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Genetic toxicity in vitro

Description of key information

The analogue of the substance was not mutagenic in the Ames test, a mammalian cell gene mutation test (MLA) and did cause structural chromosome changes in a Chinese Hamster Ovary test.

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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
Study period:
12 December 2006 to 06 February 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without deficiencies
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 prepared in-house from the livers of male Sprague-Dawley rats
Test concentrations with justification for top dose:
Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate.

Experiment 1: 50, 150, 500, 1500 and 5000 ug/plate
Experiment 2: 50, 150, 500, 1500 and 5000 ug/plate
Vehicle / solvent:
None
Details on test system and experimental conditions:
The test material was immiscible in both sterile distilled water and dimethyl sulphoxide at
50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks
performed in-house. Acetone was therefore selected as the vehicle.
The test material was accurately weighed and approximate half-log dilutions prepared in acetone
by mixing on a vmtex mixer on the day of each experiment. Analysis for concentration,
homogeneity and stability of the test material formulations is not a requirement of the test
guidelines and was, therefore, not determined. Prior to use, the solvent was dried using molecular
sieves (sodium alumino-silicate) ie 2 tmn pellets with a nominal pore diameter of 4 x 10·4
microns.

S9 was prepared in-house on 12 November 2006 from the livers of male Sprague-Dawley rats
weighing - 250g. These had each orally received three consecutive daily doses of
phenobarbitone/~-naphthoflavone (80/l 00 mg per kg per day) prior to S9 preparation on Day 4.
Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect
mutagens used in the Ames Test. The S9 was stored at -196°C.

A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine or tryptophan supplemented, top
agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility
of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Top agar was prepared using 0.6% Difco Bacto agar (lot number 5279941 08/1 0) and 0.5%
sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution
added to each 100 ml of top agar. Vogel-Bonner Minimal agar plates were purchased from ILS
Limited (lot numbers 954961-02 05/11 & 961443-02 06/11).
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in
revettant frequency over the dose range tested and/or a reproducible increase at one or more
concentrations in at least one bacterial strain with or without metabolic activation. Biological
relevance of the results will be considered first, statistical methods, as reconunended by the
UKEMS can also be used as an aid to evaluation, however, statistical significance will not be
the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria
are not met.
Although most experiments will give clear positive or negative results, in some instances the data
generated will prohibit a definitive judgement about the test material activity. Results of this type
will be reported as equivocal.

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the
vehicle and untreated controls.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10e9 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for
each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic
exposure and the integrity of the S9-mix. The historical control ranges for 2004 and 2005 were used.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contrunination.
Statistics:
Kirkland D J (Ed) (1989) Statistical Evaluation of Mutagenicity Test Data. UKEMS Subcommittee
on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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

Alkylated Naphthalene is not mutagenic in the Ames test.
Executive summary:

The substance was evaluated in the Bacterial Reverse Mutation Assay.

The method conforms to the guidelines for bacterial mutagenicity testing published by the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", and Method B13/14 of Commission Directive 2000/32/EC.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA' were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver 89 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose rm1ge as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Results. The vehicle (acetone) control plates gave counts of revertant colonies within the nonnal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. A precipitate (oily in appearance) was observed at and above 1500 ug/plate, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Conclusion. Alkylated Naphthalene is not mutagenic in the Ames bacterial reverse mutation test.

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
Study period:
11 April 2007 to 07 June 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with no deficiencies
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Test concentrations with justification for top dose:
Experiment 1
4(20)-hour without S9: 0*, 156.25*, 312"5*, 625*, 1250,2500, 5000, MMC 0"1 *
4(20)-hour with S9: 0*, 78.13*, 156"25*, 312.5*, 625, 1250,2500, CP 5*
Experiment 2
24-hour without S9: 0*, 39, 78.1, 156.25, 312.5*, 468.75*, 625*, MMC 0.05*
4(20)-hour with S9: 0*, 39, 78.1, 156.25, 312.5*, 468.75*, 625*, CP 5*
* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide
Vehicle / solvent:
acetone
Details on test system and experimental conditions:

Experiment 1
i) 4-hour exposure to the test material without S9-mix followed by 20-hour culture in treatment free
media prior to cell harvest. The test material dose range was 156.25 to 5000 ug/ml.
ii) 4-hour exposure to the test material with S9-mix (2% final concentration) followed by 20-
hour culture in treatment-free media prior to cell harvest. The test material dose range was
78.13 to 2500 ug/ml.

Experiment 2
i) 24-hour continuous exposure to the test material without S9-mix prior to cell harvest. The
test material dose range was 39 to 625 ug/ml.
ii) 4-hour exposure to the test material with S9-mix (1% final concentration) followed by 20-
hour culture in treatment-free media prior to cell harvest. The test material dose range was
3 9 to 625 ug/ml.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 ug/ml) two hours before the
required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture
medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCI.
After approximately twelve minutes (including centrifugation), most of the hypotonic solution
was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl
cell suspension into fresh methanol/glacial acetic acid (3: 1 v/v). The fixative was changed at least
three times and the cells stored at 4 deg C for at least four hours to ensure complete fixation.
Evaluation criteria:
Mitotic Index
A total of 1000 cell nuclei were counted and the number of cells in metaphase recorded and
expressed as the mitotic index and as a percentage ofthe vehicle control value.
Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted,
where there were approximately 50% of cells with aberrations, slide evaluation was terminated at
50 cells. If the cell had 19-23 chromosomes, any gaps, breaks or rearrangements were noted
according to the simplified system of Savage (1976).
Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was
compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: CHO-WBL
Conclusions:
Interpretation of results: negative

Alkylated Naphthalene is considered to be non-clastogenic to Chinese Hamster Ovary cells in vitro.
Executive summary:

Alkylated Naphthalene was tested for structural chromosome aberrations in Chinese Hamster Ovary cells. The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 2000/32/EC.

Methods. Duplicate eultures of Chinese Hamster Ovary cells (CHO), treated with the test

material, were evaluated for chromosome aberrations at three dose levels, together with vehicle

and positive controls. Four treatment conditions were used for the study, i.e. in Experiment I,

4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2%

final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in

the absence of metabolic aetivation (S9) with a 20-hour expression period. In Experiment 2, the

4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration); while in

the absence of metabolic activation the exposure time was increased to 24 hours.

Results. All vehicle (solvent) controls had frequencies of cells with aberrations within the range

expected for CHO cells.

All the positive control materials induced statistically significant increases in the frequency of

cells with aberrations indicating the satisfactory performance of the test and of the activity of the

metabolising system.

The test material was non-toxic and did not induce any statistically significant increases in the

frequency of cells with aberrations, in either of two separate experiments, using a dose range that

was limited by the formation of a greasy oily precipitate.

Conclusion. The test material was considered to be non-clastogenic to Chinese Hamster Ovary

cells in vitro.

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
Study period:
Start : 30 October 2012 Completion : 03 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without deficiencies
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
0.01, 0.03, 0.1, 0.33, 1, 3.3, 10 and 33 μg/ml
Vehicle / solvent:
tetrahydrofuran
Details on test system and experimental conditions:
All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 – 100% (actual range 38 – 91%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 35.5 – 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 35.5 - 36.0°C), humidity (with a maximum of 29%) and CO2 percentage (with a maximum of 1%) that occurred were caused by opening and closing of the incubator door, the duration of these deviations did not exceed 4 hours. Based on laboratory historical data these deviations are considered not to affect the study integrity.

In order to select appropriate dose levels for mutagenicity testing, cytotoxicity data were obtained by treating 8 x 106 cells (106 cells/ml for 3 hours treatment) or 5 x 106 cells (1.25 x 105 cells/ml for 24 hours treatment) with a number of test substance concentrations increasing with approximately half log steps. The cell cultures for the 3 hours treatment were placed in sterile 30 ml centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 spm. The cell cultures for the 24 hours treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test substance was tested in the absence and presence of 8% (v/v) S9-fraction.

Since Alkylated Naphthalene was poorly soluble in aqueous solutions, the highest tested concentration was 100 μg/ml exposure medium.
Cell cultures were exposed to the test substance in exposure medium for 3 hours in the presence of S9-mix and for 3 and 24 hours in the absence of S9-mix. After exposure, the cells were separated from treatment solutions by 2 centrifugation steps (216 g, 8 min) each followed by removal of the supernatant. The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the final centrifugation step the cells were resuspended in R10 medium. The cells in the final suspension were counted with the coulter particle counter.
For determination of the cytotoxicity, the surviving cells of the 3 hours treatment were subcultured twice. After 24 hours of subculturing, the cells were counted (day 1) and subcultured again for another
24 hours, after which the cells were counted (day 2). The surviving cells of the 24 hours treatment were subcultured once. After 24 hours of subculturing, the cells were counted. If less than 1.25 x 105 cells/ml were counted no subculture was performed.
The suspension growth expressed as the reduction in cell growth after approximately 24 and
48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.
Evaluation criteria:
The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severe affected mutant cells have grown at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appeared to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.
The small colonies are morphological dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphological less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.
Statistics:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.
The growth rate over the two-day expression period for cultures treated with tetrahydrofuran was between 15 and 24 (3 hours treatment) and 43 and 55 (24 hours treatment).

Mutation frequencies in cultures treated with positive control chemicals were increased by 16- and
8.2-fold for MMS in the absence of S9-mix, and by 14- and 15-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.
In the absence of S9-mix, Alkylated Naphthalene did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.
In the presence of S9-mix, Alkylated Naphthalene did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications in the composition of the S9 concentration for metabolic activation.
Alkylated Naphthalene is not mutagenic in the TK mutation test system under the experimental conditions.
Remarks on result:
other: strain/cell type: L5178Y/TK+/--3.7.2C mouse lymphoma cells

 

See attached tables.

Conclusions:
Interpretation of results: negative

It is concluded that Alkylated Naphthalene is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of this study.
Executive summary:

Alkylated Naphthalene was evaluated for mutagenicity in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells (with independent repeat). The test investigated induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 18515 of Alkylated Naphthalene was a clear amber slightly viscous liquid. The test substance was dissolved in tetrahydrofuran. In the first experiment, Alkylated Naphthalene was tested up to concentrations of 33 μg/ml in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. In the second experiment, the test substance was again tested up to concentrations of 33 μg/ml, but in the absence and presence of 12% (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test substance precipitated in the culture medium at this dose level. This is the highest concentration recommended in the guidelines.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were increased 16- and 8.2 -fold for MMS in the absence of S9-mix, and 14- and 15-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, Alkylated Naphthalene did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, Alkylated Naphthalene did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is concluded that Alkylated Naphthalene is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of this study.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The structural analogue was not genotoxic in the required testing (three different in-vitro studies). Based on these results, the substance is not classified for mutagenic effects according to CLP (Regulation EC No 1272/2008).