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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three key genotoxicity studies are available for 44PD: a K2 in vitro mutagenicity study in bacteria (Ames), a K1 in vitro chromosome aberration study and a K2 in vitro mammalian cell gene mutation study (HPRT). 44PD scored negative in all 3 tests.

Link to relevant study records

Referenceopen allclose all

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:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although the test protocol was not described in the publication (in stead, reference to another publication was made), the results described in the paper combine the results of Ames testing of 300 substances by a renowed Research institute (National Institute of Environmental Health Sciences, Genetic toxicity department) in the framework of the National Toxicology Program.
Principles of method if other than guideline:
Ames test
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
rat and hamster S-9 fractions
Test concentrations with justification for top dose:
0.000 - 0.330 - 1.000 - 3.300 - 10.000 - 33.000 - 100.000 - 200.000 - 333.000 µg/plate (as tested by lab. MIC)
0.000 - 0.300 - 1.000 - 3.000 - 10.000 - 16.000 - 33.000 - 66.000 - 100.000 - 166.000 - 333.000 µg/plate (as tested by lab. SRI)
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation assay

DURATION
- Preincubation period: 20 minutes
- Expression time (cells in growth medium): 2 days

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
The toxicity assay was performed using TA100 or the system developed by Waleh et al (Waleh, N.S., Rapport, S.J., Mortelmans, K. 1982. Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of construction of the required strains. Mutat. Res. 97:247-256.). Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.
Evaluation criteria:
Evaluations were made at both the individual trial and overall chemicals. Individual trials were judged mutagenic (+), weakly mutagenic (+W), questionable (?) or non-mutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of "+W", if only a single dose was elevated over the control, or if the increase seen was not dose related. It was not necessary for a response to reach twofold background for a chemical to be judged mutagenic.
A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible dose-related response over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a "+W" response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged non-mutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative

The test substance was not mutagenic in the Ames/Salmonella assay to strains TA97, TA98, TA100 or TA1535 in the presence or absence of metabolic activation.
Executive summary:

The substance was tested in the framework of the National Toxicology Program and coordinated by the National Institute of Environmental Health Sciences, Genetic Toxicology Department, Cellular and Genetic Toxicology Branch. The test was performed in two independent laboratories (Microbiological Associates Inc. (MIC) and SRI International), which both concluded the test substance to be non mutagenic in this assay. As such, a good interlaboratory reproducibility of the assay was shown.

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:
1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study and according to OECD guideline. No deviations. Protocol and results well described.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
This study is performed according to OECD recommendations of 1983
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
This study is performed according to US EPA guidelines of 1982
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT1
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The cells have a stable karyotype with a modal chromosome number of 22.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
without S9 mix: 7.50, 25.00, 50.00 and 75.00 ng
with S9 mix: 1.65, 4.50, 7.50, 8.50 µg
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, final concentration in medium 1%
- Justification for choice of solvent/vehicle: relatively non-toxic
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
solvent
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
postive control with metabolic activation: DMBA, 9,10-Dimethyl-1,2-benzanthracene

Migrated to IUCLID6: without metabolic activation
Details on test system and experimental conditions:
DURATION
- Exposure duration: 4 h
- Selection time (if incubation with a selection agent): 1 week

SELECTION AGENT: thioguanine

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
(1) The test substance is classified as mutagenic if it induces with one of the test substance concentrations reproducibly a mutation frequency that is 3 x higher than the spontaneous mutant frequency in this experiment (2) The test substance is classified as mutagenic if there is a reproducible concentration - related increase in the mutation frequency. Such evaluation may be considered independently from the enhancement factor for induced mutants. However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate as compared to the range normally found in this laboratory (5-45 mutants per 1e06 cells) a seemingly concentration-related increase of the mutations up to a factor of 3 within this range may be regarded as to be irrelevant. On the other hand, if a concentration-related increase of the number of mutants is very pronounced and unequivocally higher than by a factor of 4 this might well be taken as indication for a mutagenic potency.
Statistics:
Not necessary to perform as the mutation rates found in the groups treated with the test substance are not enhanced as compared with the negative controls. The values 33.0 and 38.5 mutants / 1e06 cells in experiment I and 65.8, 62.7 and 68.3 mutants / 1e06 cells in experiment II are not to be regarded higher than the negative control with solvent (23.6; 60.5). In this study the range of the negative controls is from 6.8 up to 60.5 mutants / 1e06 cells. Although the value 60.5 mutants / 1e06 cells is somewhat higher than the maximum value of our historical control: 45 mutants / 1e06 cells this observations does influence the result of this study.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 mix: 50.00 and 75.00 ng; with S9 mix: 4.50, 7.50, 8.50 µg
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Toxicity data experiment I

 

 

Conc. per mL

S9

      Number of cells

   seeded      found 

    I / II           I     II

Per flask

mean

PE% absolute

PE% relative

Column

1

2

3

4

5

6

7

8

Negative control

0.00 ng

 

496

346

365

355.5

71.7

100.0

Negative control

0.00 µg

+

496

386

367

376.5

75.9

100.0

Negative control with solvent

0.00 ng

 

496

309

401

355.0

71.6

100.0

Negative control

with solvent

0.00 µg

+

496

354

341

347.5

70.1

100.0

Positive control with EMS

1.00 mg

 

496

94

110

102.0

20.6

28.7

Positive control with DMBA

15.40 µg

+

496

112

123

117.5

23.7

33.8

Test substance

7.50 ng

 

496

384

336

360.0

72.6

101.4

Test substance

1.65 µg

+

496

361

328

344.5

69.5

99.1

Test substance

25.00 ng

 

496

366

337

351.5

70.9

99.0

Test substance

4.50 µg

+

496

335

265

300.0

60.5

86.3

Test substance

50.00 ng

 

496

223

258

240.5

48.5

67.7

Test substance

7.50 µg

+

496

329

220

274.5

55.3

79.0

Test substance

75.00 ng

 

496

131

118

124.5

25.1

35.1

Test stubstance

8.50 µg

+

496

187

128

157.5

31.8

45.3

* only colonies with more than 50 cells 7 days after seeding were scored

** PE absolute (value column 6 / value column 3 x 100)

*** PE relative (value column 6 / value column 6 of corresponding control x 100)

Conclusions:
Interpretation of results (migrated information):
negative

The results permit to draw the conclusion that the test substance is not mutagenic in the HGPRT-test with cells of the V79 Chinese hamster cell line.
Executive summary:

The test substance was assessed for its mutagenic potential in the HGPRT-test in two independent experiments. Under the conditions described in this report there is no indication of such potential. With no concentration of the test substance a reproducible enhancement of the mutation rate over the range of the negative controls was found, neither without nor with metabolic activation by S9 mix. The sensitivity of the test system was demonstrated by the enhanced mutation rates found in the groups treated with the positive control substances.

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study report is according to OECD and EPA guidelines and is well-documented. Deficiencies: There were no data in this report about polyploidy and endoreduplication.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
This study was performed according to OECD guidelines of 1983
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
This study was performed according to US EPA guidelines of 1982
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM + 10% FCS
- doubling time: 12 - 16 h in stock cultures
- plating efficiency of untreated cells: 70-90%
- model chromosome number: 22
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
without S9 mix: 3.00, 13.50, 75.00 ng / mL
with S9 mix: 1.30, 6.00, 8.50 µg / mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: relatively non-toxic, the test substance could be dissolved up to toxic concentrations
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
solvent
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
positive control with metabolic activation: cyclophosphamide

Migrated to IUCLID6: without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48h (7 h, 28h preparation interval) and 55h (18h preparation interval)
- Exposure duration: 4h
- Fixation time (start of exposure up to fixation or harvest of cells): 7h, 18h and 28h

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.2 µg / mL culture medium)
STAIN (for cytogenetic assays): aceto-orcein

NUMBER OF REPLICATIONS: 4 slides per group

NUMBER OF CELLS EVALUATED: 400 well spread metaphases were scored in the negative control and treatment groups.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and colony forming ability

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data

OTHER:
Breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural aberrations. Gaps were recorded and documented as well but not included in the calculation of the aberration rates.
Evaluation criteria:
1. The test substance can be classified as mutagenic if it induces with one of the concentrations tested a clearly increased aberration rate as compared with the negative controls.
2. The test substance can be classified as mutagenic if there is a clear test substance concentration related increase in the aberration rates.
3. The test substance is considered negative when item 1. and 2. do not apply.
Statistics:
Not necessary to perform as the aberration rates found in the groups treated with the test substance are in the range of the historical control values: 0-4% cells with aberrations.

This is considered as appropriate.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
concentrations of the test substance cover a range from no toxicity to maximum toxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: yes

Conclusions:
Interpretation of results (migrated information):
negative

The test substance was assessed for mutagenic potential in the chromosome aberration test with V79 cells. Under the conditions described in this report there is no indication of such potential. With none of the concentrations of the test substance a clear enhancement of the chromosome aberration rates over the range of the negative controls was found, neither without nor with metabolic activation by S9 mix. The sensitivity of the test system was demonstrated by the clearly enhanced chromosome aberration rates found in the groups treated with positive control substances. The results show that the test substance has no mutagenic potential in the cytogenetic in vitro test with cells of the V79 Chinese hamster cell line under the conditions described in this report.
Executive summary:

The test substance Kerobit BPD was assessed for its mutagenic potential to induce chromosome mutations (= chromosome aberrations) in V79 cells. The following structural aberration types were recorded: breaks, exchanges, partial or total disintegrations of chromosomes. Gaps were recorded but not included in the calculation of the aberration rates. Preparation of chromosomes was done after 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after the start of the treatment with the test substance. The treatment time was 4 h. The concentrations of the test substance applied has been determined in a pre-experiment using the mitotic index and the colony forming ability as indicator for toxicity response: 3.0 ng/mL (fixation time 18 h), 13.5 ng/mL (fix. time 18 h) and 75.0 ng/mL (fix. time 7, 18 and 28 h) without S9 mix; 1.3 µg/mL (fix. time 18 h), 6.0 µg/mL (fix. time 18 h); 7.5 µg/mL (fix. time 28h) and 8.5 µg/mL (fix. time 7 and 18h) with S9 mix.

In each experimental group 400 metaphases were recorded. In the positive control groups 200 metaphases were scored.

There was no enhancement of the number of cells with aberrations in the samples treated with the test substance as compared with the negative controls neither without nor with S9 mix for metabolic activation.

On the other hand, the sensitivity of the test system was demonstrated by significantly enhanced aberration rates after treatment with the mutagens EMS and CPA.

The results allow to draw the conclusions that the test substance did not induce chromosome aberrations in V79 cells under the experimental conditions described in the report mentioned above.

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

Genetic toxicity in vivo

Description of key information

No study was selected, since the 3 in vitro key studies were negative.

Additional information

Three key genotoxicity studies are available for 44PD: a K2 in vitro mutagenicity study in bacteria (Ames) (Zeiger et al. 1988), a K1 in vitro chromosome aberration study (Heidemann et al. 1987, report n° LMP 224 A) and a K2 in vitro mammalian cell gene mutation study (HPRT) (Heidemann et al. 1987, report n° LMP 224 B).

 

The bacterial reverse mutation test was carried out in the framework of the National Toxicology Program and coordinated by the National Institute of Environmental Health Sciences, Genetic Toxicology Department, Cellular and Genetic Toxicology Branch. Four Salmonella typhimurium strains were tested with concentrations ranging from 0.330 to 333000 µg/plate both with and without metabolic activation. The test was performed in two independent laboratories (Microbiological Associates Inc. (MIC) and SRI International). Both tests demonstrated that the test substance is non mutagenic in this assay.

 

The in vitro mammalian chromosome aberration test on V79 Chinese hamster lung fibroblast cells was carried out according to GLP and OECD guidelines. The test concentrations were determined in a pre-experiment addressing the toxicity response. Test concentrations in the absence of metabolic activation ranged from 3.00 to 75.00 ng/mL, whereas the tests in the presence of metabolic activation were run at concentrations ranging from 1.30 to 8.50 µg/mL. The following structural aberration types were recorded: breaks, exchanges, partial or total disintegrations of chromosomes. Under the described conditions the test substance did not induce chromosome aberrations in V79 cells.

 

In the in vitro mammalian cell gene mutation study the test substance was assessed for its mutagenic potential in an HPRT test on Chinese hamster lung fibroblasts (V79) both with and without metabolic activation. Test concentrations ranged from 7.50 to 75.00 ng/mL without metabolic activation and 1.65 to 8.50 µg/mL in the presence of S9 mix. Under the described conditions the test substance did not show enhancement of the mutation rate over the negative controls, thus demonstrating that the test substance is not mutagenic in the HPRT test with cells of the V79 Chinese hamster cell line.

 

Based on the above-described test results it can be concluded that 44PD does not induce gene mutations nor chromosome aberrations in the in vitro genotoxicity testing. As a consequence, in vivo testing is not required.

Justification for selection of genetic toxicity endpoint: No study was selected, since the three in vitro key studies were negative.




Justification for classification or non-classification

As no in vitro genotoxicity was found in the Ames, HPRT and chromosome aberration tests, classification for germ cell mutagenicity is not deemed required for Regulation 1272/2008 (CLP) or Directive 67/548 (DSD).