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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
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1994
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
D&C Red No 9
provoded by H. Kohnstamm
Batch: Z8054
purity: 89.8 %

Test animals

Species:
Drosophila melanogaster
Strain:
other: male from Canton-S, female from Basc stocks
Sex:
male/female
Details on test animals and environmental conditions:
Separate Canton-S and Basc stocks were maintained at Brown University and the University of Wisconsin. Males to be exposed were collected from theCanton-S stocks. The Basc stocks supplied the balancer X-chromosome. P1 females used in the SLRL test were collected as virgins from this stock.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
DMSO
Details on exposure:
All nongaseous compounds were first tested by feeding exposure. Two or three glass fiber filter discs were saturated with the compound carried in a 5 % sucrose solution (or other control solution) at the bottom of a standard glass vial. Solutions were renewed at 24 hr and 48 hr. After 72 hr of exposure, surviving males were mated. If feeding exposures were found to be nonmutagenic, 2-3-day-old Canton-S males were injected with 0.7 % NaCl solution containing the test chemical. At 24 hr postinjection, toxicity was noted and survivors were mated. Concurrent control males were treated with the solution used to dissolve the test chemical.
Duration of treatment / exposure:
72h (oral), in addition non-mutated survivors were treated by injection
Frequency of treatment:
feed: permanent
injection: once daily
Post exposure period:
24h (after injection)
Doses / concentrationsopen allclose all
Dose / conc.:
2 500 ppm
Remarks:
nominal in diet
Dose / conc.:
1 000 ppm
Remarks:
adult injection
No. of animals per sex per dose:
P1: one male pared with 100 females (feed, 2500 ppm), 10 males and 20 females (injection, 1000 ppm)
Control animals:
yes, concurrent vehicle
Positive control(s):
4 substances were juged to be mutagenic in D. melanogaster (Gene-Tox report, EPA) and serve as positive control: 1,2-epoxypropane, ethylene bromide, and myleran (were mutagenic in this study), 2-chloro-l,3-butadiene (was not mutagenic in this study)
- Justification for choice of positive control(s): Gene-Tox report, EPA
- Route of administration:feed in diet
- Doses / concentrations: 2500 ppm

Examinations

Evaluation criteria:
For a compound to be considered mutagenic, the mutant frequency in the treated series (treated frequency) must exceed 0.15 % with a P value of <0.05, or the treated frequency must exceed 0.10 % with a P value of <0.01. If the treated frequency is between 0.10 % and 0.15 % and the P value is between 0.1 and 0.01, or if the treated frequency is higher than 0.15 % and the P value is between 0.1 and 0.05, the result is considered equivocal. All other results are considered negative.
Translocation data for each treated sample were compared to the historical control data for that laboratory using a conditional binomial test [Kastenbaum and Bowman, 1970]. As a rule, at least two translocations are required among -5,000 tests in the treated series for a compound to be considered positive.
Statistics:
For the SLRL assay, a minimum of -5,000 chromosomes was scored from each of the treated and concurrent control groups unless the mutant frequency exceeded 1%. If two or more lethals were recovered among the progeny of one male, a Poisson analysis [Owen, 1962] was performed to
determine if these were part of a “cluster.” (A cluster is defined as a group of mutated sperm cells derived from a single mutational event occurring in a spermatogonial cell.) Clusters must be spontaneous in origin, because only meiotic and postmeiotic stages of spermatogenesis were treated. Therefore, in those cases in which a male was determined to have produced a cluster, the lethal and nonlethal tests for that PI male were removed from the data. The corrected treated and control data were compared using a normal approximation to the binomial distribution, as suggested by Margolin et al. [ 1983]. In addition, the treated data were compared to the historical control as described by Mason et al. [1992].

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
other: 3 of 4 positive controls were mutagenic
Additional information on results:
Chemicals were received coded from the NTP chemical repository. Solubility was determined by testing first in distilled water. If the chemical was not sufficiently soluble in distilled water, other solvents were tested in the following order of preference: ethanol, polysorbate, dimethyl sulfoxide (DMSO), rapeseed oil, or a mixture of these. In feeding exposures, palatability was noted based on feeding behavior and the presence of “fly specks” on the exposure vial.
Toxicity tests were run on a series of exposures and, if possible, an exposure level was chosen that resulted in -30% mortality after 72 hr of feeding or 24 hr after injections or inhalation.

Any other information on results incl. tables

Table 1: Results of the Sex-linked recessive lethal assay on coded chemicals

Dose

ROA

Mortality [%]

Sterility [%]

Lethals

Tests

Total lethals

Total tests

Lethals [%]

Br 1

Br 2

Br 3

Br 1

Br 2

Br 3

2.500

Feeding

0

0

3

3

0

2.860

2.185

1.608

6

6.653

0.09

0

2

2

1

2.691

2.262

1.616

5

6.569

0.08

1.000

Injection

15

4

2

3

3

2.214

1.592

1.449

8

5.255

0.15

0

4

1

0

2.045

1.933

1.475

5

5.453

0.09

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative