Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10-11-12 to 10-12-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity (analytical): 99.8%
Batch/Lot No: 10021006
Description: Colourless Liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
-Strain: Spraque Dawley rats (Crl:CD® (SD), 15 males, 15 females
-Source: Charles River (UK) Ltd, Margate, Kent, UK
-Age: 7-8 weeks (start of exposure: 8-9 weeks)
-Weight at arrival: 229-245g (males), 200-217g (females)
-Weight at start of exposure: 269-316g (males), 211-241g (females)
-Housing: Males and females housed separately; clinical examination within 24 hours of arrival, no formal randomisation procedure applied, Males a nd females housed separately (5 per cage), acclimatisation approximately 1 week before dosing
-Temperature: 19-23°C
-Humidity: 40-70%
-Air: 15 air changes per hour
-Light: light hours were 0700-1900 h
-Food: Rat and Mouse (modified) No.1 Diet SQC Expanded (Special Diets Services, 1 Stepfield, Witham, Essex, UK) available to the animals ad libitum, except during inhalation exposure or other routine in-life investigations
-Water: domestic mains drinking water available to the animals ad libitum, except during inhalation exposure
or other routine in-life investigations

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
other: snout only
Vehicle:
air
Details on inhalation exposure:
-Treatment period: 1 day (designated Day 0 of study)
-Observation period: 14 days
-aerosol generation: by using an atomiser (Düsen-Schlick, Coburg, Germany)
-Pre-exposure aerosol investigations: aerosol concentration assessment, particle size distribution measurements
-exposure: exposure treatment in a modular
snout only stainless steel flow-past system with continuous supply of aerosol to be delivered to each animal; gravimetric measurement of concentration (9 times during the 4 h exposure period)
-particle size distribution: measured twice for each group during the 4 h exposure period using a Marple Cascade Impactor.
Analytical verification of test atmosphere concentrations:
yes
Remarks:
gravimetric measurement (9 times during the 4 h exposure period)
Duration of exposure:
4 h
Remarks on duration:
Frequency: once
Concentrations:
0.5 mg/l, 1.0 mg/l, 5.0 mg/l
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
no
Details on study design:
-Mortality/Moribundity: in the morning and as late as possible each day
-Clinical Observations: During exposure period animals were observed for reaction; clinical signs were recorded; respiratory rate recorded if appropriate; Pre exposure (Day 0) and during the 14 day post exposure observation periods the animals were observed at least once a day
-Body Weights: recorded at immediately prior to commencement of exposure (Day 0). The surviving animals were weighed on days 1, 2, 3, 7, 10 and 14 of the post exposure observation period of the study. The recording of bodyweights on Day 2 for all groups was surplus to protocol requirements but has still been reported. Group 3 bodyweights were recorded, in error, on Day 4 and not recorded, in error, on Day 10.
-Terminal Procedures: Dead or moribund animals, or animals euthanised for humane reasons were necropsied as early as possible after discovery and a terminal body weight was taken. Animals surviving to scheduled euthanasia were necropsied after completion of the 14 day exposure observation period (on Day 15). Animals surviving until scheduled euthanasia had a terminal body weight recorded and were euthanised by exposure to carbon dioxide, followed by exsanguination.
-Necropsy: Animals were subjected to a complete necropsy examination (evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their
associated organs and tissues. The respiratory tract was closely examined for signs of gross irritation)
On completion of the necropsy, the animals’ carcasses were disposed of and no tissues were retained.
-Organ Weights:The lungs were weighed at necropsy for all animals. Lung to body weight ratio (using the terminal body weight) has been calculated for each individual animal. Absolute lung weights and lung: body weight ratios have been compared with historical control data to assess any treatment related effect.
Statistics:
No formal statistical analysis was conducted

Results and discussion

Effect levelsopen allclose all
Sex:
male
Dose descriptor:
LC50
Effect level:
>= 1.07 - <= 5.01 mg/L air (analytical)
Exp. duration:
4 h
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.01 mg/L air (analytical)
Exp. duration:
4 h
Remarks on result:
other: No overall LC50 (for the sexes combined) could be calculated as all females in all groups survived to terminal necropsy. However in the report the overall LC50 (for the sexes combined) was considered to be in excess of 5.01 mg/L.
Sex:
female
Dose descriptor:
LC50
Effect level:
> 5.01 mg/L air (analytical)
Exp. duration:
4 h
Remarks on result:
other: No LC50 for females could be calculated as all females in all groups survived to terminal necropsy. However in the report the LC50 for females was considered to be in excess of 5.01 mg/L.
Mortality:
Group 1: no mortality
Group 2: no mortality
Group 3: 3 males (one during exposure, one on day 4 , one at day 6)
Clinical signs:
other: Group 1 and 2 : no adverse clinical observations during exposure and post exposure observation period. Group 3: During exposure: one male irregular and laboured breathing, (laboured breathing recover at 150 minutes), slight gasping and a wet and unkempt
Body weight:
Group 1 and Group 2: the occasional animal had a transient body weight reduction on Day 1 but all animals gained body weight by the end of the 14 day post exposure observation period. The animals exhibiting the weight reduction on Day 1 generally did not gain as much as the animals experiencing no weight reduction.
Group 3: females had a transient and slight body weight reduction post exposure; Males surviving to terminal necropsy showed minimal body weight gain or stasis following exposure. All animals surviving to terminal necropsy gained body weight by the end of the
14 day post exposure observation period. The two males which died on Days 4 and 6 post exposure still had lower than initial body weights on their last measured body weight. The female animal which had the greatest initial body weight loss had the least gain over the course of the 14 day post exposure observation period.
Gross pathology:
Group 1: Single male is considered to have the possible treatment related finding of dark foci in the left lung lobe. The flaccid testicle in the other male is considered unlikely to be related to treatment. All other animals had no necropsy findings.

Group 2: one male had spongy lungs and a froth filled trachea which may be due to treatment but could also be due to the method of euthanasia. All other animals had no necropsy findings.

Group 3 males: The male which died during exposure had discoloured lungs, a distended ileum and an enlarged bronchial lymph node. The male found dead on Day 4 had dark lungs with dark foci, a froth filled trachea, a dark liver and lower intestines distended with gas and abnormal dark contents in the upper intestines. The male found dead on Day 6 was autolysed but had no other abnormal findings. At terminal necropsy for the surviving animals 1 male had dark foci in the lungs and 1 male had discoloured (mottled) kidneys and an enlarged mandibular lymph node.

Group 3 females (all survived to terminal necropsy): One female had slight darkening of the lung, there were some minor findings including reddened or enlarged lymph nodes in two females one of which also had a speckled thymus and two females had no findings at necropsy. The findings overall were less severe than for the males.

Lung:Body Weight Ratio
Lung:body weight ratios for all Group 1, 2 and 3 animals surviving to terminal necropsy were considered to be very close to the expected values noted for thisage and strain.
Lung weights for the animals that died prematurely have not been included in the means for Group 3 males as they may be unrepresentative due to the condition of the animals at necropsy.

Any other information on results incl. tables

-The target MMAD range was between 1-4 μm.

-Exposure Chamber Air Flow Rates: Carrier air flow 13 L/min (all groups). Chamber extract 12 L/min (all groups)

-Temperature and Relative Humidity:

Group 1 - 20.8 ± 0.21 °C, 3.0 ± 0.77 %

Group 2 - 20.8 ± 0.28 °C, 3.6 ± 1.02 %

Group 3 - 20.6 ± 0.26°C, 2.0 ± 0.90% (Group 3 chamber temperature and relative humidity levels were not recorded for the

final time point.)

-Exposure Chamber Concentrations:

Group 1 - 0.52 ± 0.046 mg/L

Group 2 - 1.07 ± 0.022 mg/L

Group 3 - 5.01 ± 0.088 mg/L

-Nominal Aerosol Concentrations:

The nominal aerosol concentrations for all groups were approximately three to five times the achieved aerosol concentrations. The difference between the achieved and nominal aerosol concentrations reflects deposition losses of test item within the exposure system.

-Particle Size Distribution:

Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD, values in brackets) for the two valid samples taken for each of the groups were as follows:

Group 1 - 1.30 (2.631) and 1.28 (2.277) μm.

Group 2 - 2.09 (2.575) and 2.15 (2.732) μm.

Group 3 - 2.26 (2.664) and 2.38 (2.622) μm.

The particle size data indicated that 49.5-79.3 % of the aerosol particles were less than 2.7 μm in diameter based on gravimetric estimate.

Applicant's summary and conclusion

Conclusions:
In conclusion, the 4 hour inhalation exposure to a mist (aerosol) of Isophorone diamine at 5.01 mg/L, resulted in respiratory difficulties for all animals with the death of one male during exposure and 2 males during the post dose observation period. For males the LC50 is estimated to be between 1.07 and 5.01 mg/L and for the females the LC50 could not be established but was considered to be in excess of 5.01 mg/L. The overall LC50 (for the sexes combined) could also not be established but was considered to be in excess of 5.01 mg/L.
Executive summary:

A study was conducted to determine the acute toxicity of Isophorone diamine in rats following a single 4 hour inhalation exposure. Surviving animals were retained for a 14 day post exposure observation period. Three groups of rats (5male and 5 female per group) were exposed to 0.5, 1.0 and 5.0 mg/L areosol of Isophorone diamine once for 4 hours.

In conclusion, the 4 hour inhalation exposure to a mist (aerosol) of Isophorone diamine at 5.01 mg/L, resulted in respiratory difficulties for all animals with the death of one male during exposure and 2 males during the post dose observation period. For males the LC50 is estimated to be between 1.07 and 5.01 mg/L and for the females the LC50 could not be established but was considered to be in excess of 5.01 mg/L. The overall LC50 (for the sexes combined) could also not be established but was considered to be in excess of 5.01 mg/L.