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Toxicological information

Genetic toxicity: in vivo

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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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
use of vinyl chloride as positive control
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Analytical purity: 99.5% mixed isomers TDI
- Radiochemical purity (if radiolabelling): 80.2% of 2,4-TDI isomer labelled, the rest being 2,6-TDI isomer
- Other: Analysed at 79.7% 2,4-TDI by test laboratory

Test animals

Species:
mouse
Strain:
C57BL
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Type: Male and female C57BL/6JfCD-l/Alpk mice
- Source: Barriered Animal Breeding Unit, ICI Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, UK.
- Age at study initiation: 6-12 weeks were used for Phase I, 8-12 weeks were used for Phase II, 8-9 weeks were used for Phase III
- Housing: 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
- Diet (e.g. ad libitum): Porton Combined Diet [PCD] (supplied by Special Diets Services Limited, Stepfield, Witham, Essex, UK; Appendix D)
- Water (e.g. ad libitum): filtered tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 21°C
- Humidity (%): range of 45-55% (relative humidity = RH, 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.)
- Air changes (per hour): The animal room was under positive pressure with respect to the access corridor and had 20-30 air changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled to provide 12 hours artificial light followed by 12 hours darkness.

Administration / exposure

Route of administration:
inhalation
Vehicle:
- Vehicle(s)/solvent(s) used: [air]
Details on exposure:
a) TDI - Phase I
The 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 a jacketed bubbler containing the test material. 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 vaporised test material and was split into two streams, each stream being passed to one of the two 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 flowmeters (KDG Flowmeters, Burgess Hi 11, Sussex, UK) and were recorded at approximately 30 minute intervals during the exposure periods.
b) TDI - Phase II and Phase III
The atmospheres for Phases II and III 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
Frequency of treatment:
Phase I: single 6-hour inhalation period
Phase II: single 6-hour inhalation period
Phase III: single 6-hour inhalation period
Post exposure period:
Phase I: four-day observation
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
18.9 ppm (males, Phase II and III)
Basis:

Remarks:
Doses / Concentrations:
3.7 ppm (females, Phase III)
Basis:

Remarks:
Doses / Concentrations:
5.9 ppm (males, Phase III)
Basis:

Remarks:
Doses / Concentrations:
7.5 ppm (females, Phase II and III)
Basis:

Remarks:
Doses / Concentrations:
11.8 ppm (males, Phase II and III) / 11.9 ppm (females, Phase II and III)
Basis:

No. of animals per sex per dose:
5
Control animals:
yes
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 II and 24 hours after the end of the exposure period in Phase III. 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 II. 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 polychromatic 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
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Atmosphere Analysis
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.

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 14.1 ppm for females and by linear log interpolation as 19.0 ppm for males. Atmosphere concentrations of 11.8 and 18.9 ppm for males and 7.5 and 11.9 ppm for females were used in Phase II 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.

Phase II and Phase III - Micronucleus Test
In Phase II of the study clinical signs were recorded for mice exposed to TDI as follows: male mice exposed to TDI at the target concentration of
11.8 ppm had a reduced response to stimulus throughout the exposure period, subdued nature, increased breathing depth, reduced breathing rate and piloerection, whereas male mice exposed to TDI at 18.9 ppm had no visible response to stimulus, very subdued nature, hunched posture, reduced breathing rate and increased breathing depth. The males exposed to TDI at 18.9 ppm were also noted to be subdued the day following exposure. In addition, one male exposed to TDI at 18.9 ppm was found dead in its cage approximately 48 hours after exposure. Female mice exposed to TDI at the target concentration of 7.5 and 11.9 ppm exhibited reduced response to stimulus, reduced breathing rate and increased breathing depth during exposure. In addition the females exposed to the target concentration of 11.9 ppm exhibited hunched posture and piloerection during exposure. After exposure females exposed to TDI at 7.5 ppm exhibited hunched posture, subdued nature and piloerection, whereas those exposed at 11.9 ppm exhibited clinical signs including subdued nature, hunched posture, piloerection and reduced temperature. In addition, one female exposed to TDI at 11.9 ppm 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, piloerection, reduced response to stimulus, reduced breathing rate and increased breathing depth during the exposure period 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 7.5 ppm TDI and 24 and 48 hours after being exposed at the target concentration of 11.9 ppm. 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 18.9 ppm but there was no clear concentration-response 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 and when the original and extended analyses were pooled prior to statistical analysis.
In order to further investigate the increases observed in both males and females exposed to TDI and the lack of concentration-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 18.9 ppm and groups of 5 female mice were similarly exposed to TDI at target concentrations of 3.7, 7.5 and 11.9 ppm. 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 were recorded for mice exposed to TDI. Clinical signs recorded for male mice exposed to TDI at 5.9, 11.8 and 18.9 ppm 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 18.9 ppm concentration groups. In addition, 4 males exposed to TDI at 11.8 ppm were found dead in their cages and the remaining male was killed in extremis. One male exposed to TDI at 18.9 ppm was killed in extremis.
Clinical signs recorded for female mice exposed to TDI at 3.7, 7.5 and 11.9 ppm included reduced response to stimulus and reduced breathing rate.
In addition, females exposed to TDI at 7.5 and 11.9 ppm exhibited hunched posture and little 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 11.9 ppm 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 11.8 ppm (62% MLC) concentration in males and the 11.9 ppm (84% MLC) concentration in females and therefore the slides from the males exposed at the 5.9 ppm target concentration and the females exposed at the 3.7 and 7.5 ppm 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 polychromatic erythrocytes, compared to the air control
values, were observed in the males exposed to TDI.
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 7.5 ppm target concentrations. 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; 18.9 ppm) and females (24 hours; 11.9ppm) on the first study and in females exposed at the 7.5 ppm 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.

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 were 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 was observed. 

Small statistically significant increase in micronucleated polychromatic erythrocytes (MPE) in females at 3.7 and 7.5 ppm occurred.  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:
Interpretation of results (migrated information): negative
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 repeated 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.
Executive summary:

According to good laboratory practice (GLP), a reliable study without restrictions (Klimisch 1) was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice (Mackay, 1992). The method used has been designed to comply with the OECD Guideline 474 and the corresponding validity criteria are considered as fulfilled.

A range-finding (Phase I, female mice: 7.5 ppm and 11.9 ppm, male mice: 11.8 ppm and 18.9 ppm) was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes.

In Phase II, male and female animals were given TDI at target concentrations of 7.5 and 11.9 ppm for females and 11.8 and 18.9 ppm for males.

Following the results of the cytogenetic analysis of Phase II, Phase III was conducted by giving male and female animals TDI at target concentrations of 3.7, 7.5 and 11.9 ppm for females and 5.9, 11.8 and 18.9 for males. Additionally, Phase II as well as Phase III was conducted by giving male and female animals vinyl chloride as positive control at a target concentration of 50000 ppm. Besides, further groups of mice were given a single 6 hour inhalation to air, to serve as vehicle, respectively.

The atmospheres for Phase I-III of the study were generated by passing clean dry air from the laboratory air supply via a flow controller and a flow meter to a jacketed bubbler containing the test material or the control. The frequency of treatment is described as a single 6 -hour inhalation period, respectively. Analytical measurements during the study period were performed by gas chromatography which confirmed 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.

Animals were killed 24, 48 or 72 hours later, the bone marrow was extracted and smear preparations were made and stained. Polychromatic and normochromic erythrocytes were scored for the presence of micronuclei.

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 repeated 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.