3,5,5-trimethylcyclohex-2-enone

Endpoint summary

Description of key information

In subchronic toxicity studies, oral administration of isophorone caused no significant toxic effects. At high doses (>= 1000 mg/kg bw/d), mortality was observed.

NOAEL (rat, 90 days) = 102.5 mg/kg bw/day;

NOAEL (rat and mouse, 13 weeks) = 500 mg/kg bw/day;

NOAEL (dog, 90 days) = 150 mg/kg bw/day (highest tested dose)

After inhalational administration nose and eye irritation and blood and liver changes were observed (NOAEL (rat, 28 days) < 250 mg/m³).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male and female CFE rats were orally treated with three different doses of the test substance administered via their diet daily for a period of 90 days.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: CFE

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

750 ppm

Remarks:

corresponds to 57 mg/kg bw/d in males and 78.9 mg/kg bw/d in females

Dose / conc.:

1 500 ppm

Remarks:

corresponds to 102.5 mg/kg bw/d in males and 163.8 mg/kg bw/d in females

Dose / conc.:

3 000 ppm

Remarks:

corresponds to 233.8 mg/kg bw/d in males and 311.8 mg/kg bw/d in females

No. of animals per sex per dose:

20
After 4 weeks, 5 animals per sex and dose group were killed for blood analysis.

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

Clinical signs: daily

Mortality: daily

Body weight: weekly

Food consumption: weekly

Water consumption: weekly

Ophthalmoscopic examination: no

Hematology: after 4 weeks and at the end of the study: determination of hemoglobin, hematocrit, erythrocyte counts, leukocyte counts, and differential leukocyte determinations

Biochemistry: after 4 weeks and at the end of the study: blood glucose, blood urea nitrogen, serum glutamic oxaloacetic transaminase, serum alkaline phosphatase, total serum protein, total serum bilirubin, serum albumin, lactic acid dehydrogenase, cholesterol, calcium, phosphate, and uric acid

Urinalysis: after 4 weeks and at the end of the study: pH, glucose, ketones, albumin, occult blood, microscopic examination of sediment

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC)
Organ weights: Organ-body weight ratios: heart, liver, kidney, adrenals, thyroid, brain, testes

Macroscopic: After 4 weeks and at study termination: lungs, heart, intestines, kidneys, spleen, liver, urinary bladder

Weights: Heart, liver, kidney, adrenals, thyroid, brain, testes (males) for 10 males and 10 females of each dose level

Microscopic: At study termination: 5 males and 5 females each from high dose and control groups: brain, pituitary, eye, thyroid, lung, heart, liver, kidney, adrenals, urinary bladder, mediastinal lymph node, pancreas, spleen, colon, bone marrow, skeletal muscle, testes and prostate (male), ovary and uterus (female). 5 males and 5 females each from medium and low dose groups: liver, kidney.

Statistics:

All data were evaluated statistically.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1 male (control group), 1 female (3000 ppm): deaths were due to intercurrent infection

Description (incidence and severity):

- 3000 ppm, males: significantly reduced body weight gain (P < 0.01); further observed changes returned to normal in the subsequent weeks

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- kidney, testes, males: slightly increased mean organ to body weight ratio (considered not compound-related by the authors; not statistically significant)

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Remarks:

corresponding to 311.8 mg/kg bw/d

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects up to and including the highest tested dose

Dose descriptor:

NOAEL

Remarks:

corresponding to 102.5 mg/kg bw/d

Effect level:

1 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: reduced body weight gain

Critical effects observed:

no

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Restrictions: no organ weight determination, no clinical biochemistry

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was administered at doses of 0- 1000 mg/kg body weight per day by gavage in corn oil to groups of 10 F344/N rats of each sex, 5 days/week, for 13 weeks.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Industries, Indianapolis IN
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: 5 animals per cage
- Diet, ad libitum: Purina Rat Chow, Ralston Purina, St. Louis, MO
- Water, ad libitum: not further specified
- Acclimation period: 15 - 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 27°C, average 23°C
- Humidity (%): 29 - 74%, average 56%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- Total volume applied: 1 mL/animal

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks (91 days)

Frequency of treatment:

5 days/week

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY
Clinical signs: weekly

Mortality: not specified

Body weight: weekly

Hematology: no

Urinalysis: no

Biochemistry: no

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC)
Organ weight: no

Macroscopic: tissues were examined for gross lesions and masses

Microscopic: Skin, mammary gland, sciatic nerve, salivary gland, mandibular lymph node, thymus, heart, lungs, trachea, thyroid gland, parathyroids, esophagus, stomach, duodenum, jejunum, ileum, colon, rectum, mesenteric lymph node, pancreas, spleen, liver, kidneys, adrenal glands, urinary bladder, seminal vesicles, prostate/testes or ovaries/uterus, brain, pituitary gland, bone marrow, spinal cord, and nasal cavity.

Statistics:

Survival, body weight, dose-related effects were statistically evaluated

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

1 female at 1000 mg/kg bw/d

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Critical effects observed:

not specified

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Restrictions: no organ weight determination, no clinical biochemistry

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was administered at doses of 0- 1000 mg/kg body weight per day by gavage in corn oil to groups of 10 B6C3F1 mice of each sex, 5 days/week, for 13 weeks.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Industries, Indianapolis IN
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: not specified
- Fasting period before study: not specified
- Housing: 5 animals per cage
- Diet, ad libitum: Purina Rat Chow, Ralston Purina, St. Louis, MO
- Water, ad libitum: not further specified
- Acclimation period: 15 - 18 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 27°C, average 23°C
- Humidity (%): 29 - 74%, average 56%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- Total volume applied: 0.5 mL/animal

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks (91 days)

Frequency of treatment:

5 days/week

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY
Clinical signs: weekly

Mortality: not specified

Body weight: weekly

Hematology: no

Urinalysis: no

Biochemistry: no

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC)
Organ weight: no

Macroscopic: tissues were examined for gross lesions and masses

Microscopic: Skin, mammary gland, sciatic nerve, salivary gland, mandibular lymph node, thymus, heart, lungs, trachea, thyroid gland, parathyroids, esophagus, stomach, duodenum, jejunum, ileum, colon, rectum, mesenteric lymph node, pancreas, spleen, liver, gallbladder, kidneys, adrenal glands, urinary bladder, seminal vesicles, prostate/testes or ovaries/uterus, brain, pituitary gland, bone marrow, spinal cord, and nasal cavity.

Statistics:

Survival, body weight, dose-related effects were statistically evaluated.

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

3 of ten 1000 mg/kg dose females and 1 of ten 1000 mg/kg dose males died before the end of the studies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of males in the 250, 500 and 1000 mg/kg groups were approximately 10% less than those of controls.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

102.5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Restriction: one dose only

Qualifier:

no guideline followed

Principles of method if other than guideline:

Four groups of male and female albino rats (10 per sex and dose) were exposed to the test substance (as vapour) in filtered room air at a concentration of 0.25 mg/L air for 6h per day, on 5 days per week, for a duration of 4 weeks. The animals were observed at frequent intervals for signs of irritation. At the end of the 4-week study period, hematological dererminations were carried out in 5 animals per sex per group. Terminal body weights were recorded and the animals were necropsied. Lung, liver, kidneys, adrenal and spleen were examined histopathologically in 3 animals per sex per dose.

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

other: Charles River Caesarian-derived albino rats

Sex:

male/female

Route of administration:

inhalation

Type of inhalation exposure:

not specified

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 L stainless steel and glass exposure chambers
- Method of holding animals in test chamber: not specified
- Source and rate of air: Air flow through the chamber was maintained by a positive pressure rotary pump located at the exhaust side of the chamber.
- Method of conditioning air: not specified
- System of generating particulates/aerosols: The vapours were generated by metering the liquid into a positive pressure spray nozzle with a Harvard infusion pump. The resultant aerosol was introduced into a large, triple necked distilling flask. A heating mantle was used to maintain the flask at a temperature adequate to vaporize the entering aerosol.
- Temperature, humidity, pressure in air chamber: not specified
- Air flow rate: The air flow rate was monitored by a rotameter
- Air change rate: not specified
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: The analytical concentration of the chamber was determined daily by analysis of samples with a Beckman BD spectrometer. Each sample was obtained by drawing a known volume of the chamber atmosphere through a scrubber containing methanol as absorbent. The concentration of test material in each sample was ascertained from a standard curve which was prepared by plotting known concentrations of standard solution against percent transmission of the solutions. The concentration of the experimental atmosphere was calculated from the volume of chamber atmosphere which had been passed through the scrubber and the total liquid volume of the sample.
- Samples taken from breathing zone: not specified

VEHICLE (if applicable)
- filtered room air

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 d

Frequency of treatment:

6 hours/day; 5 days/week

Dose / conc.:

0.25 mg/L air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

- Clinical signs: at frequent intervals

- Mortality: at frequent intervals

- Body weight: at study initiation and termination

- Haematology: at study initiation and termination, identical 50 % of animals of each group

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Macroscopic: brain, pituitary, trachea, thyroid, parathyroid, lungs, heart, liver, spleen, stomach, small intestine, large intestine, adrenals, kidneys, urinary bladder, gonads, femur
- organ weights: lungs, liver, kidneys, adrenal, spleen

- Microscopic: lungs, liver, kidneys, adrenal, spleen for three males and three females of each group

Statistics:

performed on body and organ weights

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Slight nasal bleeding on day 3, reddish-brown discoloration of the fur surrounding the nasal regions on days 6 through 8

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight only of male rats was significantly reduced compared to control.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Differential blood count: increase of the percentage of lymphocytes (pre-exposure: 81.6 and 80.2 % among males and females, respectively; post-exposure: 84.6 and 86.0 %, respectively), decrease in the percentage of segmented neutrophiles (pre-exposure: 17.4 and 18.2 % among males and females, respectively; post-exposure: 14.6 and 13.3 %, respectively).

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative liver weight only of male rats were significantly reduced compared to control.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Reference 2

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Restrictions: insufficient documentation, one dose only

Principles of method if other than guideline:

The method followed is not described in the OECD SIDS Initial Assessment Report

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: approximately 200 g

Route of administration:

inhalation

Type of inhalation exposure:

not specified

Vehicle:

air

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

18 months

Frequency of treatment:

6 hours/day; 5 days/week

Dose / conc.:

250 ppm

Remarks:

corresponds to 1.436 mg/L air

No. of animals per sex per dose:

10
The report suggests that two identical groups of males and two groups of females were exposed in addition to the unexposed control groups, corresponding to a total of six groups. As the presentation of results does not refer to absolute numbers of animals but only to percentages of total animals, their misinterpretation can be excluded.

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

Mortality and signs of irritation: daily

Body weight: weekly

Hematology: monthly during first 10 months

Urinanalysis: weekly

Sacrifice and pathology:

Macroscopic and microscopic examination.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs of irritation: Slight irritation of conjunctiva, no opacity of cornea, by end of third week bloody exsudate from nose indicating irritation of mucosa

Mortality:

mortality observed, non-treatment-related

Description (incidence):

40 % each of males and females, controls as well as exposed, died; time not reported

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

More or less pronounced hemorrhages of the lungs were observed in exposed and control animals likewise. Discoloration of the livers was also observed in the control group independent on exposure. The other organs inspected appeared normal.

Description (incidence and severity):

Lesions in lungs were found in both exposed and control animals. Microvacuolisation in livers was more pronounced in exposed animals than in controls. The other organs were normal, or changes were considered to be insignificant.

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

250 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Restrictions: insufficient documentation, one dose only

Principles of method if other than guideline:

The method followed is not described in the OECD SIDS Initial Assessment Report

GLP compliance:

not specified

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: approximately 200 g

Route of administration:

inhalation

Type of inhalation exposure:

not specified

Vehicle:

air

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

18 months

Frequency of treatment:

6 hours/day; 5 days/week

Dose / conc.:

250 ppm

Remarks:

corresponds to 1.436 mg/L air

No. of animals per sex per dose:

10
The report suggests that two identical groups of males and two groups of females were exposed in addition to the unexposed control groups, corresponding to a total of six groups. As the presentation of results does not refer to absolute numbers of animals but only to percentages of total animals, their misinterpretation can be excluded.

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

Mortality and signs of irritation: daily

Body weight: weekly

Hematology: monthly during first 10 months

Urinanalysis: weekly

Sacrifice and pathology:

Macroscopic and microscopic examination.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs of irritation: Slight irritation of conjunctiva, no opacity of cornea, by end of third week bloody exsudate from nose indicating irritation of mucosa

Mortality:

mortality observed, non-treatment-related

Description (incidence):

40 % each of males and females, controls as well as exposed, died; time not reported

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

More or less pronounced hemorrhages of the lungs were observed in exposed and control animals likewise. Discoloration of the livers was also observed in the control group independent on exposure. The other organs inspected appeared normal.

Description (incidence and severity):

Lesions in lungs were found in both exposed and control animals. Microvacuolisation in livers was more pronounced in exposed animals than in controls. The other organs were normal, or changes were considered to be insignificant.

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Restriction: one dose only

Qualifier:

no guideline followed

Principles of method if other than guideline:

Four groups of male and female albino rats (10 per sex and dose) were exposed to the test substance (as vapour) in filtered room air at a concentration of 0.25 mg/L air for 6h per day, on 5 days per week, for a duration of 4 weeks. The animals were observed at frequent intervals for signs of irritation. At the end of the 4-week study period, hematological dererminations were carried out in 5 animals per sex per group. Terminal body weights were recorded and the animals were necropsied. Lung, liver, kidneys, adrenal and spleen were examined histopathologically in 3 animals per sex per dose.

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

other: Charles River Caesarian-derived albino rats

Sex:

male/female

Route of administration:

inhalation

Type of inhalation exposure:

not specified

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 L stainless steel and glass exposure chambers
- Method of holding animals in test chamber: not specified
- Source and rate of air: Air flow through the chamber was maintained by a positive pressure rotary pump located at the exhaust side of the chamber.
- Method of conditioning air: not specified
- System of generating particulates/aerosols: The vapours were generated by metering the liquid into a positive pressure spray nozzle with a Harvard infusion pump. The resultant aerosol was introduced into a large, triple necked distilling flask. A heating mantle was used to maintain the flask at a temperature adequate to vaporize the entering aerosol.
- Temperature, humidity, pressure in air chamber: not specified
- Air flow rate: The air flow rate was monitored by a rotameter
- Air change rate: not specified
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: The analytical concentration of the chamber was determined daily by analysis of samples with a Beckman BD spectrometer. Each sample was obtained by drawing a known volume of the chamber atmosphere through a scrubber containing methanol as absorbent. The concentration of test material in each sample was ascertained from a standard curve which was prepared by plotting known concentrations of standard solution against percent transmission of the solutions. The concentration of the experimental atmosphere was calculated from the volume of chamber atmosphere which had been passed through the scrubber and the total liquid volume of the sample.
- Samples taken from breathing zone: not specified

VEHICLE (if applicable)
- filtered room air

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 d

Frequency of treatment:

6 hours/day; 5 days/week

Dose / conc.:

0.25 mg/L air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

Post-exposure period: none

Observations and examinations performed and frequency:

- Clinical signs: at frequent intervals

- Mortality: at frequent intervals

- Body weight: at study initiation and termination

- Haematology: at study initiation and termination, identical 50 % of animals of each group

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Macroscopic: brain, pituitary, trachea, thyroid, parathyroid, lungs, heart, liver, spleen, stomach, small intestine, large intestine, adrenals, kidneys, urinary bladder, gonads, femur
- organ weights: lungs, liver, kidneys, adrenal, spleen

- Microscopic: lungs, liver, kidneys, adrenal, spleen for three males and three females of each group

Statistics:

performed on body and organ weights

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Slight nasal bleeding on day 3, reddish-brown discoloration of the fur surrounding the nasal regions on days 6 through 8

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight only of male rats was significantly reduced compared to control.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Differential blood count: increase of the percentage of lymphocytes (pre-exposure: 81.6 and 80.2 % among males and females, respectively; post-exposure: 84.6 and 86.0 %, respectively), decrease in the percentage of segmented neutrophiles (pre-exposure: 17.4 and 18.2 % among males and females, respectively; post-exposure: 14.6 and 13.3 %, respectively).

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative liver weight only of male rats were significantly reduced compared to control.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

250 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

Male and female Fischer rats were administered 0, 125, 250, 500, 1000 and 2000 mg isophorone/kg bw/day in a 16 day and 0, 62.5, 125, 250, 500 and 1000 mg/kg bw/day in a 13 week investigation, the former being a dose finding study for the latter.

In the dose finding study, one of five males and four of five females that received 2000 mg/kg bw/day isophorone died.

In the 13-week study, one of ten females of the tope dose group died. The NOAEL is therefore 500 mg isophorone/kg bw/day for male and female rats (Bucher, 1986).

In a 90-day study with male and female CFE rats dosed with 750, 1000, or 3000 ppm isophorone via diet – corresponding to 57.0, 102.5, 233.8 mg/kg bw/day for males and to 73.9, 163.8 and 311.8 mg/kg bw/day for females – for 13 weeks, the only observed effect was a reduced body weight gain (-12 to -13%) in male rats at 3000 ppm (during weeks 6,7,8,9,10 and 11). The NOAEL derived from this study is 102.5 mg/kg bw/day for male and 311.8 mg/kg bw/day for female rats. (Rohm & Haas, 1972)

After administration of isophorone up to 2000 mg/kg bw/day to male and female B6C3F1 mice for 16 days, the reported effect was mortality at 2000 mg/kg.

In the 13 week study with dosages up to 1000 mg/kg bw/day, 3 of 10 females that received the top dose died. Body weights of males in the 250, 500 and 1000 mg/kg groups were approximately 10% less than those of controls. The NOAEL is 500 mg isophorone/kg bw/day. (Bucher, 1986)

In a further 90-day study (Rohm & Haas, 1972), beagle dogs (4 animals/dose/sex) were given orally gelatine capsules containing doses of 35, 75, or 150 mg isophorone per kg bodyweight. As the only minor clinical signs, incidences of soft stool were noted in the two upper dose levels.

Therefore, the NOAEL is 150 mg/kg bw/day for male and female beagle dogs.

 

Inhalation

Studies are available for rats, mice, rabbits, and guinea pigs.

In rats exposed for 4 weeks to 250 mg/m³ isophorone reduced body weights in males, changes in haematological parameters and reduced liver weights in males and females were found (Exxon, 1968).

In a study with 6 weeks duration, at doses ≥ 287 mg/m³ congested kidneys, dilated Bowman’s capsules and lung changes (irritation, congestion) were found in rats and in guinea pigs (Smyth et al., 1942). Further findings observed in this study were blood cell changes, albuminuria at doses and eye and nasal irritations at 2874 mg/m³. Eye and nose irritations have also been observed in a more recent study in Wistar rats and rabbits at 1436 mg/m³ after 18 months exposure. In addition at this concentration slightly increased microvacuolization of the livers was observed (Dutertre-Catella, 1976).

No effects were found in histopathological examinations of the respiratory tract of Swiss mice after exposure to 164 and 513 mg/m³ for up to 14 days (Zissu, 1995).

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on the available oral and inhalation repeated dose toxicity studies the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the 14th time in Regulation (EU) 2020/217.