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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
: only four strains tested
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Aroclor induced rat liver (Litton Bionetics Inc.)
Test concentrations with justification for top dose:
0, 0.1, 0.3, 1.0, 3.0, and 10.0 µl/plate (0.092 - 9.2 mg/plate)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: insolubility of test substance in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S-9: Sodium azide (TA100, TA1537), 5-Nitrofluorene (TA98), 9-Aminoacridine (TA1537); with S-9: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days
- Expression time (cells in growth medium): 3 days at 37° C

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; toxicity of the test substance was evaluated in a separate test by incubating tester strain TA100 with 10 graduate concentrations of test substance (0 - 200 µL/plate, spacing factor 2).
Evaluation criteria:
- The solvent/vehicle control has to show an adequate number of revertants.
- A dose-response relationship must be demonstrated at least for one tester strain.
- The maximum number of revertants must be at least double of spontaneous revertants.
- The result must be reproducible.
Statistics:
no data
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
no toxicity to TA 100 up to 200 µl/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance proved to be insoluble at 12.5 µl and higher, i.e. above recommended maximum test concentration of 5 mg/pl.


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The test substance did not induce mutant colonies above background in any strain at any concentration tested.

Conclusions:
Interpretation of results (migrated information):
negative

In a bacterial reverse mutation assay using four strains of S. typhimurium, there was no evidence of an increase in revertant colonies above background in any strain and at any concentration tested with and without metabolic activation. Diisopropylnaphthalene proved to be negative in the Ames test under the test conditions used.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: duplicate plating only
Principles of method if other than guideline:
Reverse mutation assay according to Ames; no method or guideline stated
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his, trp
Species / strain / cell type:
other: S. typh. TA 98, TA100, TA1535, TA1537, TA1538, E. coli WP2UVrA
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from livers of phenobarbital and benzoflavone induced SD rats (Oriental Yeast Corp.)
Test concentrations with justification for top dose:
0, 10, 50, 100, 500, 1000, and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S-9: 2-nitrofluorene (TA1538, WP2UVrA), 9-aminoacridine (TA98), N-ethyl-N'-nitro-N-nitrosoguanidine (TA100, TA1535, WP2UVrA); with S-9: 2-aminoanthracene (TA98, TA100, TA1535, TA1537, TA1538, WP2UVrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: No data

DURATION
- Exposure duration: no data
- Expression time (cells in growth medium): no data

DETERMINATION OF CYTOTOXICITY
- Method: no data
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537, TA1538, E. coli WP2UVrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
no toxicity observed at the highest concentration tested (5000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The test substance did not induce mutant colonies above background in any strain at any concentration tested.

Conclusions:
Interpretation of results (migrated information):
negative

In a bacterial reverse mutation assay using five strains of S. typhimurium and one strain of E. coli, there was no evidence of an increase in revertant colonies above background in any strain and at any concentration tested with and without metabolic activation. Diisopropylnaphthalene proved to be negative in this bacterial reverse mutation test under the test conditions used.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
no second test
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Hams F12 (from Imperial) supplemented with 5% foetal calf serum (Gibco)
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Aroclor 1254 induced livers of male SD rats
Test concentrations with justification for top dose:
Pre-test to determine 50% reduction in the mitotic index
1.9, 3.8, 7.6, 15.2, 30.4, 60.8, and 121.5 µg/mL (highest concentration soluble in test medium)
Main experiment
without S-9 mix: 0.95, 4.75, and 9.5 µg/mL
with S-9 mix: 6, 30, 45, and 60 µg/mL (preliminary test 7.5, 37.5, 75 µg/ml; highest dose was too toxic.)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S-9: mitomycin C (Sigma London Chemical Company Ltd.) at 0.4 µg/mL; with S-9: cyclophosphamide (Sigma) at 20 µg/mL
Details on test system and experimental conditions:
Test was performed with duplicate cultures for positive controls and test substance and quadruplicate cultures for solvent controls

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 20 h in cultures without S-9 mix; 4 h in cultures with S-9 mix
- Expression time (cells in growth medium): 21 h
- Fixation time (start of exposure up to fixation or harvest of cells): 21 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.25 µg/mL); inhibitor was added 2 h before the end of the 21 h incubation period
STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF CELLS EVALUATED: 100 metaphases per culture (200 or 400 total)

DETERMINATION OF CYTOTOXICITY
- Method: reduction of mitotic index (preliminary independent experiment)

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: ≥15 µg/ml (without S-9); ≥ 60 µg/ml (with S-9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES
The mitotic index was determined for 7 graduate test substance concentrations (without and with S-9 mix each). From the data an EC50 was estimated (reduction of mitotic index by 50%).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Test sustance concentration of ≥ 15 µg/mL and ≥ 60 µg/mL resulted in a reduction of the mitotic index by 50% and more (without and with S-9 mix, respectively).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The first main test (with S-9 mix, maximum concentration 75 µg/mL) was dismissed and not continued for chromosomal analysis. The reason was a reduction in the mitotic index at 75 µg/mL, higher than expected from pre-testing. Therefore, a second main trial was initiated with a somewhat reduced upper concentration (60 µg/mL). Cytotoxic screening was relinquished for the second main test because cytotoxicity had been demonstrated in prior screening.

 

No significant increase in chromosomal damage was seen in cultures treated with diisopropylnaphthalene at any dose level in either the presence or absence of metabolic activation.

 

Positive controls caused large, statistically highly significant increases in chromosomal damage, thus demonstrating the sensitivity of the test system and the efficiency of the S-9 mix.

 

In this in-vitro cytogenetic test diisopropylnaphthalene did not show any evidence of clastogenic activity.

Conclusions:
Interpretation of results (migrated information):
negative

In an in-vitro mammalian chromosome aberration test, diisopropylnaphthalene did not demonstrate any increase in chromosomal damage at any dose level either without or with metabolic activation. There was no evidence of any clastogenic activity under the conditions of the test used.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase locus TK +/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 (Sigma Cell Culture Ltd.) supplemented with various ingredients resulting in media R0p, R10p, R20p, and R30p
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
Without S-9 mix
Test 1: 5, 10, 15, 20, 25, 27, 29, 30, 40 µg/L
Test 2: 1, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25 µg/L
With S-9 mix
Test 1: 1, 5, 10, 15, 20, 25, 37.5, 50, 60 µg/L
Test 2: 1, 5, 7.5, 10, 15, 20, 25, 37.5, 50 µg/L
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: suitable solvent, already used in other tests
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S-9 mix: methyl methanesulfonate (test 1 10 µg/mL; test 2 5 µg/mL; with S-9 mix: 20-methylcholanthrene (2.5 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without S-9 mix: 3 and 24 h (test 1 and test 2, respectively); with S-9 mix: 3 h (test 1 and test 2)
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10 - 14 d

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (measured after incubation of cells in medium for 7 d)
Evaluation criteria:
Criteria for a positive response:
- Demonstration of a statistically significant increase in mutant frequency following treatment with substance.
- Evidence of a dose-response relationship over at least two dose levels, in any increases in mutant frequency.
- Demonstration of reproducibility in any increases in mutant frequency.
- Any observed increases in mutant frequency should lie outside the historical control range with a corresponding mean Day0 relative survival (RS) value of not less than 10%.
Statistics:
Statistical significance of data was analyzed by the methods described by Robinson et al (1989, in: Kirkland DJ (Ed.), UKEMS Sub-committee on Guidelines for Mutagenicity Testing. Report. Part III. Statistical Evaluation of Mutagenicity Test Data, p. 102, Cambridge University Press, Cambridge)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: >=25 µg/ml (+/-S-9): <= 20% Rel. Surv.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mean survival (day 0) in non-selective medium of the solvent control in the absence of S-9 mix was 74 and 68 % (Tab. 1 + 4), and in the presence of S-9 mix 72 - 88 % (Tab. 7 + 10).

 

In test 1 without S-9 mix (3-h treatment), a statistically significant increase in mutant frequency [MF] outside the historical control range was observed at the highest dose (40 µg/ml). Mean day-0 rel. survival was 12% and day-2 cloning efficiency CE was 19 % (Tab. 1 - 3). This effect can be explained by the corresponding decrease in day-2 CE in non-selective medium (MF = CE selective/CE non-selective). No substantive increase was seen in Test 2 under equivalent conditions, except 24-h exposure.

 

In test 1 with S-9 mix (3-h treatment), a statistically significant increase in MF outside the historical control range was observed at 20 µg/ml, but not at 50 µg/ml (survival 14 %) (Tab. 7 and 9). But this effect was not reproduced in the 2nd test.

 

Colony sizing demonstrated no difference in the ratios small-to-large colonies from the solvent control, while significant higher yields of small colonies were found in the pos. controls.

 

There was no reproducible increase in mutant frequency and no evidence of a dose relationship over at least two dose levels. Based on the results of this test, it can be concluded that DIPN did not demonstrate mutagenic potential in this in-vitro gene mutation assay.

Conclusions:
Interpretation of results (migrated information):
negative

The mutagenic potential of diisopropylnaphthalene (DIPN) was tested in an in-vitro mammalian cell gene mutation assay using mouse lymphoma L5178Y cells. There was no evidence of increases in mutant frequency according to the criteria of the test for all test substance concentrations tested without and with metabolic activation. It can be concluded that DIPN does not demonstrate mutagenic potential under the test conditions of this assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Limit-test: Only one dose tested (highest tolerated dose)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Firma Winkelmann, Versuchstierzucht, Borchen, Germany
- Age at study initiation: 3 months
- Weight at study initiation: males 30 - 37 g, females 27 - 33 g
- Housing: max. 5 per cage in Makrolon cages, type III , with softwood bedding (dedusted and sterilized)
- Diet (e.g. ad libitum): Ssniff-R standard rat diet (Ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): tap water, suited for human use, from Makrolon drinking bottles, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 50 - 80
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: none
Details on exposure:
Based on the results of a dose-range finding test, a single dose was administered intraperitoneally corresponding to about the maximal tolerated dosage.
Duration of treatment / exposure:
Bone marrow of positive and negative controls and one test group was collected 24 hours after administration of test substance. Collection of bone marrow of test group 2 and 3 was after 48 and 72 hours (page 10).
Frequency of treatment:
single i.p. administration
Remarks:
Doses / Concentrations:
2 mL/kg (ca. 1.92 g/kg)
Basis:
other: nominal injected dose
No. of animals per sex per dose:
5 animals per sex per dose (negative/positive control, three time points)
Control animals:
other: control animals received intraperitoneally 2 mL physiological saline/kg bw
Positive control(s):
- cyclophosphamide (Endoxan)
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/kg bw in a volume of 2 mL/kg (distilled water)
Tissues and cell types examined:
bone marrow of femur, polychromatic erythrocytes with and without micronuclei, normochromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Results of a dose-range finding test (maximum tolerated dosage)

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Controls and one treatment group 24 h, second and third treatment group 48 and 72 h respectively

DETAILS OF SLIDE PREPARATION:
Femura of sacrificed animals were removed and bone marrow was suspended in fetal calf serum. After centrifugation, one drop of each single residue was smeared on a slide by means of a second slide. These preparations were dried, fixed in absolute (99%) methanol for 5 min. After air drying, the slides were stained with May-Grünwald and Giemsa solution.
From each animal, two preparation were made.
Prior to analysis, all the slides were randomised and coded (blind evaluation).

METHOD OF ANALYSIS:
A total of 1000 polychromatic erythrocytes was scored from each slide, and the number of micronucleated cells in each sample was recorded.
The ratio of polychromatic erythrocytes to normochromatic erythrocytes was calculated on the basis of 1000 cells.
Evaluation criteria:
Based on laboratory historical data, an incidence of up to 0.8% of micronucleated polychromatic erythrocytes is considered to be within normal limits.
Statistics:
Test data were subjected to a one factorial analysis of variance.
Group mean values (negative controls / treated groups) were compared by the method of Scheffé. The positive control group was compared to the negative control group employing the U-Test of Man and Whitney additionally.
Sex:
male/female
Genotoxicity:
negative
Remarks:
no increase in micronucleated polychromatic erythrocytes
Toxicity:
yes
Remarks:
toxic effect on erythropoiesis
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- no data on dose-finding study but the dose selected for the definite study

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction of micronuclei for all test groups (different exposure times)
- Ratio of PCE/NCE (for Micronucleus assay): negative controls: 0.991± 0.313; positive controls: 0.758 ± 0.230; TS 24 h: 1.123 ± 0.316; TS 48h: 0.402 ± 0.095*; 0.444 ± 0.133*
* p > 0.001

Diisopropylnaphthalene did not produce any statistically significant increase in micronucleated polychromatic erythrocytes.

 

A decline of polychromatic erythrocytes associated with a concurrent increase in normochromatic erythrocytes indicated some toxic effects of diisopropylnaphthalene on erythropoiesis.

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the micronucleus test performed, diisopropylnaphthalene did not lead to any significant increase in the frequency of micronucleated polychromatic erythrocytes compared to control values at any time point of the group treated with 2 mL test substance/kg bw.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

Four valid tests are available to assess the genotoxic potential of DIPN. Two bacterial reverse mutation assays, one mammalian cell gene mutation assay and one in-vitro mammalian chromosome aberration test were performed according or similar to the respective OECD TG.

Leimbeck 1986 (Ames test)

In a bacterial reverse mutation assay using four strains of S. typhimurium, there was no evidence of an increase in revertant colonies above background in any strain and at any concentration tested with and without metabolic activation. DIPN proved to be negative in the Ames test under the test conditions used.

Kureha 1982 (bacterial reverse mutation assay)

In a bacterial reverse mutation assay using five strains of S. typhimurium and one strain of E. coli, there was no evidence of an increase in revertant colonies above background in any strain and at any concentration tested with and without metabolic activation. Diisopropylnaphthalene proved to be negative in this bacterial reverse mutation test under the test conditions used.

Huntington 1999 (mammalian cell gene mutation (TK) assay)

The mutagenic potential of DIPN was tested in an in vitro mammalian cell gene mutation assay using mouse lymphoma L5178Y cells. There was no evidence of increases in mutant frequency according to the criteria of the test for all test substance concentrations tested without and with metabolic activation. It can be concluded that DIPN does not demonstrate mutagenic potential under the test conditions of this assay.

Huntington 1988 (mammalian chromosomal aberration test)

In an in vitro mammalian chromosome aberration test, DIPN did not demonstrate any increase in chromosomal damage at any dose level either without or with metabolic activation. There was no evidence of any clastogenic activity under the conditions of the test used.

Genetic toxicity in vivo

The genotoxic potential of DIPN in vivo was investigated in one valid test.

IBR 1986 (micronucleus assay)

Under the conditions of the micronucleus test performed, DIPN administered at 2 mL/kg bw did not lead to any significant increase in the frequency of micronucleated polychromatic erythrocytes compared to control values at any of the three time point examined.


Short description of key information:
For bis(isopropyl)naphthalene (DIPN), five different tests on genetic toxicity (bacterial and mammalian cell mutation, chromosomal aberration, micronucleus test) have been performed. In all tests, there was no evidence of any genotoxic potential.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification required (see discussion above).