Reaction mass of 2-ethylhexyl (6-isocyanatohexyl)-carbamate and bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

Endpoint summary

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

Study period:

July 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1996)

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

- Stability under test conditions: A stability test in the formulation at 0.5 and 40 % (w/w) revealed no significant degradation of the test item up to at least 4 hours (A 01/0207/04 LEV).

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: HsdCpb:WU (SPF-bred)
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: males 209-213 g, females 163-166 g
- Fasting period before study: 17 +/- 2 hours
- Housing: conventionally in polycarbonate cages
- Diet and water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 5
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Administration volume: 5 mL/kg

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Appearance and behaviour were recorded several times on the day of treatment, and at least once a day thereafter. Body weights were recorded on Day 1 before administration and then weekly. All animals that died or are sacrificed are weighed.
- Necropsy of survivors performed: yes

Sex:

male/female

Dose descriptor:

discriminating dose

Effect level:

2 000 mg/kg bw

Based on:

test mat.

Mortality:

No animals died.

Clinical signs:

Diarrhoea was observed in both genders, in males up to day 2 and in females up to 5 hours post dosing. Additionally decreased  motility was observed in females 2 hours post application.

Body weight:

The body weight and the body weight development of males and females were not affected by the treatment.

Gross pathology:

No findings

Executive summary:

An acute oral toxicity study in rats, according to OECD TG 423, is available for the substance. In this study the limit dose of 2000 mg/kg bw, formulated in corn oil, was administered by gavage to each of the 3 male and 3 female rats.

The dose of 2000 mg/kg was tolerated by the animals without mortalities. Diarrhoea was observed in both genders, in males up to day 2 and in females up to 5 hours post dosing. Additionally decreased motility was observed in females 2 hours post dosing. Body weights/body weight development was not affected by the treatment and no gross pathological findings were observed. Concluding, the LD50 in this study was >2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

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

Study period:

April-May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

(2009)

Qualifier:

according to guideline

Guideline:

other: OECD GD 39 (Guidance Document on Acute Inhalation Toxicity Testing)

Version / remarks:

(2009)

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

no

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, AD Horst, Netherlands
- 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. The legal requirements for housing experimental animals (Directive 86/609 EEC) are followed.
- 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:

air

Mass median aerodynamic diameter (MMAD):

>= 1.4 - <= 1.46 µm

Geometric standard deviation (GSD):

>= 1.76 - <= 1.85

Remark on MMAD/GSD:

The respirability of the aerosol was adequate and in compliance with test guidelines.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Animals were exposed to the aerosolized test substance in Plexiglas exposure restrainers. Restrainers were chosen that accommodated the animals' size. The type of exposure principle is comparable with a directed-flow exposure design (Moss and Asgharian, Respiratory Drug Delivery IV, 1994, 197).
- Exposure apparatus: The chambers used are commercially available (TSE, Bad Homburg, Germany) and the performance as well as their validation has been published (Pauluhn, Journal of Applied Toxicology 14, 55-62, 1994, and Pauluhn & Thiel, Journal of Applied Toxicology 27, 160-167, 2007). Each inhalation chamber segment was suitable to accommodate 20 rats at the perimeter location. The ratio between supply and exhaust air was selected so that 90 % of the supplied air was extracted via the exhaust air location and, if applicable, via sampling ports. The slight positive balance between the air volume supplied and extracted ensured that no passive influx of air into the exposure chamber occurred.
- Source and rate of air: Conditioned (dry, oil free) compressed air, 15 L/min
- Method of conditioning air: Compressed air was supplied by Boge compressors and was conditioned (freed from water, dust and oil) automatically by a VIA compressed air dryer.
- System of generating particulates/aerosols: Under dynamic conditions the various concentrations of the test substance were atomized into the baffle (pre-separator) of the inhalation chamber. For atomization a binary nozzle and conditioned compressed air (15 L/min) was used. The test article was fed into the nozzle system using a digitally controlled pump (Harvard PHD 2000 infusion pump).
- Optimization of respirability: In order to increase the efficiency of the generation of fine particles and prevent larger particles from entering the chamber a pre-separator (baffle) system was used (Tillery, Environmental Health Perspectives, 16, 25-40, 1976).
- Inhalation chamber equilibrium concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour (15 L/min x 60 min/(3.8 L) > 237, continuous generation of test atmosphere). Under such test conditions 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.
- Method of particle size determination: The particle-size distribution was analysed using a BERNER critical orifice cascade impactor.
- Treatment of exhaust air: The exhaust air was purified via filter systems.
- Temperature, humidity: Temperature and humidity measurements were performed by the computerized Data Acquisition and Control System using HC-S3 sensors (Rotronic Messgeräte GmbH, Ettlingen, Germany). The position of the probe was at the exposure location of rats.

TEST ATMOSPHERE
- The integrity and stability of the aerosol generation and exposure system was measured by using a RAS-2 real-time aerosol photometer (MIE, Bedford, Massachusetts, USA).
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: glass-fibre filter, Sartorius, Gottingen, Germany; digital balance). This method was used to define the actual concentration.
- Samples taken from breathing zone: yes
- Particle size distribution: The particle size distribution was analysed using a BERNER critical orifice cascade impactor. Aerosol mass < 3 µm: 91.1 % at 307 mg/m³, 90.1 % at 454 mg/m³, and 88 % at 597 mg/m³..
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The respirability of the aerosol was adequate and in compliance with test guidelines, i.e. the average mass median aerodynamic diameter (MMAD) was 1.4 µm at 307 mg/m³, 1.45 at 454 mg/m³, and 1.46 at 597 mg/m³; GSD 1.75-1.85.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Target conc.: 300, 450, and 600 mg/m³
Analytical conc. (gravimetric): 307, 454, and 597 mg/m³

No. of animals per sex per dose:

5

Control animals:

yes

Remarks:

air control

Details on study design:

- Duration of observation period following administration: 2 weeks
- Frequency of observations and weighing: Body weights were measured before exposure (day 0), on days 1, 3, 7, and 14. The appearance and behaviour 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.
- Necropsy of survivors performed: yes
- Other examinations performed: Reflexes were tested, based on recommendations made by Irwin (Psychopharmacologica 13, 1968, 222-257). Rectal temperatures were measured shortly after cessation of exposure (approximately within ½ hour after the end 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.
Particle size analysis: described in detail in report.
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 (LC50) is possible, it is performed by computer (PC) according to the method of Rosiello et al. (J. Tox. and Environ. Health 3, 797-809, 1977) as modified by Pauluhn (1983). This method is based on the maximumlikelihood method of Bliss (Pharm. Pharmacol. 11, 192-216, 1938)
Randomization: A computerized list of random numbers served the purpose to assign animals at random to the treatment groups.
Analysis of variance (ANOVA): 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) = 95 % (p = 0.05). 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. This program was originally obtained from BCTIC.

Key result

Sex:

female

Dose descriptor:

LC50

Effect level:

ca. 521 mg/m³ air

Based on:

test mat.

Exp. duration:

4 h

Sex:

male

Dose descriptor:

LC50

Effect level:

ca. 857 mg/m³ air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Mortality occurred at 454 and 597 mg/m³ up to the first post exposure day.
Number of mortalities at conc. (onset of mortality): males - 0/5 at 0 mg/m³, 0/5 at 307 mg/m³, 0/5 at 454 mg/m³, 1/5 at 597 mg/m³ (0d); females - 0/5 at 0 mg/m³, 0/5 at 307 mg/m³, 1/5 at 454 mg/m³ (1 d), 4/5 at 597 mg/m³ (1 d).

Clinical signs:

other: All rats exposed to 307 mg/m³ and concentrations above showed clinical signs (bradypnea, labored breathing patterns, breathing sounds, motility reduced, atony, high-legged gait, tremor, hair-coat ungroomed, piloerection, cyanosis, pallor, nose and muzzle

Body weight:

Comparisons between the control and the exposure groups revealed significant changes in body weights at 307 mg/m³ and concentrations above.

Gross pathology:

Necropsy revealed treatment-related findings at 454 and 597 mg/m³ (e.g. nose: white foamy discharge; trachea: white foamy content; lung: less white coloured areas, less collapsed, dark-red coloured).

Other findings:

Differences between groups exposed to 454 and 597 mg/m³ (reduced tonus, impaired righting response and reduced grip strength) compared to the control group were recorded.
Statistical comparisons between the control and the exposure groups revealed significant changes in body temperature at 307 mg/m³ and above.

The mortality patterns were typical of an irritation-related acute lung edema (white foamy content in nose and trachea, lung findings, bradypnea, laboured breathing patterns, breathing sounds, cyanosis, and stridor). Due to the mortality pattern, female rats appear to be more susceptible than male rats.

Executive summary:

An acute inhalation toxicity study with the test substance has been conducted in accordance with OECD TG 403 and OECD GD 39. In that study groups of rats were nose-only exposed to the liquid aerosol of the test item at 0 (air control), 307, 454, and 597 mg/m³. The respirability of the aerosol was adequate and in compliance with test guidelines (MMAD 1.40 -1.46 µm, GSD 1.76 -1.85).

All rats exposed to 307 mg/m³ and above showed clinical signs (e.g. bradypnea, laboured breathing patterns, motility reduced, high-legged gait, nasal discharge (serous), nostrils with red encrustation). Significant decreased body temperatures and body weights were found at 307 mg/m³ and above. Mortality occurred at 454 and 597 mg/m³. At this concentrations necropsy revealed findings of toxicological importance (e.g. nose: white foamy discharge; trachea: white foamy content; lung: less white coloured areas, less collapsed, dark-red coloured). The mortality patterns were typical of an irritation-related acute lung edema with white foamy content in nose and trachea, lung findings, bradypnea, labored breathing patterns, breathing sounds, cyanosis, and stridor. Due to mortality pattern females appear to be more susceptible than male rats (4/5 females exposed to 597 mg/m³ died whereas only 1/5 males died at this concentration). The LC50 was approx. 857 mg/m³ for male rats and approx. 521 mg/m³ for female rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

521 mg/m³

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An acute oral toxicity study in rats, according to OECD TG 423, is available for the substance. In this study the limit dose of 2000 mg/kg bw, formulated in corn oil, was administered by gavage to each of the 3 male and 3 female rats.

The dose of 2000 mg/kg was tolerated by the animals without mortalities. Diarrhoea was observed in both genders, in males up to day 2 and in females up to 5 hours post dosing. Additionally decreased motility was observed in females 2 hours post dosing. Body weights/body weight development was not affected by the treatment and no gross pathological findings were observed. Concluding, the LD50 in this study was > 2000 mg/kg bw.

Further available is an acute inhalation toxicity study with the test substance that has been conducted in accordance with OECD TG 403 and OECD GD 39. In this study groups of rats were nose-only exposed to the liquid aerosol of the test item at 0 (air control), 307, 454, and 597 mg/m³. The respirability of the aerosol was adequate and in compliance with test guidelines (MMAD 1.40 -1.46 µm, GSD 1.76 -1.85).

All rats exposed to 307 mg/m³ and above showed clinical signs (e.g. bradypnea, laboured breathing patterns, motility reduced, high-legged gait, nasal discharge (serous), nostrils with red encrustation). Significant decreased body temperatures and body weights were found at 307 mg/m³ and above. Mortality occurred at 454 and 597 mg/m³. At this concentrations necropsy revealed findings of toxicological importance (e.g. nose: white foamy discharge; trachea: white foamy content; lung: less white coloured areas, less collapsed, dark-red coloured). The mortality patterns were typical of an irritation-related acute lung edema with white foamy content in nose and trachea, lung findings, bradypnea, laboured breathing patterns, breathing sounds, cyanosis, and stridor. Due to mortality pattern females appear to be more susceptible than male rats (4/5 females exposed to 597 mg/m³ died whereas only 1/5 males died at this concentration). The LC50 was approx. 857 mg/m³ for male rats and approx. 521 mg/m³ for female rats.

No acute dermal toxicity study is available for the substance. However, an assessment of acute dermal toxicity is possible based on the weight of evidences.

The substance has a molecular weight of 298 g/mol, which is not contradictive for systemic availability after dermal exposure. Due to the reactive isocyanate groups the substance is unstable towards nucleophiles, e. g. water, alcohol and amino groups.

For acute inhalation the available study on rats reveal indications for “port-of-entry” irritant toxicity (e.g.  laboured breathing patterns, serous nasal discharge, irritation-related acute lung edema; 4h-LC50 approx. 521 mg/m³ based on female rats), which is typical for aliphatic isocyanates and thus confirm the characteristic isocyanate-mode of action.

After acute oral exposure the substance is practically non-toxic (LD50 > 2000 mg/kg). In the respective guideline study (OECD TG 423) the test substance caused no mortalities, no effects on body weight development and no gross pathological changes. Transient clinical signs like diarrhoea (onset and duration: at maximum from 2 hours up to 2 days post administration) and decreased motility (only females; 2 hours post administration) were observed; these symptoms do not prove systemic toxicity, and might also be related to local effects at the gastrointestinal tract.

The available data on skin/eye irritation/corrosion and sensitisation (LLNA) confirm an irritant property for the substance. With the exception of skin sensitisation (the LLNA exhibited a potential for skin sensitization and an acute irritation/inflammation response) none of the available studies reveal indications for systemic availability, however, due to the positive LLNA it cannot be completely ruled out. Nevertheless, despite the irritant effect which may diminish the protective function of the epidermis (worst case assumption) it is not expected for a substance with such a profile (i.e. irritancy at the port-of-entry due to isocyanate-reactivity) that systemic availability after dermal exposure is higher than after oral exposure.

Taken together, with the mode of action outlined above, the oral LD50 > 2000 mg/kg and no overt signs of toxicity after oral exposure, it is not expected that testing on acute dermal toxicity would reveal adverse systemic effects. Local effects were already assessed by the respective skin irritation/corrosion studies. Thus, based on the weight of evidence a conclusion can be drawn that for acute dermal toxicity no hazard is expected.

This assessment of acute dermal toxicity is in line with the amendment to REACH Annex VIII (8.5.3) (Reg. (EU) 2016/863) that considers toxicity testing by the dermal route as not needed if a substance does not meet the criteria for classification as acute toxic by the oral route. Further publications justify this approach.[1][2]

[1] Moore, Regulatory Toxicology and Pharmacology, 2013, 66, 30-37

[2] Creton, St. et al, Critical reviews in Toxicology, 2010, Vol. 40 No.1, pages 50-83

Justification for classification or non-classification

According to Regulation (EC) No. 1272/2008, Annex I, classification as acute toxic for oral or dermal exposure is not justified.

Respective to dermal toxicity: With the mode of action outlined above, the expectation that systemic availability after dermal exposure will not exceed systemic availability after oral exposure, the oral LD50 > 2000 mg/kg and no overt signs of toxicity after oral exposure, it is not expected that data on acute dermal toxicity would lead to a hazard classification for the substance. Therefore, it can be concluded for acute dermal toxicity that the available data are conclusive for non-classification.

According to Regulation (EC) No. 1272/2008, Annex I, classification as acute toxic Cat. 3 for inhalation exposure is justified (H331: Toxic if inhaled).