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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ditolyl ether was investigated in the Salmonella/microsome test (Ames test) in 1982 using 4 strains of S. typhimurium and in 2015 using 5 strains of S typhimurium. In both studies, no evidence of mutagenic activity of ditolyl ether was seen with and without metabolic activation. Additional, ditolyl ether was evaluated as inactive in the in vitro rat primary hepatocyte UDS assay.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
The S9 fraction was prepared from the livers of Aroclor 1254-induced male Sprague Dawley rats
Test concentrations with justification for top dose:
-----plate incorporation methodology,
S. typh TA1535, TA100, TA1537, TA98, TA102 with and without S9-mix
0, 50, 160, 500, 1600, 5000 µg/plate
--additionally S. typh TA100, TA1537 with and without S9-mix
0, 1.6 , 5, 16 µg/plate
-----preincubation methodology
S. typh TA 1535 with and without S9-mix
0, 1.6, 5, 16, 50, 160, 500, 1600 µg/plate
S. typh TA100, TA1537 with and without S9-mix
0, 1.6, 5, 16, 50, 160, 500 µg/plate
S typh TA98 with and without S9-mix
0, 16, 50, 160, 500, 1600 µg/plate
S typh TA102 with and without S9-mix
0, 50, 160, 500, 1600. 5000 µg/plate
-- additionally S typh TA1535, TA98 without S9-mix
0, 1.6, 5 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
cumene hydroperoxide
mitomycin C
other: 4-Nitro-1,2-phenylenediamine, 2-aminoanthracene
Details on test system and experimental conditions:
According to OECD TG 471 ( plate incorporation methodology and pre-incubation methodology
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100, TA1537 and TA98 this increase should be about twice that of solvent controls. For TA102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
no data
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: plate incorporation test strain-specific at 160 µg/plate and above bacteriotoxic, precipitation at 5000 µg/plate: pre-incubation: stain specific bacteriotoxic at 16 µg/plate and above; precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results: negative
Executive summary:

The principal aim of this study was the investigation of the test item for point mutagenic effects using a plate incorporation test and a repeat test with preincubation modification. For this purpose, the test item, dissolved in DMSO, was administered in initially doses of up to and including 5000 μg per plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA1535, TA100, TA1537, TA98 and TA102, according to the OECD guideline 471 under GLP conditions. In the plate incorporation test strain-specific bacteriotoxic were seen at 160 μg per plate.and above and in the pre-incubation test at 16 µg/plate and above, In both methodologies substance precipitation occurred at 5000 μg per plate.

Under the experimental conditions reported (direct plate incorporation procedure and preincubation modification) the test item did not induce gene mutations by base-pair changes or frame-shifts in the genome of the S. typhimurium strains used, when tested up to a maximum recommended dose of 5000 μg/plate in the absence and presence of S9 mix.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: scientific acceptable and well documented
Principles of method if other than guideline:
Ditolyl ether was evaluated for mutagenic effects in the in vitro rat primary hepatocyte Unscheduled DNA Synthesis (UDS) assay. Freshly prepared rat hepatocytes were exposed to 6 concentrations of ditolyl ether ranging from 5 µg/ml to 100 µg/ml in the presence of 10 µCi/ml ³HTdr (18 Ci/mmole). UDS was measured by counting nuclear grains.
GLP compliance:
yes
Type of assay:
other: DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Species / strain / cell type:
other: primary rat-hepatocytes
Metabolic activation:
with
Metabolic activation system:
Rat hepatocytes are self metabolically active cells.
Test concentrations with justification for top dose:
5, 10, 20, 40, 80, 100 µg/ml (the highest dose group was not used for evaluation due to cytotoxicity of the test article).
Vehicle / solvent:
Ditoylether was dissolved in DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
Unscheduled DNA synthesis.
Evaluation criteria:
- the viability of the hepatocytes collected by this process normally exceeds 70%. although values between 50% and 70% viability can also been acceptable. trials below 50% are considered unacceptable
- the viability of the monolayer cell cultures used for the UDS assay must be 80% or greater. normally, the viability of attached cells is about 90%
- the number of viable cells in the negative (vehicle) control cultures should remain reasonably stable over the experimental time period because rapidly declining (dying) cultures may not respond in a representative manner to the test article treatments. therefore, the number of viable cells in the negative control cultures must be 60% or greater after 16-24 hours.
- for each of the 50 cells on each slide, the number of nuclear grains is scored, as well as numbers of three cytoplasmatic grain counts from nuclear-sized areas adjacent to each nucleus
- for the conditions described here, greater than or equal to 5 net nuclear grains is chosen as a conservative estimate as to whether a particular cell is responding or is in "repair"
- the avarage net nuclear grain counts (NG) in the negative control cultures should range between -8 to +1. no more than 10% of the cells should be in repair
- a minimum of 4 to 5 dose levels will be analyzed for NG
- only cells viable at the time of fixation wand with nuclei evenly coated with emulsion will be scored
- S-phase cells having dense NG will be excluded
Statistics:
An evaluation will be made of the percentage of cells in repair per dose groups compared to the negative control using a one-sided 2x2-chi²-test corrected for continuity (Yater, 1934; Armitage, 1971). to assess the statistical significance of a result, the square root of the test statistic is compared to the upper 95% quantile of the normal standard distribution.
Species / strain:
hepatocytes:
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100 µg/ml was cytotoxic
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Ditolyl ether was evaluated as inactive in the in vitro rat primary hepatocyte UDS assay.

Ditolyl ether was evaluated as inactive in the in vitro rat primary hepatocyte UDS assay.

Conclusions:
Interpretation of results: negative
Executive summary:

Method: ditolyl ether was evaluated for mutagenic effects in the in vitro rat primary hepatocyte Unscheduled DNA Synthesis (UDS) assay. Freshly prepared rat hepatocytes were exposed to 6 concentrations of ditolyl ether ranging from 5 µg/ml to 100 µg/ml in the presence of 10 µCi/ml ³HTdr (18 Ci/mmole). UDS was measured by counting nuclear grains.

Result: negative, ditolyl ether was evaluated as inactive in the in vitro rat primary hepatocyte UDS assay.

Reference: Lehn (Bayer AG), 1990

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in-vivo micronucleus assay according OECD TG 475 no indication for a mutagenic effect was found.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: scientific acceptable and well documented
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Principles of method if other than guideline:
10 mice (5 male + 5 female) received a single oral application of 0.1 ml/kg bw of the test substance. Animals were killed after 24, 48 and 72 hours and bone marrow was reprocessed. 1000 polychromatic erytrocytes were evaluated and the number of normochromatic erythrocytes counted. Additionally micronucleated cells per 1000 normochromatic and polychromatic erythrocytes were determined.
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
Male + female NMRI-mice, SPF, ca. 8 - 12 weeks old, weight 24- 34 g, husbandry: conventional in makrolon cages typ II, maximal 5 animals per cage, separated by sex and test groups.
Route of administration:
oral: gavage
Vehicle:
Test substance was applicated in peanut oil, positive controls were applied in deionized water, negative controls reveived peanut oil.
Details on exposure:
No further data.
Duration of treatment / exposure:
24, 48 or 72 hours.
Frequency of treatment:
single application
Post exposure period:
animals were sacridied after 24, 48 or 72 hours
Remarks:
Doses / Concentrations:
103.5 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals (5 males + 5 females)
Control animals:
yes, concurrent vehicle
Positive control(s):
10 animals received 29.5 mg endoxan/kg bw
Tissues and cell types examined:
1000 polychromatic erytrocytes were evaluated and the number of normochromatic erythrocytes counted. Additionally micronucleated cells per 1000 normochromatic and polychromatic erythrocytes were determined
Statistics:
According Wilcoxon.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Treated animals revealed no signs of poisoning and survived until sacrifice.

No signs of a mutagenic effect after a single oral application of 0.1 ml/kg of the test substance.

Conclusions:
Interpretation of results: negative
Executive summary:

Method: 10 mice (5 male + 5 female) received a single oral application of 0.1 ml/kg bw of the test substance. animals were killed after 24, 48 and 72 hours and bone marrow was reprocessed. 1000 polychromatic erytrocytes were evaluated and the number of normochromatic erythrocytes counted. Additionally micronucleated cells per 1000 normochromatic and polychromatic erythrocytes were determined.

Result: no indication for a mutagenic effect was found. treated animals revealed no signs of poisoning and survived until sacrifice.

Reference: Herbold (Bayer AG), 1984.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

According to the OECD guideline 471 under GLP conditions ditolyl ether, dissolved in DMSO was administered in initially doses of up to and including 5000μg per plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA1535, TA100, TA1537, TA98 and TA102 using the plate incorporation and the pre-incubation methodology (Meyer 2015). Under the experimental conditions the test item did not induce gene mutations by base-pair changes or frame-shifts in the genome of the S. typhimurium strains used, when tested up to a maximum recommended dose of 5000μg/plate in the absence and presence of S9 mix. Thus this actual test confirms the result of the Ames test from 1982 with only 4 Salmonella typhimurium strains in test. Taking into account the negative result of the in vivo micronucleus test in addition with the negative in vitro UDS assay in primary hepatocytes ditolyl ether showed no evidence of mutagenic activity.


Justification for classification or non-classification

All available genetic toxicity tests (in-vitro as well as in-vivo) were negative - therefore a non-classification of ditolyl ether is justified.