Tris(p-isocyanatophenyl) thiophosphate

Endpoint summary

Description of key information

For tris(p-isocyanatophenyl) thiophosphate an LD50 of > 675 mg/kg bw  can be calculated as worst-case assumption. The aerosolized test substance proved to have low acute inhalation toxicity in rats. 

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

SPF-bred Wistar rats of the strain HdsCpd:WU
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 206-220 g (males) and 158-165 g (females)
- Fasting period before study: about 17 h

Route of administration:

oral: gavage

Vehicle:

corn oil

Doses:

2000 mg/kg

No. of animals per sex per dose:

3

Control animals:

no

Sex:

male/female

Dose descriptor:

discriminating dose

Effect level:

> 675 mg/kg bw

Based on:

act. ingr.

Remarks on result:

other: calculated from the LD50 of the trade product (27% active ingredient in ethyl acetate)

Sex:

male/female

Dose descriptor:

other: LD50 (cut off value)

Effect level:

> 2 500 mg/kg bw

Remarks on result:

other: based on: the trade product (solution of 27% tris(p-isocyanatophenyl)thiophosphate in ethyl acetate); a dose of 2000 mg/kg bw was tolerated without any findings

Mortality:

no deaths

Clinical signs:

other: none

Gross pathology:

no findings

Other findings:

none

Dose (mg/kg bw)	Toxicological results	Onset and duration of signs	Onset of mortality
2000	0 / 0 / 3	---	---
2000	0 / 0 / 3	---	---

Toxicological results:

number of dead animals / number of animals with signs after treatment / number of animals treated

Executive summary:

The acute oral toxicity of the test item (27 % active ingredient in ethyl acetate) was evaluated in a GLP-compliant study on male and female Wistar rats according to OECD TG 423 (Krötlinger, 2002). The oral dose of 2000 mg/kg bw was tolerated without mortalities, clinical signs, effects on weight gain or gross pathological findings. According to OECD TG 423 the LD50 cut-off level of the test item was determined with > 2500 mg/kg bw. Thus, for the active ingredient the discriminating dose can be calculated with > 675 mg/kg bw (27% of 2500 mg/kg bw).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

675 mg/kg bw

Quality of whole database:

The study is GLP compliant and has Klimisch score 1.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Hsd Cpb:WU (SPF)
- Source: Harlan-Nederland (NL), AD Horst
- Age at study initiation: approximately 2 months
- Weight at study initiation: At the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex
- Housing: singly in conventional Makrolon® Type IIIH cages
- Diet and water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 40 - 60 %
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Animals were exposed to the aerosolized test article in restrainers made of Plexiglas. The design of the directed-flow inhalation chamber minimizes rebreathing of the exhaled test atmosphere.
- Generation of atmosphere: Under dynamic conditions the targeted concentrations were achieved by atomization using the nozzle-baffle-system and inhalation chamber shown in Fig. 1. For atomization a binary nozzle (Schlick water jacketed nozzle which was connected to a thermostat, 1 DoC using a digitally controlled water bath connected to a cryostat) and conditioned compressed air was used (15 Umin). The representative dispersion pressure was approximately 500 kPa (for more details see Table 1, result section). The test article was fed into the nozzle system using a digitally controlled pump (Harvard PHD 2000 infusion pump).
- Inhalation chamber: One segment of the aluminum inhalation chamber has the following dimensions: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 L). Details of this modular chamber and its validation have been published previously (Pauluhn, Journal of Applied Toxicology, 14, 55-62, 1994).
- Optimization of respirability: In order to increase the efficiency of the generation of fine particles likely to evaporate and to prevent larger particles from entering the chamber a pre-separator (baffle) system was used (Tillery et aI., 1976).
- Inhalation chamber equilibrium concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour [15 Llmin x 60 min/{3.B L) = 237, continuous generation of test atmosphere]. Based on OECD GD39 the equilibrium concentration (t9S) can be calculated after McFarland, 1976. Thus, under the test conditions used chamber equilibrium is attained in less than one minute of exposure. At each exposure port a minimal air flow rate of 0.75 L/min was provided. The test atmosphere can by no means be diluted by bias-air-flows.
- Conditioning the compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically by a VIA compressed air dryer. Adequate control devices were employed to control supply pressure.
- Exhaust air treatment: The exhaust air was purified via filter systems.
- Temperature and humidity measurements were performed by a computerized Data Acquisition and Control System using HC-S3 sensors (Rotronic). The posture of the probe was at the exposure location of rats (cf. Fig. 1). Temperature and humidity data are integrated for 30-seconds and displayed accordingly. The humidity sensors are calibrated using saturated salt solutions according to Greenspan (1977) and Pauluhn (1994) in a two-point calibration at 33% (MgCI2) and at 75% (NaCI) relative humidity. The calibration of the temperature sensors is also checked at two temperatures
using reference thermometers.

TEST ATMOSPHERE
- Nominal concentration-atomization: The nominal concentration was calculated from the ratio of the total quantity of test article consumed and not captured by the pre-separating system during the exposure period and the total throughput of air through the inhalation chamber.
- Samples taken from breathing zone: yes
- Brief description of analytical method used: gravimetric analysis of filter samples (filter: Glass-Fibre-Filter, Sartorius, Göttingen, Germany; digital balance).
- Particle size distribution: The particle-size distribution was analyzed using a BERNER critical orifice cascade impactor. Specifications and evaluations are provided in the Appendix (pp. 37). Each impactor stage was covered by a combination of glass fiber (top) and aluminum foil to prevent any run-off of impacted liquid. Accordingly, an adhesive stage coating (silicone spray) was not used to minimize particle bounce due to the adhesive properties of the test article. Gravimetric analyses of filters used a digital balance.
- MMAD (Mass median aerodynamic diameter): 2.4 µm (mean MMAD = 1.5-1.8 µm) / GSD (Geometric st. dev.): 1.9

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

1554, 3622, and 6597 mg/m³ (actual concentration for active ingredient)

No. of animals per sex per dose:

5

Control animals:

other: Comparison with an appropriate historical control were performed. This control was exposed to an atmosphere using essentially similar exposure conditions as were used for the test substance (15 L air/min; conditioned air)

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Body weights were measured before exposure, on days 3 and 7, and weekly thereafter. The appearance and behavior of each rat were examined carefully several times on the day of exposure and at least once daily thereafter. Weekend assessments were
made once a day (morning). Assessments from restraining tubes were made only if unequivocal signs occurred (e.g. spasms, abnormal movements, and severe respiratory signs).
- Necropsy of survivors performed: yes
- Other examinations performed: The rectaI temperatures were measured shortly after cessation of exposure using a digital thermometer with a rectal probe for rats.

Statistics:

Body weights: Means and single standard deviations of body weights are calculated. Mean body weights are also depicted graphically as a function of time. Since in acute studies individual group means may differ prior to commencement of the first exposure, the body weight gain was statistically evaluated for each group. For these evaluations a one-way ANOVA (vide infra) is used.
Physiological data: Data of rectal temperature measurements are statistically evaluated using the ANOVA procedure (vide infra).
Calculation of the LCso: If calculation of a median lethal concentration (LCSO) is possible, it is performed by computer (PC) according to the method of Rosiello et al. (1977) as modified by Pauluhn (1983). This method is based on the maximumlikeli hood method of Bliss (1938). If only 2 pairs of values with greater than 0% lethality and less than 100% are available then the first linear approximation is based on these values and a l-homogeneity test is not performed. In this case the interpolated concentration at SO% lethality is designated the approximate LCSO. Additionally, the moving average interpolation according to Schaper et al. (1994) is used for calculation, if applicable.
Analysis of variance (A NO VA): This parametric method checks for normal distribution of data by comparing the median and mean. The groups are compared at a confidence level of (1-a) = 9S% (p = O.OS). The test for the between-group homogeneity of the variance employed Box's test if more than 2 study groups were compared with each other. If the above F-test shows that the intra-group variability is greater than the inter-group variability, this is shown in the Appendix as "no statistical difference between the groups". If a difference is found then a pairwise post-hoc comparison is conducted (1- and 2-sided) using the Games and Howell modification of the Tukey-Kramer significance test.

Sex:

male

Dose descriptor:

LC50

Effect level:

5 721 mg/m³ air

Based on:

act. ingr.

95% CL:

3 634 - 9 008

Exp. duration:

4 h

Sex:

female

Dose descriptor:

LC50

Effect level:

> 6 597 mg/m³ air

Based on:

act. ingr.

Exp. duration:

4 h

Sex:

male/female

Dose descriptor:

other: NOAEL

Effect level:

< 1 554 mg/m³ air

Based on:

act. ingr.

Exp. duration:

4 h

Mortality:

Mortality did occur in the group 3 (3622 mg/m³) and 4 (6597 mg/m³). Animals were found dead on the exposure day or up to the first postexposure day. Marked gender-specific differences indicative of sex-specific susceptibilities were not observed. However, at the high exposure level, the mortality in male rats was higher than in female rats.
Animals that died (onset of mortality): Data for males in respective dose group 0/5 at 1554 mg/m³; 1/5 at 3633 mg/m³ (1d); 3/5 at 6597 mg/m³ (0d). Data for females in respective dose group 0/5 at 1554 mg/m³; 1/5 at 3622 mg/m³ (0d); 1/5 at 6597 mg/m³ (1d).

Clinical signs:

other: Dose group 1554 mg/m³: all rats tolerated the exposure without specific signs. Rats exhibited clinical signs in dose group 3622 mg/m³ and above. The following signs occured, e.g.: Labored breathing pattern, irregular breathing pattern, bradypnea, tachypne

Body weight:

Comparisons between the control and the exposure groups revealed significant, although transient changes (decreases) in body weights.

Gross pathology:

Animals sacrificed at the end of the observation period: The macroscopic findings of extrapulmonary organs were essentially indistinguishable between the test article exposure and control groups. In the previous lungs showed a higher incidence of discolorations.
Animals that succumbed during the observation period: Nostrils with foamy and/or white powder depositions, larynx: white deposits, lung less collapsed with foamy whitish content in trachea, hydrothorax, stomachlintestines with white pasty/yellowish content and reddish discoloration of mucosa, walls of ventricle thickened, and discoloration/ blood-less appearance of parenchymatous organs.

Other findings:

A battery of reflex measurements was made on the first post-exposure day. In comparison to the rats of the control group, some rats of group 2 to 4 displayed impaired reflexes.
Statistical comparisons between the control and the exposure groups revealed significant changes in body temperatures indicative of hypothermia.

Pre-test data demonstrated that this substance had no potential to inhibit cholinesterases (plasma, brain) to any appreciable extent. Therefore, this endpoint was not considered further in the main study.

Executive summary:

A study on the acute inhalation toxicity on rats has been conducted in accordance with OECD TG 403. Three groups of 5 male and female rats were nose only exposed (4 h) to the liquid aerosol of the test article in actual concentrations (active ingredient) of 1554, 3622, and 6597 mg/m³. The aerosol was generated so that it was respirable to rats (average MMAD of 2.4 µm / GSD 1.9). At 3622 and 6597 mg/m³ some deaths were recognized. Distinct deposits of test article were observed in the respiratory tract of these animals. At the high exposure level the mortality was higher in male than in female rats. Clinical observation showed evidence of respiratory irritation at 3622 mg/m³ and above. Based on the results the LC50 was determined with 5721 mg/m³ for males (Probit method) and > 6597 mg/m³ for females. In summary, the aerosolized test substance (solid aerosol, active ingredient) proved to have a low acute inhalation toxicity in rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

5 721 mg/m³ air

Quality of whole database:

The study is GLP compliant and has Klimisch score 1.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Tris(p-isocyanatophenyl) thiophosphate is marketed and handled as solution in ethyl acetate containing approximately 27% of tris(p-isocyanatophenyl) thiophosphate. Removal of the solvent from a solution of 27% of tris(p-isocyanatophenyl) thiophosphate in ethyl acetate does invariably lead to generation of higher molecular weight species. This is due to the inherent reactivity of the isocyanate moieties and the process can thus be monitored via the decrease of the isocyanate content (see IUCLID section 1.4: analytical material balances before/after solvent removal). Therefore, a solution of 27% of tris(p-isocyanatophenyl) thiophosphate in ethyl acetate was employed as test substance for all toxicological tests as this was believed to best represent the substance to be registered. In toxicological studies with inhalative exposure to Desmodur RFE the solvent evaporated during the exposure procedure so that the samples taken from the breathing zone were nearly solvent free. Thus, in the inhalation studies the exposure can be considered as exposure to the neat active ingredient tris(p-isocyanatophenyl)thiophosphate.

Acute oral toxicity

The acute oral toxicity of the test item (27 % active ingredient in ethyl acetate) was evaluated in a GLP-compliant study on male and female Wistar rats according to OECD TG 423 (Krötlinger, 2002). The oral dose of 2000 mg/kg bw was tolerated without mortalities, clinical signs, effects on weight gain or gross pathological findings. According to OECD TG 423 the LD50 cut-off level of the test item was determined with > 2500 mg/kg bw. Thus, for the active ingredient the discriminating dose can be calculated with > 675 mg/kg bw (27% of > 2500 mg/kg bw). The available data do not indicate that tris(p-isocyanatophenyl) thiophosphate is systemically available after oral uptake.

Acute inhalation toxicity:
A study on the acute inhalation toxicity on rats has been conducted in accordance with OECD TG 403 (Pauluhn, 2011a). Three groups of 5 male and female rats were nose only exposed (4 h) to the liquid aerosol of the test item (27% of tris(p-isocyanatophenyl) thiophosphate in ethyl acetate in actual concentrations of 1554, 3622, and 6597 mg/m³ (gravimetric concentrations of the active ingredient). The aerosol was generated so that it was respirable to rats (average MMAD of 2.4 µm / GSD 1.9).At high concentrations of ≥ 3622 mg/m³ concomitant with mortality and signs of respiratory irritation distinct test article deposits were observed in the respiratory tract.Based on the results the LC50 was determined with 5721 mg/m³ for males (Probit method) and > 6597 mg/m³ for females. In summary, the aerosolized test substance (solid aerosol, active ingredient) proved to have a low acute inhalation toxicity in rats.

A pulmonary irritant potency study on male rats according to OECD TG 403 and TRGS 430* has been conducted in order to analyze the concentration- and time-dependence onset of alveolar damage following a single 6-hour nose-only exposure to the aerosolized test item (Pauluhn, 2011b). The aerosol was gererated in gravimetric concentrations of 5.6, 25.8, and 158.3 mg/m³ (active ingredient). Bronchoalveolar lavage fluid (BAL-fluid) of the rat's lungs was examined on postexposure days 1, 3, and 7. At 5.6 mg/m³ the exposure was tolerated without specific effects while the rats exposed to 25.8 and 158.3 mg/m³ experienced clinical signs related to a slight pulmonary irritation up to the fourth postexposure day. All endpoints were fully reversible at the end of the 7-day postexposure period. The magnitude of PMN (polymorphonuclear cells) recruitment, the most sensitive parameter in BAL fluid, observed in at 25.8 mg/m³ was still in a range considered to be non-adverse. These findings suggest that the test article is a mild pulmonary irritant at concentrations of about 26 mg/m³ and above. The exposure level considered to be the NOAEL for pulmonary irritation is 5.6 mg/m³.

(*Technical Rule for Hazardous Substances 430, related to handling of isocyanates; published by the German Federal Ministry of Labour and Social Affairs.)

Acute dermal toxicity

Acute dermal toxicity studies are not available for tris(p-isocyanatophenyl)thiophosphate.

Justification for selection of acute toxicity – oral endpoint
Only one study available

Justification for selection of acute toxicity – inhalation endpoint
Only one study available

Justification for classification or non-classification

The acute oral discriminating dose of tris(p-isocyanatophenyl) thiophosphate is calculated with > 675 mg/kg bw.

Consequently, tris(p-isocyanatophenyl) thiophosphate should be regarded as harmful if swollowed and classified with Acute Toxicity Category 4 according to 1272/2008 EC (H302).

The acute inhalation toxicity of tris(p-isocyanatophenyl) thiophosphate was proven to be low with an LC50 in male rats of 5721 mg/m³. Thus, no classification with regard to acute inhalation toxicity is required.