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Description of key information

The LD50 after oral application to male rats is 1030 mg/kg bw and the kidney is the potential target organ (Klimmer 1965). Acute inhalation toxicity: The LC50 after inhalation was determined in male rats (LC50 1.07-5.01 mg/L) and in female rats (LC50 > 5.01 mg/L) resulting in a LC50 of > 5.01 mg/L for both sexes (CR 2011). Acute dermal toxicity: The LD50 after dermal exposure is > 2000 mg/kg bw in rats (Moon 2010).

Key value for chemical safety assessment

Acute toxicity: via oral route

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Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 1965-03-04 and 1965-06-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Comparable to guideline study with acceptable restrictions: no data on mortality / dose group and how LD50 was calculated, only male animals used. Evidence from repeated dose studies indicates that there is no significant difference in sensitivity between males and females and that the acute oral toxicity is not higher by an order of magnitude or more (chapter 7.5.1 entry # 1: 13 week LOAEL ca. 150 mg/g bw/day for males and females).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
yes
Remarks:
no data on mortality / dose group and how LD50 was calculated, only male animals used.
Principles of method if other than guideline:
see Test Conditions
GLP compliance:
no
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
- Weight at study initiation: 110-130 g
- Fasting period before study: since day before
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
- Concentration in vehicle: 50 % (v/v)
- Amount of preparation: 0.5, 1.0, 1.5, 2.0 and 2.5 ml per kg b.w.
Doses:
50 % v/v solution in water, 0.5, 1.0, 1.5, 2.0 and 2.5 ml per kg b.w.
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
Post dose observation period: 14 days
Urine control for proteine
EXAMINATIONS: organs not listed
Statistics:
no data
Preliminary study:
not applicable
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
1 030 mg/kg bw
Mortality:
Time of death: after 12 to 18 hours in lateral position, no data on mortality / dose group
Clinical signs:
1 hour after dosing, animals showed restlessness, thirst, rough fur and tiredness.
Body weight:
no data
Gross pathology:
irriation of the intestinal mucosa, with a few animals showing a slight increase in kidney weight and protein in the urine
POTENTIAL TARGET ORGANS: kidney
Other findings:
no other findings
no further remarks
Conclusions:
The LD50 value of acute oral toxicity in male rats of the test substance isophorone diamine was determined to be 1030 mg/kg bw.
Executive summary:

The acute oral toxicity LD50 value in male Sprague-Dawley rats was determined to be 1030 mg/kg b.w.. Doses of 0.5, 1.0, 1.5, 2.0, or 2.5 ml/kg bw of a 50 % v/v solution in water were applied by gavage followed by a post dose observation period of 14 days.Clinical signs observed from 1 hour after dosing were restlessness, thirst, rough fur and tiredness. At necropsy, irritation of the intestinal mucosa was observed. A few animals (no further data) showed a slight increase in kidney weight and protein in the urine, which may indicate that the kidney is a target organ.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
1 030 mg/kg bw
Quality of whole database:
Klimisch 2

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:
10-11-12 to 10-12-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
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
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
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
Sex:
male
Dose descriptor:
LC50
Effect level:
>= 1.07 - <= 5.01 mg/L air (analytical)
Exp. duration:
4 h
Key result
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.

-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.

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 (5 male 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
Value:
5 010 mg/m³
Quality of whole database:
Klimisch 1

Acute toxicity: via dermal route

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Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-06-08 to 2010-08-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
- Strain: Sprague-Dawley (Crl:CD(SD)), SPF
- Sex, number, age and body weight range (at receipt)
12 males, 7 weeks old, 197.9 - 210.7 g
12 females, 8 weeks old, 198.8 - 212.1 g
- Sex, number, age and body weight range (on administration)
10 males, 8 weeks old, 263.5 - 288.9 g
10 females, 9 weeks old, 215.6 - 235.9 g
- All animals were observed for general condition and clinical signs daily and body weights were on Day 7 after receipt. All animals were quarantined for 3 days, and acclimated for 4 days.
- Animal husbandry:
Type & size of a cage: stainless wire mesh cages, 260Wx350Dx210H (mm)
Number of animals per cage: one animal/cage (during the study)
Temperature: 21.0 - 23.8 °C
Relative humidity: 40.3 - 53.4%
Air changes: 10 - 15 clean, fresh, filtered air changes per hour
Lighting: 12 hours light/dark
Intensity of illumination: 150 - 300 Lux
- Feed:
Type: Pelleted rodent chow
Method: The diet was placed in feeders and provided ad libitum
- Water:
Type /method: Public tap water was filtered and irradiated; provided ad libitum
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
- Method of administration:
The subscapular dorsa surface (approx. 5 cm x 6 cm) of each animal`s back was clipped with an electric clipper approx. 24 hours prior to dosing. 4 cm x 5 cm of these shaved areas were designated as the treated sites. After the treatment of the test substance to lint tape, the treated sites were covered with lint tape and plastic film. Each animals back was over-wrapped with Soft Cloth Tape with Liner. At the end of a 24-hour exposure period, lint tape, plastic film and Soft Cloth Tape with Liner were removed and any residual test substance was removed using adsorbent cotton moistened with tepid water. The shaved treated sites of the control animals were dressed in the same manner as the treated animals.
Duration of exposure:
24 hours
Doses:
The dose level of 2,000 mg/kg was selected for this study, which was expected to show low toxicity.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
Parameters Evaluated:
- Clinical signs: All animals were observed for mortality, general condition and clinical signs (time onset, severity and recovery) for 30 minutes after dosing and at 1, 2, 4 and 6 hours after dosing on Day 0 and once saily thereafter for 14 days (Days 1 to 14).
- Body weights: Body weights were recovered once on Day 0 prior to treatment on Day 3 and 7 and 0n the day of necropsy, day 14.
- Necopsy: On Day 14, all surviving animals were anesthetized with CO2 and exsanguinated from the abdorminal aorta. Complete gross postmortem examinations were performed on all animals in the study.
- Histopathology: In necropsy findings, crust was observed on the treated sites of all animls in the 2,000 mg/kg dosing group. Therefore, histopathological examinations were performed.
Statistics:
Statistical analysis: using SAS Programm;
Body weights: Folded-F test for homogeneity of variance (significant level: 0.05);
Student t-test was employed on homogeneous data (significance level: 0.05);
Aspin-Welch t-test was employed for heterogeneous data (significant level: 0.01)
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortalities
Mortality:
No mortality was observed at 2,000 mg/kg treatment throughout the course of the study.
Clinical signs:
On the treated sites at 2,000 mg/kg treatment, discoloration of skin (black) in all males and females and crust formation in two males and five females were observed on Days 1 and/or 2. In addition, discolorations of skin and crust formations were observed in all males and females from Days 3 to 14 after dosing. Scar was observed on the treated sites of three males and females at 2,000 mg/kg treatment on Days 11 to 14.
Body weight:
Normal body weight gains were observed in all animals at 2,000 mg/kg treatment.
Gross pathology:
Necropsy and histopathological findings:
Crust were observed on the treated sites of all animals at 2,000 mg/kg treatment. In histopathological findings, scar as mild to moderate was observed on the treated sites of all animals.
Other findings:
No other findings

No further information

Conclusions:
Based on the results of this study, the acute toxicity of isophorone diamine after dermal application to male and female rats is low: the LD50 value was determined to be > 2000 mg/kg bw.
Executive summary:

This study was conducted to assess the potential toxicity of the test substance isophorone diamine, following a single dermal treatment to Sprague-Dawley rats.

All animals at 2,000 mg/kg treatment survived the duration of the study.

Discoloration of skin and crust formation from Days 1 to 14 after dosing and scar from Days 11 to 14 were observed on the treated sites of all animals at 2,000 mg/kg treatment. These were considered to be test substance-related effects. No test substance-realted affected on body weights were observed.

In necropsy findings, crust was observed on the treated sites of all animals at 2,000 mg/kg treatment. In histopatholocical findings, scar as mild to moderate was observed on the treated sites of all animals. These were considered to be skin wounds caused by the test substance.

Based on the results of this study, Acute toxicity of isophorone diamine, the dermal LD50 was > 2,000 mg/kg in male and female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Klimisch 1

Additional information

Studies in animals:

Oral acute toxicity

Cited from SIAR for SIAM 18 (Paris, April 2004):

"The oral LD50 in male Sprague-Dawley rats was determined to be 1,030 mg/kg b.w. (Klimmer,

1965). Doses of 0.5, 1.0, 1.5, 2.0, or 2.5 ml/kg bw of a 50 % v/v solution in water were applied by

gavage followed by a post dose observation period of 14 days. Clinical signs observed from 1 hour

after dosing were restlessness, thirst, rough fur and tiredness. At necropsy, irritation of the intestinal

mucosa was observed. A few animals (no further data) showed a slight increase in kidney weight

and protein in the urine, which may indicate that the kidney is a target organ."

Acute inhalation toxicity

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 aerosol 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.

Acute dermal toxicity

A study was conducted to assess the potential toxicity of the test substance isophorone diamine, following a single dermal treatment to Sprague-Dawley rats.

All animals at 2,000 mg/kg treatment survived the duration of the study.

Discoloration of skin and crust formation from Days 1 to 14 after dosing and scars from Days 11 to 14 were observed on the treated sites of all animals at 2,000 mg/kg treatment. These were considered to be test substance-related effects. No test substance-realted affected on body weights were observed.

In necropsy findings, crust was observed on the treated sites of all animals at 2,000 mg/kg treatment. In histopatholocical findings, scar as mild to moderate was observed on the treated sites of all animals. These were considered to be skin wounds caused by the test substance.

Based on the results of this study, Acute toxicity of isophorone diamine, the dermal LD50 was > 2,000 mg/kg in male and female rats.

Justification for classification or non-classification

Because of acute oral toxicity the substance isophorone diamine is classified in Category 4 (H302: Harmful if swallowed) according CLP regulation (1272/2008, self classification).