2,4-dimethylpentan-3-one

Endpoint summary

Description of key information

For the endpoint repeated dose toxicity, there are two whole body inhalation studies with subacute exposure available (OECD 421, OECD 412). Both show the same pattern of systemic toxicity and in addition, the local and narcotic effects already known from the acute inhalation toxicity studies were observed at the high concentrations. The overeall NOAEL is 100 ppm (471 mg/m3).

Local effects consist of dose-dependent lacrimation. The acute narcotic effects (STOT SE Cat 3) are probably causative of the reduced food consumption and as a consequence, the dose-dependent reduction in body weight.

Systemic effects comprise a strong, dose-dependent increase in absolute and relative liver weights accompagnied by centrilobular hypertrophy, but no degenerative changes other than possibly a grade 1 periportal vacuolation in four of ten females of the high dose (1009 ppm) group after 56 days exposure. In the screening study for reproductive toxicity, effects were more pronounced because exposures were done 7 days per week and exposures were done for 35 (m) and 56 days (f), whereas in the subacute study, exposures were done 5 days per week for a period of 28 days. The effects on body weight were also observed for the pups, which is consistent with their directly exposure to the vapours and the reduced maternal care.

Males rats showed the histopathology findings of the rat specific alpha-2u-nephropathy.

In the GLP-compliant Reproduction / Developmental Toxicity Screening Test in Wistar rats (OECD 421) the NOAEL for general systemic toxicity was 100 ppm (ca. 471 mg/m³) based on adverse clinical findings and adverse effects on food consumption, body weight development as well as liver toxicity..

The NOAEC determined in a 4 week inhalation toxicity study (OECD 412) was 386 ppm (ca. 1832.3 mg/m³) based on the reduction in activity levels which was observed during exposure. The NOAEC was less than 128 ppm because of prophyrin tears in males. At this concentration as well as in the OECD 421 study at 100 ppm only the rat specific alpha-2u-nephropathy was observed in male rats.

For direct comparison of the observed effects it is referred to the table below.

Repeated dose toxicity (OECD 412)	Reproductive / Developmental toxicity (OECD 421 plus functional observation battery and liver effects)	Acute inhalation toxicity
Inhalation (vapour, whole body)	Inhalation (vapour, whole body)	Inhalation (vapour, whole body)
28 days 5 days per week	35 days (males) and 56 days (females) 7 days per week	4h exposure
128 ppm   Porphyrin tears (m) Red discoloration of the facial haircoat (m)   Slightly lower mean prothrombin time (m) Males only: Accumulation of hyaline droplets	99.1 ppm(471 mg/m^3)       Males only: Alpha2u-nephropathy with correlating increased absolute and relative kidney weights
386 ppm   Sialorrhea (f)         Slightly lower mean prothrombin time (m)   Increased mean absolute and relative liver weight (14%f, 17% m)   Accumulation of hyaline droplets in kidney (m) Cytoplasmic vacuolation (m) Basophilic cytoplasmic changes (m)	306 ppm(1414 mg/m^3)   Salivation (both sexes) Reddish discolored fur (both sexes)   Reduction in food consumption (- 7 % m, - 13 % f) Trend: Lowered body weight (-3%) of males           Alpha2u-nephropathy with correlating increased absolute and relative kidney weights
1221 ppm   Sialorrhea (f) Brown discoloration of the facial haircoat (f) Porphyrin tears (f) Excessive tearing (f)                 Slightly lower mean prothrombin time (m) Slightly lower mean haematocrit value (f) Slightly higher mean total protein and albumin (f)   Increased mean absolute and relative liver weight (+57%m & +47%f) Hypertrophy of hepatocytes (4/5 m, 5/5 f)       Adrenal gland Cytoplasmic vacuolation (m) Basophilic cytoplasmic changes (m)       Males only: Accumulation of hyaline droplets in kidney	1009 ppm(4715 mg/m^3)   Salivation (both sexes) Reddish discoloured fur (both sexes)     Lacrimation (both sexes)     Reduced attention (both sexes)   Insufficient maternal care Plough nose-first into bedding (both sexes) Reduction in food consumption (- 24 % m, - 30 % f) Body weight loss (- 8 % m, - 10 % f)             Increased absolute (+ 33 % m, + 21 % f) and relative (+ 44 % m, + 27 % f) liver weights Minimal to moderate centrilobular hepatocellular hypertrophy in 10/10 m & f and slight (grade 1) periportal vacuolation (4/10 f)       Males only: Alpha2u-nephropathy with correlating increased absolute and relative kidney weights
		1462 ppm moderate lethargy and minor dyspnea (resolved within 2 days after exposure)   Immediately after the exposure, all male and female animals had unkempt facial haircoats and 9 of 10 had minimal to minor lethargy.   A very slight transient body weight loss was observed (2/5 males, 3/5 females) after the second day post exposure.
		2765 ppm severe narcosis and moderate dyspnea (resolved within 2 days after exposure)   Immediately after the exposure, all male and female animals from the high exposure group had unkempt facial haircoats and exhibited minor to severe narcosis, hypothermia, and excessive tearing. Fewer high exposure level animals exhibited porphyrin tears (1/5 males), dyspnea (3/5 males, 4/5 females), gasping (2/5 females), rales (3/5 females), or diarrhea (2/5 males).   A very slight transient body weight loss was observed (5/5 males, 3/5 females) after the second day post exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

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:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Remarks:

NOAEL systemic toxicity inh.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

May, 1981

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

September, 1984

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test substance( as cited in report): Diisopropyl ketone / 2.4-Dimethyl-3-pentanone; DIPK
- Purity: The purity of the test substance was determined to be 100 ± 0.00% (mean ± SD) prior to and 99.8 ± 0.006% after the study when analyzed by gas chromatography.

Species:

rat

Strain:

other: CD (SD) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: Males 46 days, Females 51 days
- Weight at study initiation: Males 231 ± 6 g, Females 194 ± 9 g (mean ± SD)
- Housing: Singly housed in multicompartmented stainless steel mesh cages
- Diet: Certified Rodent Diet (Agway Prolab RMH 3000, pellets), ad libitum during non-exposure periods
- Water: ad libitum during non-exposure periods
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 23.3 (reported as 70-74 °F)
- Humidity (%): 50 - 56
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

The inhalation exposures were conducted in 420 L stainless steel and glass inhalation chambers at target concentrations of 1200, 380, 120, and 0 ppm. Exposures were conducted five days per week, 6 hour per day, for a total of 22 exposures over a four-week period.
The chambers were maintained at pressures of -0.035 to -0.05 inch water (gauge) relative to room air and at 12 air changes per hour. For each exposure level, male and female rats were singly housed and exposed simultaneously.
A vapor was produced by metering the test substance from a reservoir into a glass bead-packed column where it evaporated. The column was supplied with metered, dried, oil-free compressed air. The resultant vapor was directed into the turret of the chamber where it was mixed with filtered, conditioned air.
Chamber vapor concentrations were analytically determined by infrared spectroscopy at least 8 times during each exposure for each exposure level.
The samples were taken from a fixed reference position in the inhalation chamber. Chamber temperature and relative humidity were recorded twice per hour. Chamber air flow was set at 84 liters per minute. The nominal chamber concentration was calculated from the mass of chemical consumed and total air flow.
The concentration of background non-gaseous material was measured for the high-exposure and control chamber at least once each week during the exposure to ensure that the exposures were to a vapor and not an aerosol.

The vapor distribution of the test substance was not measured for positional variations within the inhalation chambers for this study. However, vapor distribution was determined during an LC50 study (see section Acute toxicity: inhalation) of the test substance about two months earlier using the same lot of the test material. For the samples taken from the nine cage positions in the LC50 study, the concentration (mean ± coefficient of variation) was 1300 ppm ± 7%, and from the fixed reference position was 1264 ppm ± 1%. The results indicated that positional effects were minimal and the fixed reference position selected provided a representative sample for chamber vapor concentration. The same fixed reference position was selected for the present study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber vapor concentrations were analytically determined by infrared spectroscopy at least 8 times during each exposure for each exposure level.

Duration of treatment / exposure:

Six hours per day

Frequency of treatment:

Five days per week for a total of 22 exposures over a 4-week period

Dose / conc.:

128 ppm (analytical)

Remarks:

Target concentration 120 ppm

Dose / conc.:

386 ppm (analytical)

Remarks:

Target concentration 380 ppm

Dose / conc.:

1 221 ppm (analytical)

Remarks:

Target concentration 1200 ppm

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

BODY WEIGHT:
Body weights were measured on days 0, 4, 7, 14, 21, and 28

CLINICAL OBSERVATIONS:
Rats visible through chamber windows were observed during exposure for changes in activity and compared subjectively to study controls. Immediately before and after each exposure, each rat was removed from its cage and examined by a trained technician. On the morning of necropsy, cage-side observations were conducted. Observations included, but were not limited to, examination of behavior patterns, motor activity, respiratory patters, hair, skin, eyes, feces, and urine. On weekends, rats were observed for mortality.

HEMATOLGY AND CLINICAL CHEMISTRY:
At the time of necropsy, blood was collected from the posterior vena cava while the rats were under C02 anesthesia. All hematology and clinical chemistry assays were conducted by the Animal Clinical Analysis Group, HAEL. Hematology tests included: hemoglobin concentration, hematocrit, red blood cell count, white blood cell count, differential white blood cell count, platelet count, red blood cell indices, prothrombin time, and examination of blood smears for cellular morphology. Clinical chemistry tests included: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea nitrogen, glucose, creatinine, sorbitol dehydrogenase, total bilirubin, total protein, albumin, and albumin/globulin ratio.

Sacrifice and pathology:

PATHOLOGY:
Rats were fasted overnight, anesthetized with C02, and exsanguinated by severing the posterior vena cava after collecting blood for hematology and clinical chemistry examination. The lungs, liver, kidneys, adrenal glands, testes, spleen, and thymus were weighed. Paired organs were weighed together. Organ/body weight ratios were calculated. The following tissues were fixed in 10% buffered formalin: nasal passages, trachea, lungs, heart, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, pancreas, liver, salivary glands, kidneys, urinary bladder, pituitary gland, adrenal glands, thyroid glands, parathyroid glands, thymus, spleen, mesenteric lymph nodes, bone marrow (femoral), brain, testes, epididymides, male accessory sex glands, ovaries, vagina, uterus, and Fallopian tubes. All tissues were examined microscopically from the control and high-exposure groups, target organs and gross lesions were examined from the middle- and low-exposure group.

Statistics:

Mean values were calculated for chamber concentration, chamber temperature, chamber relative humidity, body weight, organ weights, hematology, and clinical chemistry. Body weight, organ weight, hematology, and clinical chemistry data were evaluated using the following computer generated statistical tests to indicate statistical significance: Bartlett’s test (p ≤ 0.01), one-way analysis of variance (ANOVA) (p ≤ 0.05), and Duncan’s multiple range test (p≤0.05).
When the variance of means were not considered equal by the Bartlett’s test (p≤0.01), the data were evaluated using Wilk-Shapiro test for normality (p≤0.05) and the F-test for variance ratio (p≤ 0.05), after which the means of the test groups were compared individually to the control using a two-tailed t-test (p≤0.05). When the data were not considered normally distributed by the Wilk-Shapiro test (p≤0.05), the data were evaluated using Ansari-Bradley test: adjusting median (p≤0.05), after which the data were compared individually to the control using the Mann-Whitney test (p≤0.05)
W

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During exposure, those animals near the chamber windows were observed for activity levels. Exposed animals appeared to be less active (lethargic) than control animals. Whether this was because of a depressant effect of the substance or because of the animals were avoiding the vapors due to the odor or other sensory input could not be determined. Following the second week of exposure, the animals in the low-concentration group no longer exhibited reduced activity levels during the exposure periods. Activity levels appeared normal in all groups shortly after exposures ceased, except for the animals in the high-exposure group on Days 1 and 2 when they remained less active during the post-exposure observation periods.

Other clinical signs that were seen during the post-exposure periods of the chamber included sialorrhea, discoloration of the facial haircoat, porphyrin tears, and excessive tearing. Sialorrhea was frequently observed post-exposure in one female rat from the high exposure group and in two female rats from the middle-exposure group. It was occasionally observed post-exposure in two female rats from the high-exposure group. Brown discoloration of the facial haircoat was observed periodically in most female rats from the high-exposure group. Single incidences of porphyrin tears and excessive tearing in high-exposure female rats, and a single incidence of porphyrin tears and two incidences of red discoloration of the facial haircoat in low-exposure male rats were also observed. Alopecia was not considered related to the exposure since it was not observed in rats from the high-exposure group and is frequently seen in untreated rats of this strain.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights for the high-exposure male group were consistently slightly lower than that those of the control group. At study termination this difference was 5%. The body weight differences were not statistically significant when compared to the control group.
Mean body weights for all other groups were comparable to the control group throughout the study.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The mean prothrombin time was slightly lower (p ≤ 0.05) for all male rats exposed to the test substance when compared to the control group. Prothrombin time was comparable among the exposed groups with no evidence of a substance-concentration-related effect. The mean prothrombin time for female rats from all exposure groups comparable to the control group. The mean hematocrit value for female rats from the high-exposure group was slightly but significantly (p≤ 0.05) lower when compared to the control group. Mean hematocrit values for female rats from the middle- and low-exposure group, and for all male exposure groups were comparable to the control group. The mean white blood cell count for the female low-exposure group was significantly (p ≤ 0.05) higher than the control group, but was not increased relative to expected values. Mean white blood cell counts for all other exposure groups were comparable to the control groups. All other hematologic parameters were comparable to those of the control groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean glucose levels for the male rats from the high- and middle-exposure groups were significantly lower (p≤0.05) than those of the control group. However, compared to historical data on glucose values from male control animals, the glucose levels of all male rats in the middle-exposure group and all but one male rat in the high-exposure group were within the range of the historical control (70-201 mg/dL). The mean glucose level for the low-exposure male rats was also somewhat lower when compared to those of the study control group, but the difference was not statistically significant. The mean glucose levels for the female rats from all exposure groups were comparable to the control group. The mean sorbitol dehydrogenase level was significantly (p≤0.05) lower for the high-exposure female group and significantly higher for the middle-exposure female group when compared to the control group. In the absence of a substance concentration-dependent change the sorbitol dehydrogenase values were considered spurious. Mean sorbitol dehydrogenase levels for female rats from the low-exposure group and for male rats from all exposure groups were comparable to their respective control group. Mean total protein and albumin levels were slightly but significantly (p≤0.05) higher for the female rats from the high-exposure group when compared to the control group. Mean total protein and albumin levels for the female rats from the middle- and low-exposure groups and for male rats from all exposure groups were comparable to their respective control group. The mean urea nitrogen level for female rata from the middle-exposure group was significantly higher (p≤0.05) when compared to the control group. Since the mean urea nitrogen level for female rata from the high-exposure group was comparable to the control group, the finding was not considered exposure-related. Mean urea nitrogen levels for female rata from the low-exposure group and for male rats from all exposure groups were comparable to their respective control group. All other clinical chemistry parameters for rats from all exposure groups were comparable to those of the control groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean terminal body weight for male rats from the high-exposure group was slightly, although not significantly, lower than the control group weight. Mean terminal body weights for all other male and female exposure groups were comparable to the control group.

Mean absolute and relative liver weights were significantly (P≤0.05) increased for the high-exposure male and female groups. Although mean absolute (females) and relative (males and females) liver weights were significantly (P≤0.05) higher for the middle-exposure groups when compared to control groups, the differences were very slight. No statistically significant difference was noted between the mean absolute liver weight of the middle-exposure group males and the controls. Mean absolute and relative liver weights for the low-exposure male and female groups were comparable to controls.
The mean relative kidney weight, but not the mean absolute kidney weight, for the male high-exposure group was significantly higher (P≤0.05) than the control group. This difference was primarily due to the kidney weight of one rat. There were no statistically significant differences when this rat's weight data was eliminated from the analysis. The mean absolute and relative kidney weights for the middle- and low-exposure male groups and for all female exposure groups were comparable to their respective control group. Mean absolute and relative adrenal gland weights for female rats from the high-exposure group were higher, but not significantly (P≤0.05) different, from those of the control group. Mean absolute and relative adrenal gland weights for female rats from the middle- and low-exposure groups and for male rats from all exposure groups were comparable to their respective control group. The mean absolute spleen weight, but not the mean relative spleen weight, for the low-exposure male group, was higher (P≤0.05) than the control group spleen weight. This finding was considered spurious since mean absolute and relative spleen weights for the middle- and high-exposure male groups were comparable to the control group. All other organ weight measurements for rats from all exposure groups were comparable to the control groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pallor of the external surface of the kidneys was observed for three male rats from the high-, three male rats from the middle-, and four male rats from the low-exposure groups. Pallor of kidneys was not observed for female rats from any exposure group.
A small number of other potential gross changes were noted during the necropsy examination. These included enlarged kidney (males) enlarged adrenal gland (females), and edema of the mucosa of the glandular stomach. On follow-up examinations of these observations including organ weight and histopathology, none of these potential changes could be verified except that the kidney weight of one rat (male high-exposure group) was higher than that of all other rats in this study.
No other exposure-related lesions were detected for rats of either sex from any exposure group on necropsy examination.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Accumulation of hyalin droplets was observed intracellularly in the proximal convoluted tubules of the kidneys of all male rats, but the number and severity of droplets formed was greatest in the exposed rats. The severity of droplet formation appeared to increase with increasing concentrations. The droplets were morphologically similar to alpha-2µ-globulin droplets which have been described by others in the kidneys of male rats. Cytoplasmic vacuolation and basophilic cytoplasmic changes in the proximal convoluted tubules were seen in one or two male rats from the high- and middle-exposure groups, but not the low-exposure group. None of these findings were observed in female rats from any exposure level. Hypertrophy of hepatocytes was observed in four of five male rats and all five female rats from the high-exposure group, but not in rats from either of the other exposure groups.
Increased cytoplasmic vacuolization and hypertrophy of the cells in the zona fasciculata of the adrenal glands were observed in four female rats from the high-exposure group, while increased cytoplasmic vacuolization was present in two female rats from the middle-exposure group. The vacuoles were predominantly small or fine, but sometimes the cells were coarsely vacuolated. In a few cells, extensive accumulation of vacuoles resulted in the occurrence of large ballooned cells. In the control rats, the vacuoles were fine and involved a smaller number of cells. Adrenal glands from control rats typically are more heavily vacuolated than the controls in this study. The apparent increase in severity of this lesion in treated rats may have been due more to an unusually low severity of vacuolization in the concurrent control rats, rather than from a true increased incidence in the exposed rats. Therefore, this lesion may not represent a true treatment-related effect. No other exposure-related changes were observed for rats of either sex from any exposure group during the histopathology examinations.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEC

Effect level:

386 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Dose descriptor:

NOEC

Effect level:

< 128 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Absolute and relative liver weights for males

	Test Group 0/ M 0 ppm	Test Group 1/ M 128 ppm	Test Group 2/ M 386 ppm	Test Group 3/ M 1221 ppm
liver weight (g)	11.61	12.01	13.6	15.66
relative liver weight	3.27	3.35	3.84	4.8
rel liver increase (%)	100	102	117	147

Absolute and relative liver weights for females

	Test Group 0/ F 0 ppm	Test Group 1/ F 128 ppm	Test Group 2/ F 386 ppm	Test Group 3/ F 1221 ppm
liver weight (g)	7.2	7.65	8.18	11.45
relative liver weight	3.27	3.42	3.72	5.14
rel liver increase (%)	100	105	114	157

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017 - 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

OECD 421 plus functional observation battery and investigation of liver and kidney toxicity

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Sex:

male/female

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

6h/day

Frequency of treatment:

daily

Dose / conc.:

100 ppm (nominal)

Remarks:

99.1 ppm / 471 mg/m3 (analytical)

Dose / conc.:

300 ppm (nominal)

Remarks:

306 ppm / 1414 mg/m3 (analytical)

Dose / conc.:

1 000 ppm (nominal)

Remarks:

1009 ppm / 4715 mg/m3 (analytical)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Dose descriptor:

NOAEC

Effect level:

99 ppm (analytical)

Basis for effect level:

body weight and weight gain

clinical signs

organ weights and organ / body weight ratios

other: alpha-2u nephropathy in male rats

Critical effects observed:

yes

Lowest effective dose / conc.:

1 009 ppm (analytical)

System:

other: body weight loss, strong increse in liver weight, clinical signs

Treatment related:

yes

Dose response relationship:

yes

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

471 mg/m³

Study duration:

subacute

Species:

rat

System:

other: liver and general toxicity (narcotic effects)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Remarks:

NOAEL systemic toxicity inh.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

May, 1981

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

September, 1984

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test substance( as cited in report): Diisopropyl ketone / 2.4-Dimethyl-3-pentanone; DIPK
- Purity: The purity of the test substance was determined to be 100 ± 0.00% (mean ± SD) prior to and 99.8 ± 0.006% after the study when analyzed by gas chromatography.

Species:

rat

Strain:

other: CD (SD) BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: Males 46 days, Females 51 days
- Weight at study initiation: Males 231 ± 6 g, Females 194 ± 9 g (mean ± SD)
- Housing: Singly housed in multicompartmented stainless steel mesh cages
- Diet: Certified Rodent Diet (Agway Prolab RMH 3000, pellets), ad libitum during non-exposure periods
- Water: ad libitum during non-exposure periods
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 23.3 (reported as 70-74 °F)
- Humidity (%): 50 - 56
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

The inhalation exposures were conducted in 420 L stainless steel and glass inhalation chambers at target concentrations of 1200, 380, 120, and 0 ppm. Exposures were conducted five days per week, 6 hour per day, for a total of 22 exposures over a four-week period.
The chambers were maintained at pressures of -0.035 to -0.05 inch water (gauge) relative to room air and at 12 air changes per hour. For each exposure level, male and female rats were singly housed and exposed simultaneously.
A vapor was produced by metering the test substance from a reservoir into a glass bead-packed column where it evaporated. The column was supplied with metered, dried, oil-free compressed air. The resultant vapor was directed into the turret of the chamber where it was mixed with filtered, conditioned air.
Chamber vapor concentrations were analytically determined by infrared spectroscopy at least 8 times during each exposure for each exposure level.
The samples were taken from a fixed reference position in the inhalation chamber. Chamber temperature and relative humidity were recorded twice per hour. Chamber air flow was set at 84 liters per minute. The nominal chamber concentration was calculated from the mass of chemical consumed and total air flow.
The concentration of background non-gaseous material was measured for the high-exposure and control chamber at least once each week during the exposure to ensure that the exposures were to a vapor and not an aerosol.

The vapor distribution of the test substance was not measured for positional variations within the inhalation chambers for this study. However, vapor distribution was determined during an LC50 study (see section Acute toxicity: inhalation) of the test substance about two months earlier using the same lot of the test material. For the samples taken from the nine cage positions in the LC50 study, the concentration (mean ± coefficient of variation) was 1300 ppm ± 7%, and from the fixed reference position was 1264 ppm ± 1%. The results indicated that positional effects were minimal and the fixed reference position selected provided a representative sample for chamber vapor concentration. The same fixed reference position was selected for the present study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber vapor concentrations were analytically determined by infrared spectroscopy at least 8 times during each exposure for each exposure level.

Duration of treatment / exposure:

Six hours per day

Frequency of treatment:

Five days per week for a total of 22 exposures over a 4-week period

Dose / conc.:

128 ppm (analytical)

Remarks:

Target concentration 120 ppm

Dose / conc.:

386 ppm (analytical)

Remarks:

Target concentration 380 ppm

Dose / conc.:

1 221 ppm (analytical)

Remarks:

Target concentration 1200 ppm

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

BODY WEIGHT:
Body weights were measured on days 0, 4, 7, 14, 21, and 28

CLINICAL OBSERVATIONS:
Rats visible through chamber windows were observed during exposure for changes in activity and compared subjectively to study controls. Immediately before and after each exposure, each rat was removed from its cage and examined by a trained technician. On the morning of necropsy, cage-side observations were conducted. Observations included, but were not limited to, examination of behavior patterns, motor activity, respiratory patters, hair, skin, eyes, feces, and urine. On weekends, rats were observed for mortality.

HEMATOLGY AND CLINICAL CHEMISTRY:
At the time of necropsy, blood was collected from the posterior vena cava while the rats were under C02 anesthesia. All hematology and clinical chemistry assays were conducted by the Animal Clinical Analysis Group, HAEL. Hematology tests included: hemoglobin concentration, hematocrit, red blood cell count, white blood cell count, differential white blood cell count, platelet count, red blood cell indices, prothrombin time, and examination of blood smears for cellular morphology. Clinical chemistry tests included: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea nitrogen, glucose, creatinine, sorbitol dehydrogenase, total bilirubin, total protein, albumin, and albumin/globulin ratio.

Sacrifice and pathology:

PATHOLOGY:
Rats were fasted overnight, anesthetized with C02, and exsanguinated by severing the posterior vena cava after collecting blood for hematology and clinical chemistry examination. The lungs, liver, kidneys, adrenal glands, testes, spleen, and thymus were weighed. Paired organs were weighed together. Organ/body weight ratios were calculated. The following tissues were fixed in 10% buffered formalin: nasal passages, trachea, lungs, heart, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, pancreas, liver, salivary glands, kidneys, urinary bladder, pituitary gland, adrenal glands, thyroid glands, parathyroid glands, thymus, spleen, mesenteric lymph nodes, bone marrow (femoral), brain, testes, epididymides, male accessory sex glands, ovaries, vagina, uterus, and Fallopian tubes. All tissues were examined microscopically from the control and high-exposure groups, target organs and gross lesions were examined from the middle- and low-exposure group.

Statistics:

Mean values were calculated for chamber concentration, chamber temperature, chamber relative humidity, body weight, organ weights, hematology, and clinical chemistry. Body weight, organ weight, hematology, and clinical chemistry data were evaluated using the following computer generated statistical tests to indicate statistical significance: Bartlett’s test (p ≤ 0.01), one-way analysis of variance (ANOVA) (p ≤ 0.05), and Duncan’s multiple range test (p≤0.05).
When the variance of means were not considered equal by the Bartlett’s test (p≤0.01), the data were evaluated using Wilk-Shapiro test for normality (p≤0.05) and the F-test for variance ratio (p≤ 0.05), after which the means of the test groups were compared individually to the control using a two-tailed t-test (p≤0.05). When the data were not considered normally distributed by the Wilk-Shapiro test (p≤0.05), the data were evaluated using Ansari-Bradley test: adjusting median (p≤0.05), after which the data were compared individually to the control using the Mann-Whitney test (p≤0.05)
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Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During exposure, those animals near the chamber windows were observed for activity levels. Exposed animals appeared to be less active (lethargic) than control animals. Whether this was because of a depressant effect of the substance or because of the animals were avoiding the vapors due to the odor or other sensory input could not be determined. Following the second week of exposure, the animals in the low-concentration group no longer exhibited reduced activity levels during the exposure periods. Activity levels appeared normal in all groups shortly after exposures ceased, except for the animals in the high-exposure group on Days 1 and 2 when they remained less active during the post-exposure observation periods.

Other clinical signs that were seen during the post-exposure periods of the chamber included sialorrhea, discoloration of the facial haircoat, porphyrin tears, and excessive tearing. Sialorrhea was frequently observed post-exposure in one female rat from the high exposure group and in two female rats from the middle-exposure group. It was occasionally observed post-exposure in two female rats from the high-exposure group. Brown discoloration of the facial haircoat was observed periodically in most female rats from the high-exposure group. Single incidences of porphyrin tears and excessive tearing in high-exposure female rats, and a single incidence of porphyrin tears and two incidences of red discoloration of the facial haircoat in low-exposure male rats were also observed. Alopecia was not considered related to the exposure since it was not observed in rats from the high-exposure group and is frequently seen in untreated rats of this strain.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights for the high-exposure male group were consistently slightly lower than that those of the control group. At study termination this difference was 5%. The body weight differences were not statistically significant when compared to the control group.
Mean body weights for all other groups were comparable to the control group throughout the study.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The mean prothrombin time was slightly lower (p ≤ 0.05) for all male rats exposed to the test substance when compared to the control group. Prothrombin time was comparable among the exposed groups with no evidence of a substance-concentration-related effect. The mean prothrombin time for female rats from all exposure groups comparable to the control group. The mean hematocrit value for female rats from the high-exposure group was slightly but significantly (p≤ 0.05) lower when compared to the control group. Mean hematocrit values for female rats from the middle- and low-exposure group, and for all male exposure groups were comparable to the control group. The mean white blood cell count for the female low-exposure group was significantly (p ≤ 0.05) higher than the control group, but was not increased relative to expected values. Mean white blood cell counts for all other exposure groups were comparable to the control groups. All other hematologic parameters were comparable to those of the control groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean glucose levels for the male rats from the high- and middle-exposure groups were significantly lower (p≤0.05) than those of the control group. However, compared to historical data on glucose values from male control animals, the glucose levels of all male rats in the middle-exposure group and all but one male rat in the high-exposure group were within the range of the historical control (70-201 mg/dL). The mean glucose level for the low-exposure male rats was also somewhat lower when compared to those of the study control group, but the difference was not statistically significant. The mean glucose levels for the female rats from all exposure groups were comparable to the control group. The mean sorbitol dehydrogenase level was significantly (p≤0.05) lower for the high-exposure female group and significantly higher for the middle-exposure female group when compared to the control group. In the absence of a substance concentration-dependent change the sorbitol dehydrogenase values were considered spurious. Mean sorbitol dehydrogenase levels for female rats from the low-exposure group and for male rats from all exposure groups were comparable to their respective control group. Mean total protein and albumin levels were slightly but significantly (p≤0.05) higher for the female rats from the high-exposure group when compared to the control group. Mean total protein and albumin levels for the female rats from the middle- and low-exposure groups and for male rats from all exposure groups were comparable to their respective control group. The mean urea nitrogen level for female rata from the middle-exposure group was significantly higher (p≤0.05) when compared to the control group. Since the mean urea nitrogen level for female rata from the high-exposure group was comparable to the control group, the finding was not considered exposure-related. Mean urea nitrogen levels for female rata from the low-exposure group and for male rats from all exposure groups were comparable to their respective control group. All other clinical chemistry parameters for rats from all exposure groups were comparable to those of the control groups.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean terminal body weight for male rats from the high-exposure group was slightly, although not significantly, lower than the control group weight. Mean terminal body weights for all other male and female exposure groups were comparable to the control group.

Mean absolute and relative liver weights were significantly (P≤0.05) increased for the high-exposure male and female groups. Although mean absolute (females) and relative (males and females) liver weights were significantly (P≤0.05) higher for the middle-exposure groups when compared to control groups, the differences were very slight. No statistically significant difference was noted between the mean absolute liver weight of the middle-exposure group males and the controls. Mean absolute and relative liver weights for the low-exposure male and female groups were comparable to controls.
The mean relative kidney weight, but not the mean absolute kidney weight, for the male high-exposure group was significantly higher (P≤0.05) than the control group. This difference was primarily due to the kidney weight of one rat. There were no statistically significant differences when this rat's weight data was eliminated from the analysis. The mean absolute and relative kidney weights for the middle- and low-exposure male groups and for all female exposure groups were comparable to their respective control group. Mean absolute and relative adrenal gland weights for female rats from the high-exposure group were higher, but not significantly (P≤0.05) different, from those of the control group. Mean absolute and relative adrenal gland weights for female rats from the middle- and low-exposure groups and for male rats from all exposure groups were comparable to their respective control group. The mean absolute spleen weight, but not the mean relative spleen weight, for the low-exposure male group, was higher (P≤0.05) than the control group spleen weight. This finding was considered spurious since mean absolute and relative spleen weights for the middle- and high-exposure male groups were comparable to the control group. All other organ weight measurements for rats from all exposure groups were comparable to the control groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pallor of the external surface of the kidneys was observed for three male rats from the high-, three male rats from the middle-, and four male rats from the low-exposure groups. Pallor of kidneys was not observed for female rats from any exposure group.
A small number of other potential gross changes were noted during the necropsy examination. These included enlarged kidney (males) enlarged adrenal gland (females), and edema of the mucosa of the glandular stomach. On follow-up examinations of these observations including organ weight and histopathology, none of these potential changes could be verified except that the kidney weight of one rat (male high-exposure group) was higher than that of all other rats in this study.
No other exposure-related lesions were detected for rats of either sex from any exposure group on necropsy examination.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Accumulation of hyalin droplets was observed intracellularly in the proximal convoluted tubules of the kidneys of all male rats, but the number and severity of droplets formed was greatest in the exposed rats. The severity of droplet formation appeared to increase with increasing concentrations. The droplets were morphologically similar to alpha-2µ-globulin droplets which have been described by others in the kidneys of male rats. Cytoplasmic vacuolation and basophilic cytoplasmic changes in the proximal convoluted tubules were seen in one or two male rats from the high- and middle-exposure groups, but not the low-exposure group. None of these findings were observed in female rats from any exposure level. Hypertrophy of hepatocytes was observed in four of five male rats and all five female rats from the high-exposure group, but not in rats from either of the other exposure groups.
Increased cytoplasmic vacuolization and hypertrophy of the cells in the zona fasciculata of the adrenal glands were observed in four female rats from the high-exposure group, while increased cytoplasmic vacuolization was present in two female rats from the middle-exposure group. The vacuoles were predominantly small or fine, but sometimes the cells were coarsely vacuolated. In a few cells, extensive accumulation of vacuoles resulted in the occurrence of large ballooned cells. In the control rats, the vacuoles were fine and involved a smaller number of cells. Adrenal glands from control rats typically are more heavily vacuolated than the controls in this study. The apparent increase in severity of this lesion in treated rats may have been due more to an unusually low severity of vacuolization in the concurrent control rats, rather than from a true increased incidence in the exposed rats. Therefore, this lesion may not represent a true treatment-related effect. No other exposure-related changes were observed for rats of either sex from any exposure group during the histopathology examinations.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEC

Effect level:

386 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Dose descriptor:

NOEC

Effect level:

< 128 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Absolute and relative liver weights for males

	Test Group 0/ M 0 ppm	Test Group 1/ M 128 ppm	Test Group 2/ M 386 ppm	Test Group 3/ M 1221 ppm
liver weight (g)	11.61	12.01	13.6	15.66
relative liver weight	3.27	3.35	3.84	4.8
rel liver increase (%)	100	102	117	147

Absolute and relative liver weights for females

	Test Group 0/ F 0 ppm	Test Group 1/ F 128 ppm	Test Group 2/ F 386 ppm	Test Group 3/ F 1221 ppm
liver weight (g)	7.2	7.65	8.18	11.45
relative liver weight	3.27	3.42	3.72	5.14
rel liver increase (%)	100	105	114	157

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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

In the Reproductive / Developmental Toxicity Screening Test, groups of 10 male and 10 female Wistar rats (F0 animals) per test group were exposed in a whole-body inhalation system to dynamic atmosphere of the test substance up to 1000 ppm for 6 hours per day on each day. The age of the male animals at the beginning of the study was 10 - 11 weeks, the age of the female animals 9 weeks.

The NOAEL for general systemic toxicity was 100 ppm in females based on adverse clinical findings and adverse effects on food consumption, body weight development as well as liver toxicity (high-dose only). For the males, a NOAEL could not be established in this study since a alpha2u-nephropathy with correlating increased absolute and relative kidney weights was observed down to the lowest concentration of 100 ppm. As this finding is not relevant for humans, it should not be taken into account for risk assessment and thus, 100 ppm is used as the starting point for the DNEL derivation.

In a 4 week inhalation toxicity study (Eastman Kodak 1990) performed similar to OECD Guideline 412, groups of rats (5 sex/dose) were whole body exposed to 1121, 386, 128, or 0 ppm of the test substance 5 days per week, 6 hr per day, for a total of 22 exposures over a 4-week period. The age of the animals at the beginning of the study was about 7 weeks.

Chamber vapor concentrations were analytically determined by infrared spectroscopy at least 8 times during each exposure for each exposure level. No mortality occurred during the study. Clinical signs associated with exposure to the test substance were mild and reversible. Exposed animals were less active during exposure, although, all animals were normally active following cessation of exposures. There were no other clinical signs consistently associated with exposure to the test substance. The mean body weights of the 1221 ppm male groups were consistently, but not significantly, lower than control weights. Minor statistically significant differences between exposed and control groups in hematology and clinical chemistries were not diagnostic of any toxicologic effect. The differences included: prothrombin time (males, all exposure groups), hematocrit (females, high-exposure group), white blood cell count (females, low-exposure group), glucose (males, middle- and high-exposure groups), sorbitol dehydrogenase (females, middle- and high-exposure groups), total protein and albumin (females. high exposure group), and urea nitrogen (females, middle-exposure group). No significant lesions were detected during necropsy examinations. Histopathologic changes noted in exposed animals included an increase in the number and severity of intracellular hyalin droplets accumulating in the proximal convoluted tubules of the kidneys of all exposed males. The significance of this finding, which was also seen in control rats and has been described by others in the kidneys of rats exposed to other branched-chain hydrocarbons, is unclear since the lesion does not appear to have a counterpart in man. An apparent increase in the severity of cytoplasmic vacuolization in the zona fasciculata of the adrenal glands was observed in females at both the 1221 and 386 ppm exposure levels. This difference was considered to be the result of an unusually low severity of vacuolization in the concurrent control rats, rather than a toxic response to exposure. Mean absolute and relative liver weights were significantly(p ≤0.05) heavier for the male and female 1221 ppm groups. In the 1221 ppm groups, hepatocyte hypertrophy was observed in for male and all female rats. Although mean absolute (female) and relative (male and female) liver weights were significantly (p≤ 0.05) heavier for the 316 ppm groups in comparison to the control groups, the differences were very slight, and no histopathological findings were evident in the livers. Overall, the changes observed following exposure to the test substance were mild. The no-observed-effect concentration for this study was considered to be close to, but below 128 ppm because at this concentration level, only the rat specific alpha-2u-nephropathy occured. Due to the reduction in activity levels during exposure the no-observed-adverse-effect concentration was considered to be 386 ppm.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. 100 ppm (471 mg/m3) was identfifed as NOAEC. and only slight effects were obsered at 306 and 36 ppm. Therefore, the criteria for STOT RE Cat 1 are not fulfilled.
Significant toxic effects (strong reduction in food consumption and body weight loss, reduced attention and maternal care) were observed at 1009 ppm upon subacute inhalative exposure in rats. The strong absolute and relative increase in liver weights were not accompagnied by liver degeneration. The guidance value for assigning STOT RE Cat 2 after subacute exposure is 750 ppm. This value is between the mildly adverse dose (386 ppm) and the clearly adverse dose (1009 ppm). It is not possible to assign a specific target organ; however the body weight loss is indicatative of a significant general toxicity. It is expected that this would also be observed at the dose level of 750 ppm. As a result the substance is considered to be classified for repeated dose toxicity in Category 2 under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No. 2015/1221.