Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro genotoxicity studies are available: an Ames test, an in vitro HPRT test, a chromosome aberration test in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
December 1989 - January 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP, however, study was carried out according to the previous version of the guideline. Only 4 Salmonella strains were used.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
Only four Salmonella strains were used, but not one of the strains having AT base pairs at the primary reversion site (such as TA 102 or E. coli WP2 strains).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 mix.
Test concentrations with justification for top dose:
Concentrations in the RF test: 5, 50, 500 and 5000 µg/plate
Concentrations in the main test (with and without metabolic activation): 50, 150, 500, 1500, 5000 µg/plate
Vehicle / solvent:
Solvent: Dimethylsulphoxide
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine, N-ethyl-N'-nitro-N-nitrosoguanidine, 2-nitrofluorene
Remarks:
without S-9 mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S-9 mix
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:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance was negative in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 with and without metabolic activation.
Conclusions:
Interpretation of results: negative

When tested up to dose levels of 5000 µg/plate, Perkalink 900 was not mutagenic in this bacterial test system.
Executive summary:

The test substance (solved in DMSO) was tested in an Ames Test performed according to OECD TG 471 in 1990 with the 4 Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. Doses of up to and including 5000 µg/plate did not show mutagenic effects in this bacterial system with and without external metabolization.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April-May 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
The effects on chromosomal structure of exposure to N,N -Bis(citraconimido)-m Xylylene (1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene) were investigated in cultured human lymphocytes. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activating System (S-9 mix): without S-9 mix cells were exposed for 24 hours, with S-9 mix e exposure was limited to three hours and cells were harvested 21 and 45 hours later. Treatments were established by the addition of test solutions (in dimethyl sulphoxide: DMSO) to 48-hour cultures established from whole, human blood. Cell division was arrested by the addition of the spindle poison, Colcemid (to a final concentration of 0.4 µg/ml), three hours before the cells were harvested; slides were then prepared for microscopic analysis.
Mitotic indices were calculated for each culture: these were based on the number of metaphases observed per 1000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture, and the frequencies of cells with one or more aberrations were calculated both including and excluding gap type aberrations.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: primary culture obtained from a human volunteer
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
Concentration in the RF test (with and without S9-mix), 1, 3, 10, 33 and 100 µg/mL
Concentrations in the main test (with metabolic activation): 2.5, 5, 10, 15 µg/ml
Concentration range in the main test (without metabolic activation): 1, 2, 3 µg/ml
Vehicle / solvent:
Dimethyl sulphoxide
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: chlorambucil
Remarks:
without S9-mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 3 hours , harvest 21 and 45 hours later
Exposure period (without metabolic activation): 24 hours

Fixation time:
6th Amendment fixation time : 3
Evaluation criteria:
Criteria for a positive response
Statistically significant increases in aberrant cell frequency, compared to concurrent solvent control values, will be considered indicative of clastogenic activity. The significant elevation of aberrant cell frequency at only one concentration will be considered on a case-by-case basis, but in order to designate the material as clastogenic the following criteria will be met:
- the increase is reproducible between replicate cultures
- the mean number of aberrant metaphases is significantly higher than the historical control value.
Statistics:
The Fisher Exact Probability test is a useful technique for analysing data when comparing two independent samples. It is used when the observed events all fall into one or other of two mutually exclusive classes. The test determines whether the two groups differ in the proportions with which they fall into the two classifications.
The difference between two groups is considered statistically significant if the p-value obtained is less than 0.05.
The frequency of aberrant metaphases for each treatment group was compared with the corresponding solvent control group value using
a one-tailed test.
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(>= 10 µg/ml)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 1 µg/ml)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Both in the presence and in the absence of metabolic activation an increase in aberrant cell frequencies was observed.
Conclusions:
Interpretation of results:
positive with metabolic activation
positive without metabolic activation

Under the conditions of the test, Perkalink showed clastogenic activity in both the presence and absence of metabolic activation.
Executive summary:

The effects the test substance (solved in DMSO) on chromosomal structure was investigated in cultured human lymphocytes. Tests were conducted with inclusion of rat liver S9 mix for 3 hours (harvest 21 and 45 hours later, test concentrations 5, 10, and 15 µg/ml) and without for 24 hours (test concentrations 1, 2, and 3 µg/ml). Dose related toxicity was seen as reduction in mitotic index in the cultures without S9 mix starting at 1 µg test substance/ml and with S9 mix starting at 2.5 µg/ml.

In the absence of S9 mix dose-related and statistically significant increases in mean aberrant cell frequencies were apparent at 3 µg/ml. In the presence of S9 mix high significance was seen at 15 µg/ml in the 24 hour cultures whereas no statistically enhanced clastogenicity was seen in the 48 hour cultures at this dose.

It is concluded that the test substance under the conditions of the test showed clear evidence of clastogenic activity in both the presence and absence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The study was performed to investigate the potential of 1,3-Bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The study was performed in two independent experiments, using general identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
The first experiment with and without metabolic activation was not analysable due to bacterial contamination of the cultures. Therefore, the experiment was repeated under identical experimental conditions. The data of the repeat experiment are included as experiment I.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared of the liver of phenobarbital/ß-naphthoflavone induced rats
Test concentrations with justification for top dose:
The highest applied concentration in the pre-tests on toxicity (2000 μg/ml active ingredient) was chosen with regard to the solubility properties of the test item in an appropriate solvent with respect to the current OECD Guideline 476. The concentration range of the main experiments was limited by cytotoxic effects. DMSO was used as solvent.
The dose range of the first main experiment was set according to data generated in the second pre-experiment. The dose range of the second experiment was set based on data generated in the pre-experiment (24 hour treatment without metabolic activation) and in the first experiment (4 hour treatment with metabolic activation). The individual concentrations were spaced by a factor of 2.0. Narrower spacing was used at higher concentrations to cover the toxic range more closely.
Based on the results of the pre-experiment the following concentrations were selected for the main experiments:

exposure S9
period mix
concentrations in µg/mL
Experiment I
4 hours - 0.031 0.063 0.13 0.19 0.25
4 hours + 2.0 3.0 4.0 6.0 8.0
Experiment II
24 hours - 0.031 0.063 0.13 0.19 0.25
4 hours + 2.0 4.0 6.0 8.0 10.0


Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
ethylmethane sulfonate used as positive control in cultures without S9-mix (150 µg/mL); dimethylbenzanthracene used as positive control in cultures with S9-mix (1.1 µg/mL).
Details on test system and experimental conditions:
Influence of test item at the maximum concentration on pH and osmolality was assessed.
Three or four day after treatment 1.5x10 E6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Fowowing the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3-5x10 E5 cells each in medium containing 6-Thioguanine (11 µg/ml). Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
Stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as folIows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the laboratory's historical control range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No substantial and reproducible dose dependent increase of the mutation frequency was observed. Under the experimental conditions the test item did not induce gene mutations at the HPRT locus in V79 cells.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls.

The threshold of three times the mutation frequency of the corresponding solvent control was reached in the first culture of the first experiment at 0.063 μg/mL active ingredient without metabolic activation. This effect however, was judged as biologically irrelevant as there was no dose dependent increase of the mutation frequency as indicated by a lacking statistical significance and the mutation frequency remained within the historical solvent control range.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in the first culture of the second experiment without metabolic activation. This trend however, was judged as irrelevant as it was not reproduced in the parallel culture and the mutation frequency remained within the historical range of solvent controls. Another trend was noted in the second culture of the second experiment with metabolic activation. This trend was irrelevant as it actually was reciprocal, going down versus increasing concentrations.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% occurred in experiment I at 0.031 μg/mL active ingredient and above in the absence of metabolic activation and at 8.0 μg/mL active ingredient in the presence of metabolic activation (4 hours treatment). In the second experiment cytotoxic effects as described above were noted at 0.063 μg/mL active ingredient and above in the absence of metabolic activation (24 hours treatment), and at 8.0 and 10.0 μg/mL active ingredient in the presence of metabolic activation (4 hours treatment). The recommended cytotoxic range of approximately 10-20% relative cloning efficiency 1 or relative cell density was covered with and without metabolic activation.

Conclusions:
Interpretation of results: negative
Executive summary:

The study was performed to investigate the potential of 1,3-Bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The highest applied concentration in the pre-tests on toxicity (2000 μg/ml active ingredient) was chosen with regard to the solubility properties of the test item in an appropriate solvent with respect to the current OECD Guideline 476. The concentration range of the main experiments was limited by cytotoxic effects. DMSO was used as solvent.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, 1,3-Bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two in vivo genotoxicity studies are available: a micronucleus test in vivo and a DNA repair test (UDS) in vivo.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June-July 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
The effect of 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene on chromosome structure in bone marrow cells was investigated following acute oral administration to mice. Chromosome damage was measured indirectly by counting micronuclei.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
corn oil
Duration of treatment / exposure:
single application
Frequency of treatment:
single application
Post exposure period:
24h after treatment for all treatment groups and additionally 48 and 72 h after treatment for 2500 and 3000 mg/kg bw groups
Dose / conc.:
0 other: mg/kg
Remarks:
vehicle control; Sacrifice time: 24, 48, 72 hours
Dose / conc.:
750 other: mg/kg
Remarks:
Sacrifice time: 24 hours
Dose / conc.:
1 500 other: mg/kg
Remarks:
Sacrifice time: 24 hours
Dose / conc.:
2 500 other: mg/kg
Remarks:
Sacrifice time: 24, 48, 72 hours
Dose / conc.:
3 000 other: mg/kg
Remarks:
Sacrifice time: 24, 48, 72 hours
No. of animals per sex per dose:
Range finding: 750, 1500, 3000 and 5000 mg/kg bw

Main study: 5 mice/sex/group
0 mg/kg (vehicle control); Sacrifice time: 24, 48, 72 hours
750 mg/kg; Sacrifice time: 24 hours
1500 mg/kg; Sacrifice time: 24 hours
2500 mg/kg; Sacrifice time: 24, 48, 72 hours
3000 mg/kg; Sacrifice time: 24, 48, 72 hours
Control animals:
yes, concurrent vehicle
Positive control(s):
30 mg/kg chlorambucil
Tissues and cell types examined:
Bone marrow smears were prepared and stained for examination; 2000 erythrocytes per animals were analysed for the presence of micronuclei; the ratio of polychromatic versus normochromatic cells was calculated.
Evaluation criteria:
The test material will be designated an in vivo clastogen if a statistically and biologically significant increase in micronucleated polychromatic cells, compared to vehicle contro values, is seen in at least one treatment group, particularly if supported by evidence of a dose-related response.
Statistics:
Mann-Whitney U procedure, two-tailed test
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
see below
Vehicle controls validity:
valid
Positive controls validity:
valid

One male animal treated with BCI-MX at 3000 mg/kg was killed in extremis approximately 2 hours after dosing as a result of hunched posture, piloerection, inactivity and slow respiration. Another male animal dosed with BCI-MX at 2500 mg/kg was found dead approximately 4 hours after dosing, having previously showed signs of hunched posture and inactivity. All other animals survived to scheduled termination, although some animals dosed at 1500, 2500 and 3000 mg/kg showed intermittent signs throughout the study, inclUding hunched posture, piloerection, ungroomed fur at the base of the tail, reduced activity, and redness and swelling in the anal region.

There was some evidence of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, on slides prepared from animals dosed with BCI-MX at 3000 mg/kg and sacrificed 48 or 72 hours later. Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of BCI-MX were similar to those in concurrent controls. This lack of treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis. Statistically significant increases over controls were, however, seen in positive control group animals given chlorambucil at 30 mg/kg (p<0.01). It is concluded that, under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of BCI-MX. The test procedure was highly sensitive to the chromosome-damaging action of chlorambucil.

Conclusions:
Interpretation of results: negative
Executive summary:

In an in vivo mouse bone marrow micronucleus test performed according to OECD 474 (1983) male and female mice were given a single oral dose of the test substance in corn oil at 750, 1500, 2500 or 3000 mg/kg bw. Five males and five females from each group were killed 24 hours after treatment; further lots of five males and- five females given corn oil or 2500 or 3000mg/kg bw were killed 48 and 72 hours after treatment.

Besides on male in the 3000 and 2500 mg group each, all other animals survived to scheduled termination, although some animals dosed at 1500, 2500 and 3000 mg/kg showed intermittent clinical signs throughout the study, including hunched posture, piloerection, reduced activity, and redness and swelling in the anal region.

There was some evidence of bone marrow toxicity, as seen by depression of bone marrow proliferation on slides prepared from animals dosed with 3000 mg/kg and sacrificed 48 or 72 hours later. Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of the test substance were similar to those in concurrent controls. It is concluded that, under the conditions of test, there was no evidence of induced chromosomal leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of the test substance.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June - July 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP.
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Principles of method if other than guideline:
Rats were dosed orally with Perkalink 900. After 2-4 or 12-16 h the hepatocytes were isolated and labelled for 4 h with tritiated thymidine (³H-TdR) and cultured for 14-18 h. During the labelling period some of the DNA damage that occurred in the animal will be repaired by excision repair, resulting in the incorporation of ³HTdR (UDS). Incorporation of ³HTdR was measured by an autoradiographic method. UDS was determined by counting the number of silver grains resulting from ³HTdR incorporation in the hepatocyte nucleus.
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Test system: Male Wistar rat
Source: Charles River Wiga, Sulzfeld, Germany.
No of animals: Pilot study: 3 male rats, Main study: 25 male rats.
All animals: 234 ± 15 g, range: 211-289 g
Identification: By unique number on the tail.
Acclimatization: Six days under laboratory conditions.
Allocated to treatment groups as they came to hand from delivery boxes.
On arrival and prior to final assignment to study, all animals were clinically examined to ensure that the selected animals were in a good state of health.

The animals were housed in an air-conditioned room with approximately 15 air changes per hour and controlled environment with optimal conditions being a temperature of 21 ±3°C and a relative humidity of 30-70%. The room was illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.
Animals were housed in groups of 3 animals per cage in labeled polycarbonate cages (lxbxh=56x35x20 cm) containing purified sawdust as bedding material (SAW I, Jelu Werk, Rosenberg, Germany). Certificates of analysis were examined and accepted and then retained in the NOTOX archives. Cages were changed once a week.
The animals were provided with standard pelleted animal diet (from Altromin (code VRF 1), Lage, Germany) and free access to tap water. Certificates of analysis were examined and accepted and then retained in the NOTOX archives.
Feeding regimen: Animals for the 12-16 hours sampling time: The feed was withheld 4 hours before dosing (late in the afternoon) until perfusion, but the
animals had free access to tap water. Animals for the 2-4 hours sampling time: The animals received a limited quantity of food during the night before dosing (ca. 7 g/rat) and had free access to tap water. This feeding regimen was chosen to make the process of perfusion a little easier with less food in the.
stomach, with a minimum influence on metabolism or uptake.
Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
Three dose groups received a single oral dose of Perkalink 900. Hepatocytes were sampled 2-4 or 12-16 hours after dosing with 2000, 1500 or 1000 mg Perkalink 900/kg bw. Three slides per animal and 3 animals per data point were examined.
Corresponding vehicle treated groups (corn oil) served as negative controls. Positive control groups treated with single oral doses of dimethylnitrosamine (DMN, 10 mg/kg bw) or 2-acetylaminofluorene (2-AFF, 50 mg/kg bw) were harvested 2-4 or 12-16 hours after dosing.
Duration of treatment / exposure:
single application
Frequency of treatment:
single application
Post exposure period:
0 mg/kg: 2-4 h (1) and 12-16 h (1)
1000 mg/kg: 2-4 h (3) and 12-16 h (3)
1500 mg/kg: 2-4 h (3) and 12-16 h (3)
2000 mg/kg: 2-4 h (3) and 12-16 h (3)
10 mg/kg DMN: 2-4 h (1)
50 mg/kg 2-AAF: 12-16 h (1)
Between brackets the number of animals examined for UDS are indicated.
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
see above for the number of animals examined per dose and time point
Control animals:
yes, concurrent vehicle
Positive control(s):
Dimethylnitrosamine (DMN; CAS-no. 62-75-9, purity 99%, Acros); 10 mg/kg body weight, dissolved in Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass. USA) (used for 2-4 h treatment time).
2-Acetylaminofluorene (2-AAF; CAS-no. 53-96-3, purity >99%, Fluka AG, Switzerland); 50 mg/kg body weight, administered in corn oil (used for 12-16 h treatment time).
Solutions of reference substances were prepared within 4 hours prior to dosing. The route and frequency of administration and the volume administered was consistent with those of the test article.
Tissues and cell types examined:
hepatocytes
Details of tissue and slide preparation:
See below.
Evaluation criteria:
A DNA~repair assay is considered acceptable if it meets the following criteria:
a) The positive control substances should produce significant increases in the number of net nuclear grain counts.
b) The net nuclear grain count found in the negative controls should reasonably be within the laboratory historical control data range,
c) The selected dose range should include a toxic dose as demonstrated by the preliminary toxicity range-finding test. In case of a non-toxic test substance the dose range should extend to 2000 mg/kg body weight or to the limit of solubility.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: unscheduled DNA synthesis in the liver
Additional information on results:
PERKALINK 900 was not genotoxic in the DNA repair assay using hepatocytes obtained from male rat liver following in vivo exposure to the test substance.

Mortality / signs of toxicity:

For the dose group of 2000 mg/kg b.w., additional animals were used to correct for possible deaths. One animal orally dosed with PERKALINK 900 (2000 mg/kg body weight) died within one hour after treatment. All other animals survived the sampling period.

Viability of the hepatocytes:

The viability of the hepatocytes, used for the evaluation of DNA repair inducing ability of PERKALINK 900, was at least 54%. A viability of at least 65% was found for the control cultures

Results DNA repair assay:

As a result of orally dosing PERKALINK 900 the net nuclear grain count (N-C), per coverslip, per animal and the group average revealed no positive response in this assay. The percentage of cells in repair (repair taken as N-C >=5), both per individual animal and the group average, revealed no increase at any dose.

Acceptability

The net nuclear grain count (N-C) in the solvent-treated control cultures fell within the historical control data range (<0).

Oral dosing of a male rat with dimethylnitrosamine (DMN) resulted in a net nuclear grain count (N-C) of 32.8 with 96% of the cells in repair (N-C >=5). Oral dosing of a male rat with 2-acetylaminofluorene (2AAF) resulted in a net nuclear grain count (N-C) of 24.0 with 100% of the cells in repair (N-C >=5).

In the scored coverslips the mean background of a single area of the same size as the corresponding nuclear area, located outside the cytoplasm, was always less than 8 grains. It was concluded that the test system was functioning correctly.

Conclusions:
Interpretation of results: negative
PERKALINK 900 was not genotoxic in the DNA repair assay using hepatocytes obtained from male rat liver following in vivo exposure to the test substance under the conditions described in this report.
Executive summary:

When treated orally with the test substance at doses of 1000, 1500 and 2000 mg/kg body weight in the in vivo UDS Test (OECD 486) male Wistar rats showed no induction of DNA repair in hepatocytes isolated 2-4 or 12-16 hours after dosing, respectively. In conclusion, the test substance was not genotoxic in the DNA repair assay using hepatocytes obtained from male rat liver following in vivo exposure to the test substance under the conditions described in this report.

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

Additional information

The test substance (solved in DMSO) was tested in an Ames test performed according to OECD TG 471 in 1990 with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. Doses of up to and including 5000 µg/plate did not show mutagenic effects in this bacterial system with and without external metabolization.

The ECHA guidance "Three recently approved in vivo genotoxicity test guidelines (revised in February 2018)" stated: 'When there is a positive result from an in vitro gene mutation study in bacteria (Ames test, OECD TG 471) or from an in vitro gene mutation study in mammalian cells (OECD TG 476 for tests using the Hprt and xprt genes; OECD TG 490 for tests using the thymidine kinase gene), adequate somatic cell in vivo tests to investigate gene mutations are TGR (OECD TG 488), comet assay (OECD TG 489) or, if justified, Unscheduled DNA Synthesis (UDS) test with mammalian liver cells in vivo (OECD TG 486)'.

https://echa.europa.eu/documents/10162/21650280/oecd_test_guidelines_genotoxicity_en.pdf/56ab5788-0103-4716-8903-59ab0c942efe

In 4 Salmonella typhimurium strains 1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene was negative. In the hypothetical case the test substance would be positive in strains such as TA 102 or E. coli WP2 an UDS test is recommended. An UDS test however is acutally available with a negative result. Therefore an additional Ames test with 5 strains (including TA 102 or E. coli WP2) is unnecessary due to the negative UDS test.

A gene mutation test on mammalian cells (HPRT locus in V79 cells) was performed with the test substance (solved in DMSO). The highest applied concentration in the pre-tests on toxicity (2000 μg/ml active ingredient) was chosen with regard to the solubility properties of the test item in an appropriate solvent with respect to the current OECD Guideline 476. The concentration range of the main experiments was limited by cytotoxic effects. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments with and without external metabolization. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

The effect of the test substance (solved in DMSO) on chromosomal structure was investigated in cultured human lymphocytes. Tests were conducted with inclusion of rat liver S9 mix for 3 hours (harvest 21 and 45 hours later, test concentrations 5, 10, and 15 µg/ml) and without for 24 hours (test concentrations 1, 2, and 3 µg/ml). Dose related toxicity was seen as reduction in mitotic index in the cultures without S9 mix starting at 1 µg test substance/ml and with S9 mix starting at 2.5 µg/ml.

In the absence of S9 mix dose-related and statistically significant increases in mean aberrant cell frequencies were apparent at 3 µg/ml. In the presence of S9 mix high significance was seen at 15 µg/ml in the 24 hour cultures whereas no statistically enhanced clastogenicity was seen in the 48 hour cultures at this dose.

It is concluded that the test substance under the conditions of this in vitro test showed clear evidence of clastogenic activity in both the presence and absence of metabolic activation.

In an in vivo mouse bone marrow micronucleus test performed according to OECD 474 (1983) male and female mice were given a single oral dose of the test substance in corn oil at 750, 1500, 2500 or 3000 mg/kg bw. Five males and five females from each group were killed 24 hours after treatment; further lots of five males and five females given corn oil or 2500 or 3000mg/kg bw were killed 48 and 72 hours after treatment.

Besides one male in the 3000 and 2500 mg group each, all other animals survived to scheduled termination, although some animals dosed at 1500, 2500 and 3000 mg/kg showed intermittent clinical signs throughout the study, including hunched posture, piloerection, reduced activity, and redness and swelling in the anal region.

There was some evidence of bone marrow toxicity, as seen by depression of bone marrow proliferation on slides prepared from animals dosed with 3000 mg/kg and sacrificed 48 or 72 hours later. Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of the test substance were similar to those in concurrent controls. It is concluded that, under the conditions of test, there was no evidence of induced chromosomal leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of the test substance.

When treated orally with the test substance at doses of 1000, 1500 and 2000 mg/kg body weight in the in vivo UDS Test (OECD 486) male Wistar rats showed no induction of DNA repair in hepatocytes isolated 2-4 or 12-16 hours after dosing, respectively. In conclusion, the test substance was not genotoxic in the DNA repair assay using hepatocytes obtained from male rat liver following in vivo exposure to the test substance under the conditions described in this report.

In conclusion: When tested up to dose levels of 5000 µg/plate, the test substance was not mutagenic in the Ames test. A HPRT test in V79 Chinese hamster cells (gene mutation test in mammalian cells) also showed no mutagenic effects for the test substance. In contrast, the test substance showed clastogenic activity in the in vitro chromosome aberration test both the presence and absence of metabolic activation. However, in the in vivo micronucleus test, at levels up to 3000 mg/kg bw, even in the presence of some evidence of bone marrow toxicity as evidenced by depression of bone marrow proliferation, frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after treatment were similar to those in concurrent controls. It was concluded that, under the conditions of this in vivo test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of mice after oral administration of the test substance. Finally, the test substance was not genotoxic in the DNA repair assay using hepatocytes obtained from male rat liver following in vivo exposure to the test substance at a level up to 2000 mg/kg bw.


Endpoint Conclusion: No adverse effect observed (negative).

Justification for classification or non-classification

Based on the overall picture of genotoxicity tests no classification for mutagenicity is warranted.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.