p-phenylene diisocyanate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

p-phenylene diisocyanate

EC Number:

203-207-6

EC Name:

p-phenylene diisocyanate

Cas Number:

104-49-4

Molecular formula:

C8H4N2O2

IUPAC Name:

1,4-diisocyanatobenzene

Test material form:

solid: flakes

Details on test material:

Identification: Paraphenylene diisocyanate (PPDI)
Appearance: White Chips
Batch: 20171126004
CAS number: 104-49-4
Test item storage: At room temperature
Stable under storage conditions until: 25 November 2018 (expiry date)

Additional information
Test Facility test item number: 209403/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Molecular structure:
Molecular formula: C8H4N2O2
Molecular weight: 160.0
Irritant or corrosive: Yes
Volatile: Not indicated
Solubility in water: Not available
Stability in water: Not stable

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

Structural chromosome aberrations.

Species / strain

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.

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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
Control^a)	54	1018	100
3.13	46	1032	85
6.25	48	1019	89
12.5	60	1012	111
25^b)	54	1039	100
50^b)	35	1035	65
100^b)	43	1013	80
48 h exposure time, 48 h fixation time
Control^a)	76	1026	100
3.13	57	1023	75
6.25	58	1018	76
12.5	52	1012	68
25	46	1015	61
50^b)	50	1025	66
100^b)	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 metaphases^a)
	Absolute	Number of cells scored	Percentage of control
Without metabolic activation (-S9-mix)
3 h exposure time, 24 h fixation time
Control^b)	54 – 67	1011 – 1024	100
12.5	49 – 60	1011 – 1007	90
25	50 – 52	1018 – 1005	84
50^c)	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
Control^b)	70 – 57	1009 – 1014	100
12.5	53 – 47	1019 – 1025	79
25	60 – 54	1005 – 1019	90
50^c)	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 metaphases^a)
	Absolute	Number of cells scored	Percentage of control
Without metabolic activation (-S9-mix)
24 h exposure time, 24 h fixation time
Control^b)	49 – 54	1007 – 1012	100
6.25	47 – 48	1016 – 1008	92
12.5	45 – 43	1017 – 1007	85
25^c)	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
Control^b)	57 – 45	1009 – 1032	100
1	51 – 44	1014 – 1019	93
5	52 – 51	1023 – 1013	101
25	48 – 44	1016 – 1016	90
50^c)	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.

 

Applicant's summary and conclusion

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.