Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: non-mutagenic

OECD473: negative

OECD490: negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
The bacterial reverse mutation assay (Ames test1-4) to
detect mutagenicity in vitro is of crucial importance in drug
discovery and development as an early alerting system for
potential carcinogenicity and/or teratogenicity. Existing commercial
tools suitable for predicting the outcome of the Ames
test, such as DEREK5 for Windows and MultiCASE,6
provide promising results on several evaluation data sets5,7-9
and the possibility to derive structure-activity and/or even
mechanistic information from the predictions. Still, these two
commercial tools are limited in terms of statistical performance,
technical accessibility for bench chemists, and
adaptability to a company’s chemical space.
Using the benchmark data set, we evaluated four noncommercial
implementations of machine learning techniques and
three commercial prediction tools. For the noncommercial
predictors compounds were represented as numerical vectors
of molecular descriptors. Molecular descriptors were selected
from blocks 1, 2, 6, 9, 12, 15, 16, 17, 18, and 20 of DragonX
version 1.220 based on a 3D structure generated by Corina
version 3.4.21 These DragonX blocks provide a wide variety
of descriptor types including constitutional, topological,
geometrical, functional group count, and atom-centered
fragments descriptors as well as various molecular properties,
representing a starting point for unbiased modeling attempts
on the new benchmark data set.
In the following we provide details on algorithms and
implementations, i.e. for the noncommercial implementations
of Support Vector Machines, Gaussian Processes, Random
Forests, and k-Nearest Neighbors as well as for the commercial
tools DEREK and MultiCase.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The methods and databases used are described in the article.
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
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
The mutagenicity of the substance is considered negative.
Executive summary:

The mutagenicity of the substance is considered negative.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Jun 2018 to 02 Sep 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) (migrated information)
Version / remarks:
OECD Guideline 473. In Vitro Mammalian Chromosome Aberration Test (adopted 29 July 2016).
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
No further details specified in the study report.
Target gene:
Structural chromosome aberrations.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Test System
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (e.g. OECD, EC).
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2017) are presented below:
Dose-range finding study / First cytogenetic assay: age 28, AGT = 14.2 h
Second cytogenetic assay: age 26, AGT = 14.7 h

Cell Culture
Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.

Culture medium
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was added.

Environmental conditions
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 53 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 - 37.4°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, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Due to migration, the value was transferred to one of the current document's attachments
Test concentrations with justification for top dose:
Dose-range Finding Test / First Cytogenetic Assay
At a concentration of 50 μg/mL PPDI precipitated in the culture medium. At the 3 h exposure time, blood cultures were treated in duplicate with 12.5, 25 and 50 μg test item/mL culture medium with and without S9-mix (first cytogenetic assay).
At the 24 hour and 48 hour exposure time single blood cultures were treated with 3.13, 6.25, 12.5, 25, 50 and 100 μg PPDI/mL culture medium without S9-mix (dose-range finding test).

Second Cytogenetic Assay
Without S9-mix :
6.25, 12.5 and 25 μg/mL culture medium (24 h exposure time, 24 h fixation time).
1, 5, 25 and 50 μg/mL culture medium (48 h exposure time, 48 h fixation time).

The highest tested concentration was determined by the solubility of PPDI in the culture medium.
Vehicle / solvent:
The vehicle for the test item was dimethyl sulfoxide.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Dose-range Finding Test / First Cytogenetic Assay
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose-range finding test. PPDI was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 ± 2 h and thereafter exposed to selected doses of PPDI for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.
The highest tested concentration was determined by the solubility of PPDI in the culture medium.
The test item precipitated at concentrations of 50 μg/mL and upwards. The lymphocytes were cultured in duplicate at the 3 h exposure time and appropriate positive controls were included. The cytogenetic assay was carried out as described by Evans, 1984 with minor modifications. PPDI was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate.
After 3 h exposure to PPDI in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).
Cytotoxicity of PPDI in the lymphocyte cultures was determined using the mitotic index. No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for chromosome aberrations. The pilot study (short term exposure period) was used as the first cytogenetic assay.
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the second cytogenetic assay considering the highest dose level was determined by the solubility. As clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary.

Second Cytogenetic Assay
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of PPDI for 24 h and 48 h in the absence of S9-mix.
The cells were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time). Appropriate negative and positive controls were included in the second cytogenetic assay.

Chromosome Preparation
During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/mL medium) (Acros Organics, Geel, Belgium).
Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).

Preparation of Slides
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

Mitotic Index/Dose Selection for Scoring of the Cytogenetic Assay
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analyzable concentrations were used for scoring of the cytogenetic assay. PPDI was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium.

Analysis of Slides for Chromosome Aberrations
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with Charles River Den Bosch study identification number and code was placed over the marked slide. One hundred and eight to one hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations were calculated. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.
Rationale for test conditions:
In accordance with test guidelines.
Evaluation criteria:
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).
d) An adequate number of concentrations covering the appropriate concentration range is analyzable.
e) The criteria for selection of the top concentration are fulfilled.
f) All experimental conditions are tested unless one resulted in clear positive results.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

A test item is considered positive (clastogenic) in the chromosome aberration test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose-range Finding Test / First Cytogenetic Assay
At a concentration of 50 μg/mL PPDI precipitated in the culture medium. At the 3 h exposure time, blood cultures were treated in duplicate with 12.5, 25 and 50 μg test item/mL culture medium with and without S9-mix (first cytogenetic assay).
At the 24 hour and 48 hour exposure time single blood cultures were treated with 3.13, 6.25, 12.5, 25, 50 and 100 μg PPDI/mL culture medium without S9-mix (dose-range finding test).
All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix, PPDI did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
Both in the absence and presence of S9-mix, PPDI did not show a biologically relevant increase in the number of polyploid cells and cells with endoreduplicated chromosomes.

Second Cytogenetic Assay
To obtain more information about the possible clastogenicity of PPDI, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to PPDI in the absence of S9-mix for 24 or 48 hours. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix : 6.25, 12.5 and 25 μg/mL culture medium (24 h exposure time, 24 h fixation time).
1, 5, 25 and 50 μg/mL culture medium (48 h exposure time, 48 h fixation time).

Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix : 6.25, 12.5 and 25 μg/mL culture medium (24 h exposure time, 24 h fixation time).
5, 25 and 50 μg/mL culture medium (48 h exposure time, 48 h fixation time).
PPDI did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
PPDI did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

Evaluation of the Results
The potential of PPDI to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analyzed was selected based on the solubility of the test item in the culture medium.
The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Both in the absence and presence of S9-mix PPDI did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
No biologically relevant effects of PPDI on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.
Therefore it can be concluded that PPDI does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in the report.

Mitotic Index of Human Lymphocyte Cultures Treated with PPDI in the Dose-Range Finding Test

PPDI concentration (µg/mL)

Number of metaphases

Absolute

Number of cells scored

Percentage of control

Without metabolic activation (-S9-mix)

 

 

 

24 h exposure time, 24 h fixation time

 

 

 

Controla)

54

1018

100

3.13

46

1032

85

6.25

48

1019

89

12.5

60

1012

111

25b)

54

1039

100

50b)

35

1035

65

100b)

43

1013

80

48 h exposure time, 48 h fixation time

 

 

 

Controla)

76

1026

100

3.13

57

1023

75

6.25

58

1018

76

12.5

52

1012

68

25

46

1015

61

50b)

50

1025

66

100b)

46

1012

61

a)Dimethyl sulfoxide

b)PPDI precipitated in the culture medium


Mitotic Index of Human Lymphocyte Cultures Treated with PPDI in the First Cytogenetic Assay

PPDI concentration (µg/mL)

Number of metaphasesa)

Absolute

Number of cells scored

Percentage of control

Without metabolic activation (-S9-mix)

 

 

 

3 h exposure time, 24 h fixation time

 

 

 

Controlb)

54 – 67

1011 – 1024

100

12.5

49 – 60

1011 – 1007

90

25

50 – 52

1018 – 1005

84

50c)

54 – 48

1013 – 1008

84

MMC-C; 0.5 µg/mL

37 – 45

1009 – 1008

68

MMC-C; 0.75 µg/mL

37 – 45

1009 – 1008

68

With metabolic activation (+S9-mix)

 

 

 

3 h exposure time, 24 h fixation time

 

 

 

Controlb)

70 – 57

1009 – 1014

100

12.5

53 – 47

1019 – 1025

79

25

60 – 54

1005 – 1019

90

50c)

60 – 50

1030 – 1014

87

CP; 10 µg/mL

43 – 42

1031 – 1019

67

a)Duplicate cultures

b)Dimethyl sulfoxide

c)PPDI precipitated in the culture medium


Chromosome Aberrations in Human Lymphocyte Cultures Treated with PPDI in the Absence of S9-Mix in the First Cytogenetic Assay (3 H Exposure Time, 24 H Fixation Time)

Conc

DMSO

(1.0% v/v)

12.5

µg/mL

25

µg/mL

50

µg/mL

MMC-C

0.5 µg/mL

Culture

A   B   A+B

A   B   A+B

A   B   A+B

A   B   A+B

A   B   A+B

Mitotic

Index (%)

100

90

84

84

68

No. of

Cells scored

150   150   300

150   150   300

150   150   300

150   150   300

150   150   300

No. of

Cells with

aberrations

(+ gaps) a)

0

0

0

0

0

0

0

0

0

1

0

1

36

39

***)

75

 

No. of

Cells with

aberrations

(- gaps)

0

0

0

0

0

0

0

0

0

0

0

0

34

39

***)

73

 

g’

 

 

 

 

 

 

 

 

 

 

 

 

1

1

 

g”

 

 

 

 

 

 

 

 

 

1

 

 

4

 

 

b’

 

 

 

 

 

 

 

 

 

 

 

 

19

14

 

b”

 

 

 

 

 

 

 

 

 

 

 

 

14

23

 

m’

 

 

 

 

 

 

 

 

 

 

 

 

 

1

 

m”

 

 

 

 

 

 

 

 

 

 

 

 

2

1

 

exch.

 

 

 

 

 

 

 

 

 

 

 

 

5

4

 

dic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

d’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

misc.

 

 

 

 

 

 

2poly

 

 

 

total aberr

(+ gaps)

0

0

 

0

0

 

0

0

 

1

0

 

45

44

 

total aberr

(- gaps)

0

0

 

0

0

 

0

0

 

0

0

 

40

43

 

a)Abbreviations used for various types of aberrations are listed below. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above. The numerical variation polyploidy (poly) was not counted as an aberration.

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.


Chromosome Aberrations in Human Lymphocyte Cultures Treated with PPDI in the Presence of S9-Mix in the First Cytogenetic Assay (3 H Exposure Time, 24 H Fixation Time)

Conc

DMSO

(1.0% v/v)

12.5

µg/mL

25

µg/mL

50

µg/mL

CP

10 µg/mL

Culture

A   B   A+B

A   B   A+B

A   B   A+B

A   B   A+B

A   B   A+B

Mitotic

Index (%)

100

79

90

87

67

No. of

Cells scored

150   108   258

150   150   300

150   150   300

150   150   300

150   150   300

No. of

Cells with

aberrations

(+ gaps) a)

1

0

1

1

1

2

1

1

2

0

0

0

27

27

***)

54

 

No. of

Cells with

aberrations

(- gaps)

1

0

1

1

1

2

1

1

2

0

0

0

27

27

***)

54

 

g’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

g”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

b’

1

 

 

1

1

 

 

 

 

 

 

 

10

10

 

b”

 

 

 

 

 

 

1

 

 

 

 

 

16

15

 

m’

 

 

 

 

 

 

 

1

 

 

 

 

1

2

 

m”

 

 

 

 

 

 

 

 

 

 

 

 

1

1

 

exch.

 

 

 

 

 

 

 

 

 

 

 

 

5

2

 

dic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

d’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

misc.

 

 

 

 

poly

 

poly

 

 

 

total aberr

(+ gaps)

1

0

 

1

1

 

1

1

 

0

0

 

33

30

 

total aberr

(- gaps)

1

0

 

1

1

 

1

1

 

0

0

 

33

30

 

a)Abbreviations used for various types of aberrations are listed below. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above. The numerical variation polyploidy (poly) was not counted as an aberration.

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.


Mitotic Index of Human Lymphocyte Cultures Treated with PPDI in the Second Cytogenetic Assay

PPDI concentration (µg/mL)

Number of metaphasesa)

Absolute

Number of cells scored

Percentage of control

Without metabolic activation (-S9-mix)

 

 

 

24 h exposure time, 24 h fixation time

 

 

 

Controlb)

49 – 54

1007 – 1012

100

6.25

47 – 48

1016 – 1008

92

12.5

45 – 43

1017 – 1007

85

25c)

35 – 42

1022 – 1006

75

MMC-C; 0.2 µg/mL

22 – 24

1022 – 1017

45

MMC-C; 0.3 µg/mL

12 – 11

1012 – 1011

22

48 h exposure time, 48 h fixation time

 

 

 

Controlb)

57 – 45

1009 – 1032

100

1

51 – 44

1014 – 1019

93

5

52 – 51

1023 – 1013

101

25

48 – 44

1016 – 1016

90

50c)

47 – 44

1011 – 1029

89

MMC-C; 0.1 µg/mL

39 – 37

1029 – 1018

75

MMC-C; 0.15 µg/mL

22 – 25

1017 – 1015

46

a)Duplicate cultures

b)Dimethyl sulfoxide

c)PPDI precipitated in the culture medium


Chromosome Aberrations in Human Lymphocyte Cultures Treated with PPDI in the Absence of S9-Mix in the Second Cytogenetic Assay (24 H Exposure Time, 24 H Fixation Time)

Conc

DMSO

(1.0% v/v)

6.25

µg/mL

12.5

µg/mL

25

µg/mL

MMC-C

0.2 µg/mL

Culture

A   B   A+B

A   B   A+B

A   B   A+B

A   B   A+B

A   B   A+B

Mitotic

Index (%)

100

92

85

75

45

No. of

Cells scored

150   150   300

150   150   300

150   150   300

150   150   300

150   150   300

No. of

Cells with

aberrations

(+ gaps) a)

0

0

0

0

1

1

0

0

0

0

0

0

24

20

***)

44

 

No. of

Cells with

aberrations

(- gaps)

0

0

0

0

1

1

0

0

0

0

0

0

23

19

***)

42

 

g’

 

 

 

 

 

 

 

 

 

 

 

 

1

1

 

g”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

b’

 

 

 

 

1

 

 

 

 

 

 

 

10

9

 

b”

 

 

 

 

 

 

 

 

 

 

 

 

5

4

 

m’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

exch.

 

 

 

 

 

 

 

 

 

 

 

 

8

8

 

dic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

d’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

misc.

 

 

 

 

 

 

 

 

p

 

total aberr

(+ gaps)

0

0

 

0

1

 

0

0

 

0

0

 

25

22

 

total aberr

(- gaps)

0

0

 

0

1

 

0

0

 

0

0

 

24

21

 

a)Abbreviations used for various types of aberrations are listed below. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.


Chromosome Aberrations in Human Lymphocyte Cultures Treated with PPDI in the Absence of S9-Mix in the Second Cytogenetic Assay (48 H Exposure Time, 48 H Fixation Time)

Conc

DMSO

(1.0% v/v)

5

µg/mL

25

µg/mL

50

µg/mL

MMC-C

0.1 µg/mL

Culture

A   B   A+B

A   B   A+B

A   B   A+B

A   B   A+B

A   B   A+B

Mitotic

Index (%)

100

101

90

89

75

No. of

Cells scored

150   150   300

150   150   300

150   150   300

150   150   300

150   150   300

No. of

Cells with

aberrations

(+ gaps) a)

0

0

0

0

1

1

0

0

0

0

0

0

24

29

***)

53

 

No. of

Cells with

aberrations

(- gaps)

0

0

0

0

1

1

0

0

0

0

0

0

23

29

***)

52

 

g’

 

 

 

 

 

 

 

 

 

 

 

 

1

 

 

g”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

b’

 

 

 

 

 

 

 

 

 

 

 

 

8

12

 

b”

 

 

 

 

1

 

 

 

 

 

 

 

3

4

 

m’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m”

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

exch.

 

 

 

 

 

 

 

 

 

 

 

 

10

12

 

dic

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

d’

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

misc.

 

 

 

 

 

 

 

 

2p

3p

total aberr

(+ gaps)

0

0

 

0

1

 

0

0

 

0

0

 

24

31

 

total aberr

(- gaps)

0

0

 

0

1

 

0

0

 

0

0

 

23

31

 

a)Abbreviations used for various types of aberrations are listed below. misc. = (miscellaneous) aberrations not belonging to the ones mentioned above.
*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

 

Historical Control Data for in vitro Chromosome Aberration Studies of the Solvent Control

 

3 hours exposure time

24 hours exposure time

48 hours exposure time

 

Gaps included

Gaps excluded

Gaps included

Gaps excluded

Gaps included

Gaps excluded

 

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

Mean number of aberrant cells per 100 cells

0.42

0.50

0.32

0.43

0.47

0.40

0.67

0.40

SD

0.61

0.76

0.53

1.69

0.64

0.95

0.95

0.62

n

102

102

102

102

100

100

96

96

Upper control limit
(95% control limits)

1.92

2.35

1.61

2.03

2.05

1.83

2.76

1.65

Lower control limit
(95% control limits)

-1.08

-1.34

-0.97

-1.17

-1.11

-1.04

-1.42

-0.85

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between October 2014 and October 2017.

 

Historical Control Data for in vitro Chromosome Aberration Studies of the Positive Control Substances

 

3 hours exposure time

24 hours exposure time

48 hours exposure time

 

Gaps included

Gaps excluded

Gaps included

Gaps excluded

Gaps included

Gaps excluded

 

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

Mean number of aberrant cells per 100 cells

23.85

26.42

23.10

25.81

30.34

29.48

35.18

34.19

SD

10.99

12.93

11.06

13.01

14.32

14.44

13.81

13.81

n

100

100

100

100

98

98

94

94

Upper control limit
(95% control limits)

44.39

48.72

43.52

47.75

56.21

55.87

64.71

63.19

Lower control limit
(95% control limits)

3.30

4.12

2.67

3.86

4.48

3.09

5.65

5.19

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between October 2014 and October 2017.

 

Historical Control Data for Numerical Aberrations for in vitro Chromosome Aberration Studies of the Solvent Control

 

3 hours exposure time

24 hours exposure time

48 hours exposure time

 

Poly

Endo

Poly

Endo

Poly

Endo

 

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

- S9-mix

Mean number numerical aberrations per 100 cells

0.07

0.03

0.01

0.02

0.08

0.03

0.06

0.02

SD

0.26

0.14

0.07

0.11

0.30

0.13

0.20

0.15

n

102

102

102

102

100

100

96

96

Upper control limit
(95% control limits)

0.39

0.21

0.04

0.09

0.42

0.13

0.36

0.13

Lower control limit
(95% control limits)

-0.24

-0.14

-0.03

-0.05

-0.26

-0.08

-0.24

-0.09

SD = Standard deviation

n = Number of observations

Poly = polyploidy

Endo = endoreduplication

Distribution historical negative control data from experiments performed between October 2014 and October 2017.

Definitions of Chromosome Aberrations Score in Metaphase Portraits

Aberration

Abbreviation

Description

Chromatid gap

g'

an achromatic lesion which appears as an unstained region in the chromatid arm, the size of which is equal to or smaller then the width of the chromatid and the apparently “broken” segments of the chromatid arm are in alignment.

Chromosome gap

g"

An achromatic lesion which appears as an unstained region in both chromatids at the same position, the size of which is equal to or smaller then the width of the chromatid and the apparently “broken” segments of the chromatids are in alignment.

Chromatid break

b'

An achromatic lesion in a chromatid arm, the size of which is larger then the width of the chromatid. The broken segments of the chromatid arm are aligned or unaligned.

Chromosome break

b"

An achromatic lesion in both chromatids at the same position, the size of which is larger than the width of the chromatid. The broken segments of the chromatids are aligned or unaligned.

Chromatid deletion

d'

Deleted material at the end of a chromatid arm.

Minute

m'

A single, usually circular, part of a chromatid lacking a centromere.

Double minutes

m"

Two, usually circular, parts of a chromatid lacking a centromere.

Dicentric chromosome

dic

A chromosome containing two centromeres.

Tricentric chromosome

tric

A chromosome containing three centromeres.

Ring chromosome

r

A ring structure with a distinct lumen.

Exchange figure

exch.

An exchange(s) between two or more chromosomes resulting in the formation of a tri- or more-armed configuration.

Chromosome intrachange

intra

A chromosome intrachange is scored after rejoining of a lesion within one chromosome.

Pulverized chromosomes

p

A fragmented or pulverized chromosome

Multiple aberrations

ma

A metaphase spread containing ten or more of the above chromosome gaps (chromatid and chromosome gaps not included). ma is counted as 10 aberrations.

Polyploidy

poly

A chromosome number that is a multiple of the normal diploid number.

Endoreduplication

endo

A form of polyploidy in which each centromere connects two or four pairs of chromatids instead of the normal one pair.

 

Conclusions:
In conclusion, this test is valid and PPDI is not clastogenic in human lymphocytes under the experimental conditions described in the report.
Executive summary:

The objective of this study was to evaluate Paraphenylene diisocyanate (PPDI) for its potential to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

 

The possible clastogenicity of PPDI was tested in two independent experiments.

 

The study procedures described in this report are in compliance with the most recent OECD guideline.

 

Batch 20171126004 of PPDI were white chips with a purity of 99.8%. The vehicle of the test item was dimethyl sulfoxide.

 

In the first cytogenetic assay, PPDI was tested up to 50 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. PPDI precipitated in the culture medium at this dose level.

 

In the second cytogenetic assay, PPDI was tested up to 25 μg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 50 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. PPDI precipitated in the culture medium at these dose levels.

 

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

PPDI did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

 

No biologically relevant effects of PPDI on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Therefore, it can be concluded that PPDI does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in the report.

 

In conclusion, this test is valid and PPDI is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Jun 2018 to 16 Jul 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
OECD Guideline 490. “Genetic Toxicology: In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene", (adopted 29 July 2016).
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
No further details specified in the study report.
Target gene:
Forward mutations at the thymidine kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Test System: L5178Y/TK+/--3.7.2C mouse lymphoma cells.
Rationale: Recommended test system in international guidelines (e.g. OECD 490).
Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
Stock cultures of the cells were stored in the freezer (-150°C). The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 106 cells/mL.

Cell Culture
Horse serum
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.

Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).

Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).

Exposure medium
For 3 hour exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine (TFT) (Sigma).

Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).

Environmental conditions
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 61 - 97%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 - 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, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Any variation to these conditions were evaluated and maintained in the raw data.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Due to migration, the value was transferred to one of the current document's attachments
Test concentrations with justification for top dose:
First Mutagenicity Test
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test in the absence and presence of S9-mix: 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL exposure medium.

Second Mutagenicity Test
Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 0.39, 0.78, 1.56, 3.1, 6.25, 12.5, 25 and 50 μg/mL exposure medium.
Vehicle / solvent:
The vehicle for the test item was dimethyl sulfoxide (DMSO, Merck Darmstadt, Germany).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Experimental Design
Cleansing
Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.

Dose-range Finding Test
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 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) with a number of test item concentrations increasing by approximately half log steps. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. The test item was tested in the absence and presence of S9-mix.
Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 100 μg/mL.
For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium).
For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter.
The surviving cells of the 3 hour treatment were subcultured twice to determine cytotoxicity.
After 24 hour of subculturing, the cells were counted and subcultured again for another 24 hours, after that the cells were counted. The surviving cells of the 24 hour 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.

Mutagenicity Test
Eight doses of the test item were tested in the mutation assay. The test item was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and 24 hour treatment periods.
Since the test item was not toxic and difficult to dissolve in aqueous solutions the highest concentration was determined by the solubility in the culture medium. The highest test item concentrations showed slight precipitate in the exposure medium.

Treatment of the Cells
Per culture 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate.
In the first experiment, cell cultures were exposed for 3 hours to the test item in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test item in exposure medium for 24 hours in the absence of S9-mix.
For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium).
For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter.

Expression Period
For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 106 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

Determination of the Mutation Frequency
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). In the first experiment in some of the treatment groups, a total number of 432 wells was used for determination of the mutation frequency instead of 480 as specified in the study plan (see deviation). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
Rationale for test conditions:
In accordance with test guidelines.
Evaluation criteria:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including 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 item 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 item 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 item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Statistics:
Not specified
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Solubility
In the solubility test, Paraphenylene diisocyanate (PPDI) precipitated directly in the exposure medium at concentrations of 50 μg/mL and above. After 5 minutes, the test item still slightly precipitated in the exposure medium at concentrations of 50 μg/mL and above.
In the dose range finding test, Paraphenylene diisocyanate (PPDI) precipitated directly in the exposure medium at concentrations of 50 μg/mL and above. After 3 hours of treatment, the test item slightly precipitated in the exposure medium at the concentration of 100 μg/mL.
After 24 hours of treatment, the test item slightly precipitated in the exposure medium at concentrations of 50 μg/mL and above.

Dose-range Finding Test
In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 3.1 to 100 μg/mL in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.
In the absence of S9-mix, the relative suspension growth was 12% at the test item concentration of 100 μg/mL compared to the relative suspension growth of the solvent control.
In the presence of S9-mix, the relative suspension growth was 26% at the test item concentration of 100 μg/mL compared to the relative suspension growth of the solvent control.
The relative suspension growth was 5% at the test item concentration of 100 μg/mL compared to the relative suspension growth of the solvent control.

First Mutagenicity Test
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test in the absence and presence of S9-mix: 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL exposure medium.
Evaluation of precipitate
Since the appearance of the stock solution was changed compared to the dose range finding test, an additional check on precipitate was performed. The test item slightly precipitated in the exposure medium at 50 μg/mL and above.
Evaluation of toxicity
No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.
Evaluation of the mutagenicity
No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with Paraphenylene diisocyanate (PPDI) either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the Paraphenylene diisocyanate (PPDI) treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second Mutagenicity Test
To obtain more information about the possible mutagenicity of Paraphenylene diisocyanate (PPDI), a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period.
Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 0.39, 0.78, 1.56, 3.1, 6.25, 12.5, 25 and 50 μg/mL exposure medium.
Evaluation of toxicity
No severe toxicity was observed and all dose levels were evaluated. The relative total growth of the highest test item was 27% compared to the total growth of the solvent controls.
Evaluation of mutagenicity
No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

DISCUSSION
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The suspension growth over the two-day expression period for cultures treated with DMSO was between 9 and 15 (3 hour treatment) and 80 and 93 (24 hour treatment).
In the absence of S9-mix, Paraphenylene diisocyanate (PPDI) did not induce a biologically relevant increase in the mutation frequency. This result was confirmed in a repeat experiment with modification in the duration of treatment.
In the presence of S9-mix, Paraphenylene diisocyanate (PPDI) did not induce a biologically relevant increase in the mutation frequency.

Dose-range Finding Test: Cytotoxicity of Paraphenylene diisocyanate (PPDI) (3 Hour Treatment)

Dose

(µg/ml)

Cell count after 24 hours of subculture

(cells/ml x105)

Cell count after 48 hours of subculture

(cell/ml x105)

SG(1)

RSG(2)

(%)

Without metabolic activation

SC

5.5

5.6

15

100

3.1

5.0

5.8

15

94

6.3

5.0

4.8

12

78

12.5

4.7

4.0

9

61

25

4.2

4.0

8

55

50

3.4

4.0

7

44

100(3)

1.2(4)

3.2

2

12

With metabolic activation

SC

5.2

6.4

17

100

3.1

4.7

6.2

15

88

6.3

5.3

6.2

16

99

12.5

4.9

5.8

14

85

25

4.5

6.0

14

81

50

3.1

5.5

9

51

100(3)

2.2

3.9

4

26

Note: all calculations were made without rounding off

SC = solvent control = Dimethyl sulfoxide

(1)= suspension growth

(2)= relative suspension growth

(3)= the test item precipitated in the exposure medium

(4)= since less than 1.25 x 105c/ml were present, no subculture was performed

SG = Suspension growth = [Cell count after 24 hours of subculture (Day 1)/1.6 x 105] x [Cell count after 48 hours of subculture (Day 2)/1.25 x 105]*

*Or appropriate cell concentration

RSG = [SG(test)/SG(control)] x 100

 

Dose-range Finding Test: Cytotoxicity of Paraphenylene diisocyanate (PPDI) (24 Hour Treatment)

Dose

(µg/ml)

Cell count after 24 hours of treatment

(cells/ml x105)

Cell count after 24 hours of subculture

(cell/ml x105)

SG(1)

RSG(2)

(%)

Without metabolic activation

SC

10.8

4.5

31

100

3.1

9.4

4.6

28

89

6.3

9.1

4.1

24

77

12.5

7.9

4.3

22

70

25

7.1

4.2

19

61

50(3)

5.3

3.6

12

39

100(3)

1.2(4)

1.9

2

5

Note: all calculations were made without rounding off

SC = solvent control = Dimethyl sulfoxide

(1)= suspension growth

(2)= relative suspension growth

(3)= the test item precipitated in the exposure medium

(4)= since less than 1.25 x 105c/ml were present, no subculture was performed

SG = Suspension growth = [Day 0 cell count/1.25 x 105] x [Day 1 cell count/1.25 x 105]*

*Or appropriate cell concentration

RSG = [SG(test)/SG(control)] x 100

 

Experiment 1: Cytotoxic and Mutagenic Response of Paraphenylene diisocyanate (PPDI) in the Mouse Lymphoma L5178Y Test System

Dose

(µg/ml)

RSG

(%)

CEday2

(%)

RCE

(%)

RTG

(%)

Mutation frequency

per 106survivors

Total

( small

large )

Without metabolic activation

3 hour treatment

SC1

100

97

100

100

119

( 50

63 )

SC2

81

139

( 65

66 )

0.78

142

115

129

183

86

( 37

45 )

1.56

125

113

127

158

110

( 44

60 )

3.13

122

111

125

153

100

( 46

48 )

6.25

85

93

104

89

163

( 86

64 )

12.5

100

115

129

128

119

( 45

67 )

25

93

93

104

97

167

( 73

81 )

50(1)

84

118

132

111

131

( 61

61 )

100(1)

82

86

97

80

145

( 68

68 )

MMS

51

41

46

23

1046

( 578

361 )

With metabolic activation

3 hour treatment

SC1

100

128

100

100

94

( 42

47 )

SC2

93

139

( 67

63 )

0.78

116

111

102

118

105

( 48

50 )

1.56

109

99

91

99

87

( 45

38 )

3.13

111

115

104

116

81

( 30

47 )

6.25

105

107

97

102

83

( 27

53 )

12.5

118

115

104

124

83

( 34

45 )

25

108

95

87

94

98

( 41

53 )

50(1)

113

101

92

104

112

( 40

66 )

100(1)

74

94

86

63

128

( 45

76 )

CP

84

88

80

67

426

( 139

250 )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cylophosphamide

(1)= the test item precipitated in the exposure medium.

 

Experiment 2: Cytotoxic and Mutagenic Response of Paraphenylene diisocyanate (PPDI) in the Mouse Lymphoma L5178Y Test System

Dose

(µg/ml)

RSG

(%)

CEday2

(%)

RCE

(%)

RTG

(%)

Mutation frequency

per 106survivors

Total

( small

large )

Without metabolic activation

24 hour treatment

SC1

100

68

100

100

62

( 19

42 )

SC2

 

129

 

 

65

( 22

40 )

0.39

85

94

95

81

59

( 15

43 )

0.79

116

102

104

121

48

( 13

35 )

1.56

95

105

107

102

53

( 21

31 )

3.13

97

118

120

116

58

( 32

24 )

6.25

72

90

91

66

75

( 33

40 )

12.5

49

93

94

47

92

( 42

46 )

25

38

84

85

32

115

( 48

61 )

50(1)

35

78

79

27

92

( 49

40 )

MMS

84

85

86

72

440

( 229

171 )

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate

(1)= the test item precipitated in the exposure medium.

 

Abbreviations used:

SC, Solvent Control (DMSO)

MMS, Methylmethanesulfonate

CP, Cyclophosphamide

RSG, Relative Suspension Growth

CE, Cloning Efficiency

SG, Suspension Growth

 

Experiment 1: Cell Counts and Subculture Data (Without Metabolic Activation)

 

DAY 0

DAY 1

DAY 2

 

Dose

(µg/ml)

Total amount of cells before treatment (x 106)

Cell count

(106c/ml)

Subculture x 106total amount(1)

Cell count (x 105c/ml)

RSG

(%)

SG

SC1

SC2

0.78

1.56

3.13

6.25

12.5

25

50(2)

100(2)

MMS

8

8

8

8

8

8

8

8

8

8

8

3.5

3.6

4.5

4.1

3.8

2.7

3.0

2.9

2.7

2.6

1.6

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

5.2

4.8

5.6

5.4

5.7

5.6

5.9

5.7

5.5

5.6

5.6

100

100

142

125

122

85

100

93

84

82

51

9

9

Note: all calculations were made without rounding off.

(1)= cell density 1.25 x 105c/ml

(2)= the test item precipitated in the exposure medium

 

Experiment 1: Cell Counts and Subculture Data (With Metabolic Activation)

 

DAY 0

DAY 1

DAY 2

 

Dose

(µg/ml)

Total amount of cells before treatment (x 106)

Cell count

(106c/ml)

Subculture x 106total amount(1)

Cell count (x 105c/ml)

RSG

(%)

SG

SC1

SC2

0.78

1.56

3.13

6.25

12.5

25

50(2)

100(2)

MMS

8

8

8

8

8

8

8

8

8

8

8

4.9

5.3

4.6

4.1

5.6

4.2

5.6

5.1

5.6

3.8

4.3

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

5.8

5.7

7.4

7.8

5.8

7.3

6.2

6.2

5.9

5.7

5.7

100

100

116

109

111

105

118

108

113

74

84

14

15

Note: all calculations were made without rounding off.

(1)= cell density 1.25 x 105c/ml

(2)= the test item precipitated in the exposure medium

 

Experiment 1: Selection Data and cloning Efficiency

Dose

(µg/ml)

mutant colonies

Cloning efficiency (at day 2)

Mutation frequency per 106survivors

Number of well with mutants per selection plate(1)

Total number of mutants

No. of empty wells per cloning plate

Total number of empty wells

CE x 100%

1

2

3

4

5

1

2

Total

Small

Large

Without metabolic activation

 

s

l

s

l

s

l

s

l

s

l

s

l

s+l

 

 

 

 

 

 

 

SC1

SC2

0.78(3)

1.56

3.13(3)

6.25

12.5

25(3)

50(2)

100(2)

12

11

11

9

8

19

9

10

18

6

12

9

9

16

6

11

17

12

14

7

6

6

7

11

6

12

11

11

11

16

11

10

10

9

5

9

12

16

15

8

4

11

2

8

11

14

4

20

12

8

16

8

8

10

13

14

12

11

14

11

10

12

8

11

6

12

7

9

12

13

7

7

6

10

11

10

13

12

14

13

12

8

7

6

11

14

16

5

11

10

9

15

9

16

9

10

14

9

7

14

44

48

35

45

42

71

47

55

65

53

55

49

42

61

44

54

68

60

64

53

99

97

77

106

86

125

115

115

128

106

46

48

29

35

35

39

35

41

28

35

27

37

32

27

28

37

26

35

31

46

73

85

61

62

63

76

61

76

59

81

97

81

115

113

111

93

115

93

118

86

119

139

86

110

100

163

119

167

131

145

50

65

37

44

46

86

45

73

61

68

63

66

45

60

48

64

67

81

61

68

MMS

19

18

8

9

15

29

14

15

12

28

20

12

24

13

15

17

25

21

15

8

204

133

337

63

64

127

41

1046

578

361

With metabolic activation

 

s

l

s

l

s

l

s

l

s

l

s

l

s+l

 

 

 

 

 

 

 

SC1

SC2

0.78

1.56

3.13(3)

6.25

12.5

25

50(2)

100(2)

11

14

5

8

5

4

6

8

6

6

14

9

9

10

16

7

4

8

10

14

7

7

10

6

9

6

7

8

11

6

9

10

10

6

7

11

10

11

14

14

6

10

18

10

6

6

3

6

6

10

12

9

7

7

9

18

13

12

15

9

9

11

9

11

5

6

9

11

9

6

9

12

12

4

8

6

10

3

9

13

15

14

7

6

4

5

11

3

5

11

10

13

13

8

4

9

10

12

12

14

48

56

49

41

29

27

36

36

37

39

54

53

51

35

44

51

47

46

60

64

102

109

100

76

73

78

83

82

97

103

22

30

32

37

33

35

34

32

32

32

32

46

31

34

28

31

27

42

38

43

54

76

63

71

61

66

61

74

70

75

127

93

111

99

115

107

115

95

101

94

94

139

105

87

81

83

823

98

112

128

42

67

48

45

30

27

34

41

40

45

47

63

50

38

47

53

45

53

66

76

CP

15

8

21

20

8

10

20

14

9

9

14

20

11

14

21

21

10

16

17

21

110

189

299

44

36

80

88

426

139

250

s = small colonies

l = large colonies

(1)= Solvent controls and treatment groups five plates with 2000 cells/well and the positive controls ten plats with 1000 cells/well

(2)= the test item precipitated in the exposure medium

(3)= for determination of the mutation frequency 432 wells were used

 

Experiment 2: Cell Counts and Subculture Data (Without Metabolic Activation)

 

24 HOUR TREATMENT

DAY 1

DAY 2

Dose

(µg/ml)

Total amount of cells before treatment

(x 106)

Total amount of cells after treatment

(x 106)

Subculture x 106total amount(1)

Cell count

(x 105c/ml)

Subculture x 106total amount(1)

Cell count

(x 105c/ml)

RSG

(%)

SG

SC1

SC2

0.39

0.78

1.56

3.13

6.25

12.5

25

50(3)

MMS

6

6

6

6

6

6

6

6

6

6

6

19.2

19.4

16.8

19.8

19.6

19.0

14.4

12.6

9.0

8.8

20.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

6.4

5.6

5.8

7.0

5.8

5.9

5.7

4.9

4.7

4.7

4.3

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

4.0

7.1

6.9

7.1

6.8

6.8

7.0

7.1

6.5

7.2

6.8

7.9

100

 

85

116

95

97

72

49

38

35

84

96

80

Note: all calculations were made without rounding off.

(1)= cell density 1.25 x 105c/ml

(2)= not used for the mutation experiment

(3)= the test item precipitated in the exposure medium

 

Experiment 2: Selection Data and cloning Efficiency

Dose

(µg/ml)

mutant colonies

Cloning efficiency (at day 2)

Mutation frequency per 106survivors

Number of well with mutants per selection plate(1)

Total number of mutants

No. of empty wells per cloning plate

Total number of empty wells

CE x 100%

1

2

3

4

5

1

2

Total

Small

Large

Without metabolic activation

 

s

l

s

l

s

l

s

l

s

l

s

l

s+l

 

 

 

 

 

 

 

SC1

SC2

0.39

0.78

1.56

3.13

6.25

12.5

25

50(2)

1

5

3

3

3

6

6

8

2

8

7

6

5

7

6

6

5

9

13

7

2

4

4

1

5

5

4

7

7

3

6

9

10

8

2

4

9

8

7

5

1

5

2

1

4

12

8

8

6

10

6

12

5

7

9

5

5

11

8

3

4

6

4

4

4

4

4

6

12

6

4

8

8

7

8

5

7

5

10

8

4

7

0

4

5

8

6

7

10

8

4

12

9

4

5

6

7

6

9

6

12

27

13

13

21

35

28

36

37

35

27

47

37

33

30

26

33

39

47

29

39

74

50

46

51

61

61

75

84

64

50

21

41

38

36

34

45

40

37

43

47

32

34

31

31

25

33

36

46

45

97

53

75

69

67

59

78

76

83

88

68

129

94

102

105

118

90

93

84

78

62

65

59

49

53

58

75

92

115

92

19

22

15

13

21

32

33

42

48

49

42

40

43

35

31

24

40

46

61

40

MMS

16

14

11

12

9

14

13

18

21

22

13

14

13

23

9

14

21

17

12

14

170

130

300

40

42

82

85

440

229

171

s = small colonies

l = large colonies

(1)= Solvent controls and treatment groups five plates with 2000 cells/well and the positive controls ten plats with 1000 cells/well

(2)= the test item precipitated in the exposure medium

 

Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 106survivors

 

-S9 mix

 

+ S9 mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

96

92

96

SD

29

30

29

n

268

248

285

Upper control limit

(95% control limits)

160

152

160

Lower control limit

(95% control limits)

32

31

32

SD = Standard deviation

n = Number of observations

 

Historical Control Data of the Spontaneous Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 106survivors

 

-S9 mix

 

+ S9 mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

808

684

1669

SD

239

206

843

n

136

124

146

Upper control limit

(95% control limits)

1465

1222

4196

Lower control limit

(95% control limits)

152

146

-859

SD = Standard deviation

n = Number of observations

 

Distribution historical positive control data from experiments performed between November 2014 and November 2017.

Conclusions:
In conclusion, Paraphenylene diisocyanate (PPDI) is not mutagenic or clastogenic in the TK mutation test system under the experimental conditions described in the report.
Executive summary:

The objective of this study was to evaluate the mutagenic potential of Paraphenylene diisocyanate (PPDI) by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

 

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

 

The study procedures described in this report were based on the most recent OECD 490 guideline.

 

Batch 20171126004 of PPDI consisted of white chips. The vehicle of the test item was dimethyl sulfoxide.

 

In the first experiment, the test item was tested up to concentrations of 100 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. The test item slightly precipitated in the culture medium at dose levels of 50 μg/mL and above.

 

In the second experiment, the test item was tested up to concentrations of 50 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The Relative Total Growth was reduced to 27%. The test item slightly precipitated in the culture medium at this dose level.

 

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

 

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, PPDI did not induce a biologically relevant increase in the mutation frequency. This result was confirmed in an independent experiment with modification in the duration of treatment.

 

In the presence of S9-mix, PPDI did not induce a biologically relevant increase in the mutation frequency.

 

In conclusion, Paraphenylene diisocyanate (PPDI) is not mutagenic or clastogenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

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

Based on the results of in vitro bacterial gene mutation study, in vitro mammalian chromosomal aberration and gene mutation studies no classification is proposed for genotoxicity according to the criteria of CLP regulation 1272/2008.