information on results:
The sex-linked recessive lethal frequency was similar between insects fed at 0 and 50000 ppm (0.09% vs 0.06%, respectively) and similar between insects injected at 0 and 10000 ppm (0.05% vs 0.08%, respectively). Mortality was in the range of 17-19% for insects treated with the test chemical. No cases of sterility were observed in the study.

Applicant's summary and conclusion

The test chemical was non-mutagenic (negative) in sex-linked recessive lethal (SLRL) mutation induction test in Drosophila melanogaster.
Executive summary:

The test chemical was tested for sex-linkedrecessive lethal (SLRL) mutation inductionin Drosophila melanogaster. Initially, the test chemical was tested by feeding, whenno significant mutagenicitywas observed,it was re-tested by injection.Doses used in the mutagenicity test was selected based on the results of a preliminary dose-finding study. The test substance was dissolved in water anddiluted with aqueous 5%sucrose for feedingand with aqueous 0.7% NaCl for injection. Toxicity tests were performed both forfeeding and injection treatments using a series of concentrations.Doses were chosen to induce mortalityof about 30% after 72 hoursof feeding or 24 hoursafter injection. The males to be treated werekept on regular culture medium until exposure. Males to befed were shaken into vials containing glass fiber filter material that was soaked withthe feeding solution ortest chemicalmixture. At 24 hoursand again at 48hours, the flies were transferredto vials with freshly prepared feeding mixture and were mated at 72 hours.Males to be injected were held on regular food for 1-3 days. They were theninjected with0.7% NaCl solution containing the test chemical. They were held for24 hoursto recover, then mated. The flies for the SLRL assay were exposed for 4hours. Treated and control males were matedindividuallyto threeharems ofBascvirgin females to produce three broods of 3,2, and 2 days.Thus,primarily post-meiotic germ cells were tested. To reduce the chances of recoveringseveral lethals from the same male, no more than 40 F1 females were mated individuallyfrom each brood of each male. Thus, no more than 120 chromosomes weretested from each P1male. F2 cultures were scored as presumptive lethals if thenumber of wild-type males recovered was 0, 1, or fewer than5% of the number ofBascmales (or Basc/+females). Presumptive lethals were confirmed by repeating the matings and scoring the F3.The test chemical was used at doses of 0 (solvent control) and 50000 ppm for feeding experiments and 10000 ppm for injection. No mutagenic response was observed in broods examined when the test chemical solution was fed or injected to male flies. The percents of lethals (total lethals) were statistically comparable in treated and control groups using both treatment methods. The mortality was 19 and 17% in experiments with injection and feeding treatment, respectively In conclusion, the test chemical was non-mutagenic (negative) insex-linkedrecessive lethal (SLRL) inductiontest in Drosophila melanogaster.