9,10-Anthracenedione, 1,4-diamino-, N,N'-mixed 2-ethylhexyl and 3-[(2-ethylhexyl)oxy]propyl and 3-methoxypropyl derivs.

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The in vivo Pig-a assay uses flow cytometry to measure phenotypic variants for antibody binding to cell surface glycosylphosphatidylinositol (GPI)-anchored proteins. There is good evidence suggesting that the absence of antibody binding is the result of a mutation in the endogenous X-linked Pig-a gene, which forms the rationale for the assay.

The study was integrated into the OECD 422 guideline study described in section 7.8 and 7.5.
Gollapudi, B. B., Lynch, A. M., Heflich, R. H., et al. (2014). The in vivo Pig-a assay: A report of the International Workshop on Genotoxicity Testing (IWGT) Workgroup. Mutation Research: Genetic Toxicology and Environmental Mutagenesis, http://dx.doi.org/10.1016/j.mrgentox.2014.09.007

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vivo pig A

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,4-bis(methylamino)anthraquinone and 1,4-bis[(2-ethylhexyl)amino]anthraquinone and 1-[(2-ethylhexyl)amino]-4-(methylamino)anthraquinone and 9,10-Anthracenedione, 1,4-bis(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-(methylamino)-4-(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-[(2-ethylhexyl)amino]-4-(pentylamino)-, branched and linear

EC Number:

911-360-1

Molecular formula:

variable structures

IUPAC Name:

Reaction mass of 1,4-bis(methylamino)anthraquinone and 1,4-bis[(2-ethylhexyl)amino]anthraquinone and 1-[(2-ethylhexyl)amino]-4-(methylamino)anthraquinone and 9,10-Anthracenedione, 1,4-bis(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-(methylamino)-4-(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-[(2-ethylhexyl)amino]-4-(pentylamino)-, branched and linear

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn Oil

Duration of treatment / exposure:

dosed daily for 14 days prior to mating and continuing throughout the mating period for at least 34 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

6 male animals were included into the OECD 422 study as a satellite group, serving as the positive control for the pig A portion of the study. These animals were exposed to N-Ehtyl-N-nitroso-urea (759-73-9) on test days 1-3 of the study at a dose level of 20 mg/kg bw/day via gavage in an acidic buffer solution (KH2PO4, Na2HPO4).

Examinations

Tissues and cell types examined:

red blood cells

Statistics:

The Pig-a assay data were analyzed by the following statistical methods as recommended in Gollapudi et al. (2014). A Bartlett’s test (alpha=0.01; Winer, 1971) was used to test for homogeneity of variance, the test was also sensitive to normality. Transformations of mutant frequencies (e.g., a log (10) transformation) were conducted to meet these assumptions when necessary. Since zeroes may have been observed occasionally, prior to the log-transformation, an offset of +0.1 (i.e., addition of 0.1 to each mutant cell frequency expressed as mutants × 10−6) may have been required. An analysis of variance (ANOVA) followed by pair-wise comparisons of mutant frequencies in treated groups to the vehicle control group was performed utilizing a one-sided (upper to detect an increase in mutant cells) Dunnett’s test at alpha=0.05 for mutant RBC and mutant RET and a 2-sided Dunnett’s test at alpha=0.05 for percent RET. If any of the pairwise tests were significant, evidence of dose-related increases were evaluated with a linear trend test. The positive control was compared to the vehicle control with an analysis of variance followed by a one-sided (upper) Dunnett’s test at alpha=0.05 for mutant RBC and mutant RET and a 2-sided Dunnett’s test at alpha=0.05 for percent RET.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

general toxixity - increased liver weights, and histopathology in the pancreas - effects decribed in Repeat dose tox section

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Two animals with pre-exposure frequency of CD59-negative reticulocytes (RET^CD59-) exceeding the reported background range of 0-5 × 10^-6(Gollapudiet al.,2014), (animal 2458 in the 300 mkd test material group and animal 2472 in the 1000 mg/kg/day test material group) were excluded from the post-exposurePig-aassay. Six male animals from eachC.I. Solvent Blue 98 (3Amine)treated group and the vehicle control group were selected for the post-exposurePig-aassay by the ascending order of animal ID among surviving animals with pre-exposure frequency of RET^CD59-not exceeding
5 × 10^-6. 

The frequencies(×10^-6)of RET^CD59-and RBC^CD59-in the vehicle control animals were within the reported background range (Gollapudiet al.,2014). The frequencies of RET^CD59-and RBC^CD59-in the positive control (ENU) group were significantly higher than those in the vehicle control group (Text Table 12). The normal frequencies of RET^CD59-and RBC^CD59-in the vehicle control animals and the significantly increased frequencies of RET^CD59-and RBC^CD59-in the positive control animals indicated acceptability of the test system for identifying a mutagen. There were no significant differences in the frequency of RET^CD59-or RBC^CD59-among the vehicle control group and theC.I. Solvent Blue 98 (3Amine)treated groups (Text Table 12). 

There were no significant differences in the percentage of reticulocytes (% RET) among the vehicle control group and theC.I. Solvent Blue 98 (3Amine)treated groups (Text Table 12); however, the gross pathology observation of blue discoloration of tissue observed at necropsy demonstrated that theC.I. Solvent Blue 98 (3Amine)and/or its metabolites were absorbed and systemically bioavailable after oral gavage (see adult gross pathology) . 

Therefore,C.I. Solvent Blue 98 (3Amine)was negative in thisin vivogene mutationPig-aassay under the experimental conditions used.

Applicant's summary and conclusion

Conclusions:

C.I. Solvent Blue 98 (3Amine) was negative in this in vivo gene mutation Pig-a assay under the experimental conditions used.