TDI Biuret

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

m-tolylidene diisocyanate

EC Number:

247-722-4

EC Name:

m-tolylidene diisocyanate

Cas Number:

26471-62-5

IUPAC Name:

2,4-diisocyanato-1-methylbenzene

Details on test material:

99.5% mixed isomers TDI, labelled 80.2% 2,4-TDI isomer, the rest being 2,6-TDI isomer
Analysed at 79.7% 2,4-TDI by test laboratory.

Test animals

Species:

mouse

Strain:

C57BL

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female C57BL/6JfCD-l/Alpk mice in the age range of 6-12 weeks were
used for Phase I, mice in the age range of 8-12 weeks were used for
Phase I1 and mice in the age range of 8-9 weeks were used for Phase I11 of
the study.
The animals were supplied by the Barriered Animal Breeding Unit,
ICI Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, UK.
On arrival the mice were housed by sex with between 5 and 10 per cage on
single sided wire mesh mouse cage racks or in the long-term inhalation
chambers and given food, Porton Combined Diet [PCD] (supplied by Special
Diets Services Limited, Stepf ield, Witham, Essex, UK; Appendix D) and
filtered tap water ad 1 ibitum.
The animal rooms were maintained at a temperature range of approximately
21°C and within a relative humidity (RH) range of 45-55%. Excursions
outside this RH range- were noted throughout the study but this is
considered not to affect the integrity of the study. The RH was measured
using a Kew Pattern wet and dry bulb hygrometer. Lighting was controlled
to provide 12 hours artificial light followed by 12 hours darkness. The
animal room was under positive pressure with respect to the access corridor
and had 20-30 air changes per hour.

Administration / exposure

Route of administration:

inhalation

Details on exposure:

a TDI - Phase I
TDI atmospheres for Phase I of the study were generated by passing clean
dry air from the laboratory air supply via a flow controller and a flow
meter to d jacketed'bubbler containing the test material (Appendix A). The
bubbler was heated by a flow of warm air at a temperature controlled by a
thermocirculator. The generation air passed through the bubbler picking up
vapourised test material and was split into two streams, each stream being
passed to one of the 2 exposure chambers being used.
Two streams of dilution air were each passed through flow controllers and
flow meters. Each of the dilution streams then joined a generation stream
prior to entry to the exposure chamber. The diluted test material stream
was then passed through the exposure chamber and was subsequently vented
into a fume cupboard. Air flows were monitored continuously using
f lowmeters (KDG Flowmeters, Burgess Hi 11, Sussex, UK) and were recorded
at approximately 30 minute intervals during the exposure periods.
b TDI - Phase I1 and Phase 111
The atmospheres for Phases I1 and 111 of the study were generated using the
system described above except that flow controllers were not fitted into
the system and as only single chambers were supplied from each generation
system, only one dilution stream was used.
C) Vinyl Chloride
Vinyl chloride was extracted from the cylinder and mixed with compressed
air at a flow rate to allow generation of 50000ppm. Both the air and vinyl
chloride flow rates were monitored using in-line flow meters with needle
valves.

Duration of treatment / exposure:

6 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 5.9, 11.8, 18.9 ppm (males): 0, 3.7, 7.5, 11.9 ppm (females)
Basis:

No. of animals per sex per dose:

5

Positive control(s):

Vinyl chloride

Examinations

Tissues and cell types examined:

Clinical observations were performed at approximately 30 minute intervals during the exposure period and at least once daily following exposure. Bone marrow smears were prepared at 24, 48 or 72 hours after exposure. One thousand polychromatic erythrocytes per slide were originally evaluated for the presence of micronuclei. An additional 2000 polychromatic erythrocytes were also evaluated for the presence of micronuclei from all slides from male animals exposed to the air control or TDI 24 hours after exposure and female animals exposed to the air control or TDI 24 and 48 hours after exposure. IN addition, 1000 erythrocytes were counted to determine the percentage of polychromatic erythrocytes in the total erythrocyte population.

Details of tissue and slide preparation:

Bone marrow smears were prepared 24, 48 and 72 hours after the end of the exposure periods in Phase I1 and 24 hours after the end of the exposure period in Phase 111. The preparations were stained with polychrome methylene blue and eosin to visualise the various
cell types. One thousand polychromatic erythrocytes per slide were originally evaluated for the presence of micronuclei. An additional 2000
polychromatic erythrocytes were also evaluated for the presence of micronuclei from all slides from male animals exposed to the air control or
TDI 24 hours after exposure and female animals exposed to the air control or TDI 24 and 48 hours after exposure in Phase 11. In addition 1000
erythrocytes were counted to determine the percentage of polychromatic erythrocytes in the total erythrocyte population. This provides an
indication of any cytotoxicity in the bone marrow.

Statistics:

The incidence of micronucleated polychromatic erythrocytes and percentage
polychromatic erythrocytes in the erythrocyte sample, were considered by
analysis of variance, regarding each combination of sampling time,
concentration and sex as a separate group. The results were examined to
determine whether any differences between air control and TDI treated
groups were consistent between sexes and across sampling times. The data
from the extended counts were similarly analysed as an independent database
and also after combination with the original counts. All analyses were
carried out after calculating the average number of micronuclei per 1000
polychromatic erythrocytes. The values for micronucleated polychromat ic
erythrocytes were transformed using a natural logarithmic transformation,
to stabilise the variance, before analysis.
All analyses were carried out using the GLM procedure in SAS (1985).
Unbiased estimates of the group means were provided by the least square
means (LSMEANS option in SAS) but for simplicity standard means are
presented. Each treatment group mean was compared with the air control
group mean at the corresponding sampling time using a one-sided Student's
t-test based on the error mean square in the analysis.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Additional information on results:

3.1 Atmosphere Analysis
The TD1 and vinyl chloride group mean atmosphere concentrations for Phases I, I1 and I11 are detailed in Tables 13, 14 and 15 respectively,

Examination of peak areas of the gas chromatograph confirms that the ratio of 2,4 to 2,6 TDI in the exposure chambers approximated closely to the
expected ratio of 80:20. There was no TDI detected in control group atmospheres and no TDI or vinyl chloride were detected in the room air
samples.

3.2 Phase I - MLC Determination
Groups of 5 male and 5 female mice were exposed to TDI at target concentrations of 7, 10, 15, 20 and 30ppm. From the resultant mortalities
the MLC over a four day observation period was calculated by logistic regression as 14.lppm for females and by linear log interpolation as
19.0ppm for males. Atmosphere concentrations of 11.8 and 18.9ppm for males and 7.5 and 11.9ppm for females were used in Phase I1 of the study. In both cases these concentrations were selected to represent 50 and 80% respectively of the median lethal concentration (MLC). Due to an error in
the original calculation of the MLC values the target concentrations used actually represented 62 and 99% of the MLC in males and 53 and 84% of the
MLC in females.

3.3 Phase I1 and Phase I11 - Micronucleus Test
In Phase I1 of the study clinical signs were recorded for mice exposed to TDI as follows: male mice exposed to TDI at the target concentration of
11.8ppm had a reduced response to stimulus throughout the exposure period, subdued nature, increased breathing depth, reduced breathing rate and piloerection, whereas male mice exposedto TDI at 18.9ppm had no visible response to stimulus, very subdued nature, hunched posture, reduced
breathing rate and increased breathing depth. The males exposed to TDI at 18.9ppm were also noted to be subdued the day following exposure. In
addition, one male exposed to TDI at 18.9ppm was found dead in its cage approximately 48 hours after exposure. Female mice exposed to TDI at the target concentration of 7.5 and 11.9ppm exhibited reduced response to stimulus, reduced breathing rate and increased breathing depth during exposure. In addition the females exposed to the target concentration of 11.9ppm exhibited hunched posture and piloerection during exposure. After exposure females exposed to TDI at 7.5ppm exhibited hunched posture, subdued nature and piloerection, whereas those exposed at 11.9ppm exhibited clinical signs including subdued nature, hunched posture, piloerection and reduced temperature. In addition, one
female exposed to TDI at 11.9ppm was found dead in its cage 24 hours after the end of the exposure period.

Males exposed to vinyl chloride were noted to have a slightly subdued nature, a reduced response to stimulus and hunched posture during exposure, and one male exhibited a subdued nature the day following exposure. Females exposed to vinyl chloride were noted to be exhibiting a subdued
nature, hunched posture, pi loerection, reduced response to stimulus, reduced breathing rate and increased breathing depth during the exposure
peri~d'and hunched posture, subdued nature and. piloerection after exposure.


Small but statistically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the air control values,
were observed in females 24 hours after being exposed at the target concentration of 7.5ppm TDI and 24 and 48 hours after being exposed at the
target concentration of 11.9ppm . These increases were small and - not concentration-related.
Extended analysis of a further 2000 polychromatic erythrocytes from these animals and the female air control animals at the 24 and 48 hour time
points was conducted. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes
were observed in these extended counts. However, when the original and extended analyses were combined prior to statistical analysis
small but statistically significant increases were observed in females 24 hours after being exposed at both target concentrations.
Small but statistically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the air control values,
were observed in males 24 hours after being exposed at the target concentrations of 11.8 and 18.9ppm but there was no clear concentrationresponse relationship. Extended analysis of a further 2000
polychromatic erythrocytes from these animals and the male air control
animals at the 24 hour time point was conducted. A small but statistically
significant increase in the incidence of micronucleated polychromatic
erythrocytes was observed only at the lower target concentration (11.8ppm)
in these extended counts (Table 3) and when the original and extended
analyses were pooled prior to statistical analysis (Table 4).
In order to further investigate the increases observed in both males and
females exposed to TDI and the lack of concentrat ion-response relationships
observed a second assay was conducted. Groups of 5 male mice were exposed
to TDI for a 6 hour period by the inhalation route at target concentrations
of 5.9, 11.8 and 18.9ppm and groups of 5 female mice were similarly exposed
to TDI at target concentrations of 3.7, 7.5 and 11.9ppm. In both cases
these concentrations were selected to represent the concentrations used in
the first study with an additional lower concentration to investigate any
concentration-response relationships. Due to error in the original
calculation of the MLC values, the target concentrations used actually
represented approximately 31, 62 and 99% of the MLC in males and 26, 53 and
84% of the MLC in females. Bone marrow samples were taken 24 hours after
the end of the exposure period for all concentrations.
Adverse reactions to treatment was recorded for mice exposed to TDI.
Clinical signs recorded for male mice exposed to TDI at 5.9, 11.8 and
18.9ppm were reduced response to stimulus, subdued nature and decreased
breathing rate during exposure, although due to misting of the inside of
the exposure chamber difficulty was experienced in carrying out the
clinical observations on the 11.8 and 18.9ppm concentration groups. In
addition, 4 males exposed to TDI at 11.8ppm were found dead in their cages
and the remaining male was killed in extremis. One male exposed to TDI at
18.9ppm was killed in extremis.
Clinical signs recorded for female mice exposed to TDI at 3.7, 7.5 and
11.9ppm included reduced response to stimulus and reduced breathing rate.
In addition, females exposed to TDI at 7.5 and 11.9ppm exhibited hunched
posture and 1 itt le movement although difficulty was experienced in carrying
out the clinical observations due to misting of the exposure chambers. In
addition, six females exposed to TDI at 11.9ppm were found dead in their
cages following exposure to TDI.
Males exposed to vinyl chloride were noted to have a reduced response to
stimulus, piloerection and hunched posture whereas females exposed to vinyl
chloride exhibited a hunched posture and reduced response to stimulus.
In this second study high levels of lethality were observed at the 11.8ppm
(62% MLC) concentration in males and the 11.9ppm (84% MLC) concentration in
females and therefore the slides from the males exposed at the 5.9ppm
target concentration and the females exposed at the 3.7 and 7.5ppm target
concentrations only were analysed. The maximum concentration in each case
is considered to represent a maximum tolerated concentration (MT.C .) in this
second study.
No statistically or biologically significant increases in the incidence of
micronucleated pol ychromat ic erythrocytes, compared to the air control
values, were observed in the males exposed to TDI (Table 9).
Small but statistically significant increases in the incidence of
micronucleated polychromatic erythrocytes, over the air control values,
were observed in females 24 hours after being exposed at both the 3.7 and
7.5ppm target concentrations (Table 10). These increases were small and
the values fell within the range of female air control values reported in
this study.
Statistically significant decreases in the percentage of polychromatic
erythrocytes were observed in both males (24 and 48 hours; 18.9ppm; Table
7) and females (24 hours; 11.9ppm; Table 8) on the first study and in
females exposed at the 7.5ppm target concentration in the second study.
The test system positive control, vinyl chloride, induced statistically and
biologically significant increases in the incidence of micronucleated
polychromatic erythrocytes in both male and female animals at the 24 hour
sampling time on both studies (~ables 1, 2, 9 and 10).
4. DISCUSSION
The criteria for a valid test system as laid down by OECD Guideline 474
(1983) for the conduct of micronucleus studies, are that the positive
control substance should induce a significant elevation in micronucleated
polychromatic erythrocytes compared to the vehicle control values, and that
the test compound should be tested at a level that causes a decrease in the
percentage of polychromatic erythrocytes (indicating a cytotoxic effect on
the bone marrow) or at the maximum tolerated dose level.
The study satisfies these criteria in that TDI was tested in excess of 80%
of a median- lethal concentration (MLC), a concentration which also induced
adverse reactions to treatment. Consideration of the percentage of
polychromatic erythrocytes showed statistically significant decreases,
compared to the air control values, in both males and females in the first
study and in females in the second study. These decreases may indicate
that TDI or a metabolite has induced a cytotoxic response in the bone
marrow resulting in a depression of cell proliferation. The positive
control substance, vinyl chloride, gave a statistically significant and
biologically meaningful increase in micronucleated polychromatic
erythrocytes, compared to air control values, in both male and female mice
in both studies.
Small but statistically significant increases in the incidence of
micronucleated polychromatic erythrocytes, over the air control values,
were observed in both males and females exposed to TDI in the first study.
The increases at the 24 hour time point were confirmed by extended analysis
of the slides and a second assay was conducted to clarify the increases
observed.
No statistically or biologically significant increases were observed in the
males in the second study, and although small but statistically significant
increases were observed in the females the values fell within the range of
female air control values reported in this study. It is therefore
considered that the increases observed in the second study are due to a low
control value rather than to any effect of TDI. The increases are
therefore considered not to be biologically significant.
In summary, although increases in the incidence of micronucleated
polychromatic erythrocytes were observed in both males and females exposed
to TDI these increases were small, not concentration-related and were not
reproducible at concentrations limited by lethality in a repeat study. It
is therefore considered that the increases observed are of no biological
significance and do not indicate any clastogenic activity of TDI in the
mouse bone marrow micronucleus assay.

Any other information on results incl. tables

Exposure levels of 11.8 ppm in males and 11.9 ppm in females were lethal.   Therefore bone marrows assessed only at 5.9  ppm in males and 3.7, 7.5  ppm in females. No effect on male bone marrow at 5.9 ppm. 

Small statistically significant increase in micronucleated polychromatic erythrocytes (MPE) in females at 3.7 and 7.5 ppm.  However, the values  were within control range, therefore changes are not considered to be  biologically significant.

Positive control, vinyl chloride, induced statistically and  biologically  significant increases in MPE, demonstrating  sensitivity of test system.

Mean Incidence of MPE/1000 PE ±SD        

Conc.	24h	48h	72h	Extended Counts	Combined + Original
Males	.	.	.	(24h)	(24h)
Control	2.0±1.2	1.4±0.9	2.0±1.6	2.8±1.1	2.5±1.2
11.8 ppm	7.4±4.5**	.	5.9±2.1**	6.4±3.0**	.
18.9 ppm	4.4±2.0*	1.8±2.9	1.8±0.8	1.9±1.1	2.7±1.8
Repeat study	.	.	.	.	.
Control	1.2±0.5	.	.	.	.
5.9 ppm	2.0±1.9	.	.	.	.
.	.	.	.	.	.
Conc.	24h	48h	72h	Extended Counts	Combined + Original
Females	.	.	.	(24h)	(24h)
Control	0.4±0.9	0.6±0.6	1.4±0.6	1.4±1.1	1.1±1.4
.	.	.	.	1.3±1.1	1.1±1.0(48h)
7.5 ppm	4.0±1.4**	.	.	2.3±2.1	2.9±2.0**
11.9 ppm	1.8±1.5*	2.0±1.4*	0.5±0.6	2.0±1.4	1.9±1.4*
.	.	.	.	(48h)	(48h)
Repeat study	.	.	.	.	.
Control	0.2±0.5	.	.	.	.
3.7 ppm	1.2±0.8*	.	.	.	.
7.5 ppm	1.4±0.9*	.	.	.	.

**Sig. at p 0.01
*Sig. at p 0.05

Mean % Polychromatic Erythrocytes ±SD   

Conc.	24h	48h	72h
Males	.	.	.
Control	38.3±3.9	40.7±2.1	41.1±4.4
11.8 ppm	38.8±7.9	.	.
18.9 ppm	27.1±7.4*	29.8±13.5**	35.3±6.0
Repeat study	.	.	.
Control	48.4±3.3	.	.
5.9 ppm	46.1±4.1	.	.
.	.	.	.
Conc.	24h	48h	72h
Females	.	.	.
Control	41.7±5.8	34.7±7.1	37.1±7
7.5 ppm	39.1±5.0	.	.
11.9 ppm	34.0±3.1*	29.5±4.3	33.2±7.1
.	.	.	.
Repeat study	.	.	.
Control	47.3±2.8	.	.
3.7 ppm	43.9±1.0	.	.
7.5 ppm	40.1±8.0*	.	.
7.5 ppm	40.1±8.0*	.	.

**Sig. at p 0.01
*Sig. at p 0.05

Applicant's summary and conclusion

Conclusions:

Although increases in the incidence of micronucleated PCEs were observed in both males and females exposed to TDI, these increases were small,
not concentration-related and were not reproducible at concentrations limited by lethality in a repeat study. It is therefore considered that the
increases observed are of no biological significance and do not indicate clastogenic activity of TDI in the mouse bone marrow micronucleus assay.
TDI is not clastogenic in the mouse micronucleus test.