m-tolylidene diisocyanate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976-10 to 1978-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1976-10 to 1978-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

2 doses investigated

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 - 9 weeks
- Housing: 6 ♂ / 6 ♀ per cage

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 9 m³ chambers

Analytical verification of doses or concentrations:

yes

Remarks:

Atmospheres monitored at least 5 times each day by two methods: Marcali test and test paper monitor.

Duration of treatment / exposure:

6 h / day

Frequency of treatment:

daily, 5 days per week, up to 113 weeks

Post exposure period:

no

Dose / conc.:

0.05 ppm (nominal)

Dose / conc.:

0.15 ppm (nominal)

No. of animals per sex per dose:

126

Control animals:

yes, sham-exposed

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, before and after exposure

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the firt 8 weeks, thereafter monthly.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 6, 12, 18 months, and termination.
- How many animals: 7♂/ 7♀
- Parameters checked: Hb, RBC, PCV, WBC

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 6, 12, 18 months, and termination.
- How many animals: 7♂/ 7♀
- Parameters checked: BUN, AlkP, GPT

URINALYSIS: Yes
- Time schedule for collection of urine: after 6, 12, 18 months, and termination.

Sacrifice and pathology:

GROSS PATHOLOGY:
Yes, interim sacrifices on 7♂ and 7♀ at 6, 12, and 18 months. Gross examination on all rats dying and at scheduled sacrifice. Organ weights on brain, liver, heart, gonads, and kidneys.

HISTOPATHOLOGY:
Yes, on 35 tissues from each rat.

Statistics:

Survival probabilities for each group were calculated by the method of Kaplan and Meier (J. Am. Statist. Ass. 53: 457 (1958)). The survival of the individual treatment groups was compared using the log rank method of Peto and Pike (Biometrics 29: 579 (1973)). All tumors observed where the difference between tumor incidence in treated animals and controls was 4 or less were not statistically significant (United States of America, National Research Council, Food Protection Committee, Nat. Acad. Sci. Publ., 749 (1959)).
Since neither in the publication (Loeser, 1983) nor in the study report (Owen, 1980+1984) statistics for tumor incidences were presented, the tumor data for rats were reassessed using both the method cited in the original protocol and the more current one-sided Fisher's exact test (Müller, 2008).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEC

Effect level:

0.15 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

The results for general toxicity of this study are described in chapter 7.5.2

With respect to the assessment of tumour formation, there were slight increases in the tumour incidence for some locations when compared to the incidence in the controls. In Table 1, specific tissues and the number of tumours in the various treatment and control groups are listed only if there were at least two more tumours in the groups treated with TDI than in the controls. The author concluded, that the type and incidence of tumours and the numbers of tumour-bearing rats were similar in both control and TDI treated groups.

A statistical analysis of the results was not carried out, but the tumour spectrum in all groups was in the range of the general experience (Loeser, 1983). A full assessment of the original tumour data by both the protocolled method and more current methodology has been reported by Müller (2008). Overall, and taking into account the statistical results, contemporary control animal data from the laboratory and the tumour profiles of the animal strain used, Müller concluded that there was no toxicologically significant increase in the tumour incidences of this long-term TDI rat study.    

The overall evaluation of the study therefore led to the conclusion, that TDI is not carcinogenic in rats under the conditions described.

 

Table 1: Tumour incidences of the 2-year rat study with TDI (80/20)

TDI (ppm)	Sex	0	0.05	0.15
Number of animals investigated	M/F	104/104	104/105	104/105
Types of tumours for which there were at least 2 more tumours in the groups treated with than in the controls:
Adenomas (skin)	M	0	0	3
Papillomas (skin)	F	1	3	3
Benign tumours (mammary gland)	F	24	27	22
Carcinomas (mammary gland)	F	9	9	14
Fibromas (subcutis/muscle/bones)	M/F	29/1	22/1	35/4
Histiocytomas (subcutis/muscle/bones)	M	1	4	1
Malignant lymphomas (haematopoietic system)	F	1	1	3
Haemangiomas (haematopoietic system)	M	1	1	4
Islet cell adenomas (pancreas)	M	1	2	3
Adenomas (pituitary gland)	F	64	62	67
Meningiomas (brain)	M	0	0	2
Lipomatous tumours (kidney)	M	1	3	0

M = male; F = female

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Reference 3

Reference Type:

publication

Title:

Unnamed

Year:

1983

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

2 doses investigated

GLP compliance:

yes (incl. QA statement)

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

Molecular formula:

C9H6N2O2

IUPAC Name:

2,4-diisocyanato-1-methylbenzene, 2,6-diisocyanato-1-methylbenzene

Details on test material:

- Name of test material (as cited in study report): 2,4/ 2,6-TDI (80:20), production grade

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 - 9 weeks

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION:
- Method of holding animals in test chamber: Animals were housed 6 ♂ / 6 ♀ per cage and exposed in 9 m³ chambers.
- Method of conditioning air: Passage of dry nitrogen through liquid TDI maintained at 21°C. The vapour produced was then diluted with air in all-glass systems to produce the desired exposure concentration.

OTHER:
Because of the known chemical reactivity and thus its potential storage instability, TDI lots were renewed every three months and fresh TDI was placed in the respective vapour-generating apparatus at the beginning of each daily exposure period. The measured mean test concentrations of TDI were 0.052 ppm and 0.146 ppm over a period of up to 113 weeks, with standard deviations of 0.007 ppm and 0.014 ppm, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

5/day, each exposure group.
U.E.I. Model 7000 TDI tape monitor and the Marcali colorimetric method.

Duration of treatment / exposure:

up to 113 weeks

Frequency of treatment:

6 hours/day, 5 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.05 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

0.15 ppm (nominal)

No. of animals per sex per dose:

126

Control animals:

yes, sham-exposed

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
BODY WEIGHT: Yes
OPHTHALMOSCOPIC EXAMINATION: Yes
HAEMATOLOGY: Yes
CLINICAL CHEMISTRY: Yes
URINALYSIS: Yes

Interim sacrifices were performed after 6, 12, and 18 months of exposure consisting of 7 animals/sex/dose.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
ORGAN WEIGHTS: Yes

Other examinations:

Special histopathology of the nasal cavity.

Statistics:

Survival probabilities for each group were calculated by the method of Kaplan and Meier (J. Am. Statist. Ass. 53: 457 (1958)). The survival of the individual treatment groups was compared using the log rank method of Peto and Pike (Biometrics 29: 579 (1973)).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Compared to control rats, there were significantly reduced body weight gains in males and in females of the highest dose groups up to study week 12 that remained significantly reduced throughout the study for female rats.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In rats sacrificed as scheduled (interim sacrifices after 6, 12 and 18 months + terminal sacrifice), lesions were localized primarily in the anterior respiratory mucosa of the nasal cavity. Rhinitis was increased in incidence and severity (grades1-4: minimal - marked rhinitis, generally characterized by squamous metaplasia and hyperplasia of the epithelium, leucocyte infiltration in the lamina propria and exudate in the lumen) in a dose-dependent manner and is considered to be due to local irritation.

Histopathological findings: neoplastic:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEC

Effect level:

0.05 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

2

Key result

Dose descriptor:

NOAEC

Effect level:

< 0.05 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no NOAEC identified

3

Dose descriptor:

LOAEC

Effect level:

0.15 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

4

Dose descriptor:

LOAEC

Effect level:

0.05 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

No exposure-related clinical signs of toxicity were observed during the study. No treatment-related changes in hematology, blood biochemistry, urinalyses, ophthalmoscopy or organ weights were recorded.

Compared to control rats, there were significantly reduced body weight gains in males and in females of the highest dose groups up to study week 12 that remained significantly reduced throughout the study for female rats. Mortality occurred in both test and control groups, with generally higher mortality rates at earlier time points in test groups than in the controls for males, providing additional supporting evidence indicating that MTD was reached.

Table 1: Body weight gain and mortality rate of rats during exposure

	Males			Females
	Control	Low dose	High dose	Control	Low dose	High dose
Group mean body weight gain (g): week 0 –12	298	296	275*	136	132	121*
Group mean body weight gain (g): week 0 - termination	554	579	525	395	378	345*
Mortality before interim sacrifice after 6 m	3/126	13/126	11/126	0/126	1/126	0/126
Mortality before interim sacrifice after 12 m	4/116	1/106	1/108	1/119	3/118	1/119
Mortality before interim sacrifice after 18 m	7/105	8/97	13/100	14/111	20/108	9/110
Mortality before terminal sacrifice	52/89	46/81	50/80	53/88	55/81	46/84

* P <0.01 when compared to the controls

In rats sacrificed as scheduled (interim sacrifices after 6, 12 and 18 months + terminal sacrifice), lesions were localized primarily in the anterior respiratory mucosa of the nasal cavity. Rhinitis was increased in incidence and severity (grades1-4: minimal - marked rhinitis, generally characterized by squamous metaplasia and hyperplasia of the epithelium, leucocyte infiltration in the lamina propria and exudate in the lumen) in a dose-dependent manner and is considered to be due to local irritation.

Table 2: Incidence of rhinitis in the anterior nasal cavity of rats

	Males			Females
	Control	Low dose	High dose	Control	Low dose	High dose
Grade 0*	30/58	30/55	6/51	46/56	27/47	17/57
Grade 1*	11/58	7/55(13 %)	5/51	7/56	8/47(17 %)	16/57(28 %)
Grade 2*	13/58	15/55 (27 %)	13/51(25 %)	3/56	9/47(19 %)	18/57(32 %)
Grade 3*	4/58	3/55	23/51 (45 %)	0	3/47(6 %)	5/57(9 %)
Grade 4*	0	0	4/51	0	0	0
No section	0	0		0	0	2
Percent rats with grade1- 4 rhinitis	48 %	45 %	88 %	18 %	43 %	70 %

* Grade 0 = unremarkable; Grade 1 = minimal rhinitis, Grade 2 = slight rhinitis, Grade 3 = moderate rhinitis, Grade 4 = marked rhinitis generally characterized by squamous metaplasia and hyperplasia of the epithelium, leucocyte infiltration in the lamina propria and exudate in the lumen