4-methylpent-3-en-2-one

Endpoint summary

Description of key information

In a combined repeated dose and reproductive/developmental toxicity study by inhalation in rats (OECD 422), a lowest-observed-adverse-effect concentration (LOAEC) for toxicity was determined to be 31 ppm based on effects on feed consumption, body weights, body weight gain, and nasal passage histopathology.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Neither deviation had a detrimental effect on the outcome of the study

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Kingston, NY)
- Age at study initiation: The male and female rats were 66 days of age at the beginning of the pre-mating phase and 80 days of age at the beginning of the mating phase
- Weight at study initiation: The male and female rats weighed 330.3 ± 10.6 or 218.5 ± 9.2 grams (mean ± SD), respectively, at the beginning of the pre-mating phase.
- Fasting period before study: no
- Housing: During the acclimation period, animals were housed by sex in groups of five in suspended, stainless-steel, wire-mesh cages. During exposure periods, the main study animals were singly housed in multicompartmented, stainless-steel, wire-mesh cages. During nonexposure period, the rats were housed in suspended, stainless-steel, wire-mesh cages in a room separate from the exposure room. Starting at study initiation and continuing through the premating period, and following the mating period, male and female animals were singly housed during nonexposure periods. During the mating period, the animals were housed one male with one female during nonexposure periods. On the 20th day of gestation, solid bottom pans containing bedding material for nesting were put in the nonexposure cages of the female rats.
- Diet: Agwaya Prolab' Animal Diet (RMH 3200 ad libitum
- Water: municipal tap water ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70-76
- Humidity (%): 36-58
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: October 7, 1991 to May 7, 1992

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 420 L stainless steel and glass inhalation chambers
- Method of holding animals in test chamber: male and female rats were singly housed and exposed simultaneously
- Air flow rate and air change rate:
Exposure Level Compressed AirFlow (Lpm) Dilution Air Flow (Lpm) Total Air Flow (Lpm) Air Changes Per
Hour
High 40 50-62 89-102 13-15
Mid 25 65-93 90-118 13-17
Low 25 62-68 89-95 12-13
Control 25 65 90 13
- Method of conditioning air: Compressed air (Kodak Park Utilities Division) was passed through silices gel (EM Science, Cherry Hill, NJ) and an oil filter (Deltech Engineering Inc., New Castle, DE).
- System of generating vapors: The test substance was metered by a Sage peristaltic pump Model No. 375A (Orion Research Inc., Cambridge, MA) [high-exposure group] from a graduated cylinder reservoir or by a Harvard syringe pump Model No. 2274 (Harvard Apparatus, South Natick, MA) [mid- and low¬exposure groups] from a 25 mL syringe into a glass distillation column (1.5" o.d. x 17" [high¬exposure group] or 1.0" o.d. x 20.5" [mid- and low-exposure groups] Ace Glass Inc., Vinland, NJ). The liquid trickled over the surface of tightly packed glass beads (4 mm diameter) within the distillation column. The beads were used to increase the surface area and enhance vaporization.
- Temperature, humidity, pressure in air chamber: Chamber temperatures for the high-, mid-, and low-exposure groups and the control group were 23.5 ± 0.6, 24.0 ± 0.6, 23.7 ± 0.7, and 23.5 ± 0.8-C (mean ± SD), respectively, and chamber relative humidity were 49.7 ± 6.9, 50.4 ± 4.4, 52.4 ± 3.7, and 43.6 ± 4.1%, respectively.
- Method of particle size determination: Nongaseous airborne material within the exposure inhalation chambers was measured using a Micro Laser Particle Counter model µLCP-301 at a sampling rate of 0.1 cmf for 10 sec. (Particle Measuring Systems, Inc., Boulder, CO). Airborne particles greater than 0.3 mm. were counted. Samples were collected from a fixed reference position within each chamber at least six days per week during the exposure. The samples collected from the test substance exposure chambers were compared to samples collected from the control chamber. The results indicated the absence of a test substance aerosol.
- Treatment of exhaust air: The effluent from all Chambers was filtered through a coarse prefilter, a HF-PA filter, and a charcoal filter.

TEST ATMOSPHERE
- Brief description of analytical method used:
Chamber vapor concentrations were monitored with an automated multipositional air sampling and analysis system. The system consisted of a MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, Cl.), a Perkin-Elmer SIGMA 15 data station, and a four-port multipositional environmental sampling valve (Valco Instruments, Houston, TX). Chamber vapor samples were continuously collected, from a fixed reference position within each chamber, through the four port valves using TEFLON tubing (3/16" i.d.). On Day 10 of the study, the microprocessor of the Pekin-Elmer SIGMA 15 data station (which controls the automatic sampling, analyses, and recording of the chamber concentrations) began to malfunction, and it failed on Day 11. This did not affect the performance of the Miran, but did make it necessary to manually sample and record the concentrations for the duration of the study.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see "Details on inhalation exposure"

Duration of treatment / exposure:

pre-mating (14 days), mating (1-14 days), gestation (21-22 days), and early lactation (4 days) (36 to 49 exposures (females) and 49 exposures (males))

Frequency of treatment:

six hours per day, seven days per week

Remarks:

Doses / Concentrations:
31, 103 and 302 ppm (124, 413 and 1211 mg/m3)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
35, 10 and 388 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
30, 100 and 300 ppm
Basis:
other: target conc.

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
A range-finding inhalation study in pregnant female rats was conducted 1) to evaluate maternal toxic effects of the test substance following repeated inhalation exposures, and 2) to establish exposure levels for this study.
- Rationale for animal assignment (if not random):
All culling and randomization of the animals was done by computer-generated lists using the Automated Animal Toxicology System (AATS). The male and female rats were assigned to the study groups on the basis of body weights so that the mean body weights were similar among the groups at the start of the study. Within an exposure group, male and female rats were randomly paired for mating. For the second week of mating, the uninseminated female rats were randomly paired with a male from the same exposure group that had inseminated a female the previous week.
- Post-exposure recovery period in satellite groups: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
For adult animals, clinical observations were made at least once daily. Observations included, but were not limited to, examination of the hair, skin, eyes, mucous membranes, behavior patterns, respiratory patterns, feces, urine, and general activity. Post-partum neonatal observations were conducted similarly. Rats visible through chamber windows were observed for clinical signs during exposure. The dams were observed for the beginning of parturition and this date was used to calculate the gestation period. The day on which the dam had completely finished delivering was considered Day 0 post-partum.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes The male rats were weighed on Days 0, 7, and weekly thereafter. Male rats were also weighed the day before euthanasia. Body weight change was calculated for Days 0-7, 7-14, 14-21, 21-28, 28-35, 35-42, 42-48, and 0-48.
The female rats were weighed on Days 0, 7, 14 and 21 of the premating/mating phase, Days 0, 7, 14, and 20 of the gestation phase, and Days 0 and 4 post-partum. Body weight change was calculated for Days 0-7, 7-14, 14-20, and 0-20 of the gestation phase and for Days 0-4 post-partum.
The unexposed female rats used in the second mating of the male high-exposure group were weighed on Days 0 and 20 of gestation. Body weight change between Days 0-20 of gestation was calculated.

FOOD CONSUMPTION: Yes
For male rats, feed consumption was determined between Days 0-7, 7-14, 28-35, 35-41, 41-42, and 42-48. Feed consumption was not determined during the mating period. The male high-exposure group did not have feed consumption determined between Days 42-48 because of the second mating period. Male rats were fasted the day before euthanasia.
For female rats from the main study, feed consumption was determined between Days 0-7, and 7-14 of the pre-mating phase, Days 0-7, 7-14, and 14-20 of the gestation phase, and Days 0-4 post-partum. Feed consumption was not determined during the mating period.
Feed consumption was not determined for the unexposed female rats from the male high¬exposure group second mating.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy, blood was collected from the posterior vena cava of all male animals while the rats were under CO2 anesthesia.
- Animals fasted: Yes
- How many animals: all
- Parameters checked : hematocrit, hemoglobin concentration, red blood cell count, and white blood cell count. Fixed blood smears were prepared and stored.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy, blood was collected from the posterior vena cava of all male animals while the rats were under CO2 anesthesia.
- Animals fasted: Yes
- How many animals:
- Parameters checked: aspartate aminotransferase (AST1.), alanine aminotransferase (ALT), glucose, urea nitrogen (UN), gamma glutamyl transpeptidase (GGTP), total bilirubin, creatinine, total protein, albumin, albumin/globulin ratio (A/G ratio), potassium, chloride, calcium, sodium, and phosphorus.

URINALYSIS: Yes
- Time schedule for collection of urine: On Day 41, male rats were placed in metabolism cages for approximately 16 hours for urine collection on Day 42
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: The osmolality and volume of the urine was measured and the urine was evaluated for color and appearance. The urine was tested using Multistix® for nitrite, urobilinogen, protein, pH, blood, ketones, bilirubin, and glucose. Microscopic examination was conducted on urinary sediment for the presence of red blood cells, white blood cells, epithelial cells, triple phosphate crystals, amorphous sediment, sperm, and bacteria.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
Wet weights of the liver, kidneys, thymus, lungs, testes, and epididymides were collected at necropsy. Paired organs were weighed together. Org/body weight ratios were calculated.

HISTOPATHOLOGY: Yes
The following tissues were collected from adult animals and were fixed in 10% buffered formalin: liver (2 sections), kidneys (1 section/kidney), adrenal glands, brain (3 sections including medulla oblongata, pons, cerebellar cortex, and cerebral cortex), heart, spleen, thymus, trachea, lungs, nasal passages, ovaries, and gross lesions. The testes and epididymides were preserved in Bouin's fixative.
Histological evaluation was conducted on the tissues listed above from control and high-exposure adult male and female animals. If abnormalities or equivocal results were seen in any of these tissues, the same tissues from lower exposure groups were examined. Histopathology was performed on significant gross lesions for all adult animals. Ovaries from any female which failed to complete its pregnancy were examined.

Statistics:

Mean values were calculated for test substance concentration, chamber temperature, chamber relative humidity, body weight, body weight change, feed consumption, organ weights, hematology, and clinical chemistries. Mean body weight, feed consumption, organ weight, hematology, and clinical chemistry data were evaluated using the following computer-generated statistical, tests: Bartlett's test (p <= 0.01), one-way analysis of variance (ANOVA) (p <= 0.05), and Duncan's multiple range test (p <= 0.05) to indicate statistical significance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During-exposure, male and female rats from the high-exposure group exhibited reduced general
activity on Days Oto 13 (Day 0 - minor, Days 1 to 13 - minimal) and partially closed eyes on
Days Oto 3 (Day 0 - minor, Days 1 to 3 - minimal). No clinical abnormalities were observe,d
during exposure for the high-exposure group from Day 14 ·to study termination. During
exposure clinical abnormalities for male and female rats from the-~ mid-exposure group were
restricted to a minimal reduction of general activity on Day 0 only. Reduced activity was not
observed for the low-exposure group.
A total of nine incidences of post-exposure sialorrhea were observed for three high-exposure
male rats. Sialorrhea was not observed for any other test substance exposed or control rats.
The incidence of post-exposure porphyrin nasal discharge was higher for the test substance
exposed male and female rats when compared to the control rats (

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

An exposure-dependent reduction in mean body weight was observed for all male and female
test substance-exposed groups from the main study.
For male rats, the reduction in mean body weights was statistically significant (p < 0.05) on
Days 7 to 35 for all test substance exposure groups, on Day 42 for the high-exposure group
only, and on Day 48 for the high- and low-exposure groups when compared to the control
group. Mean body weights for male rats from the mid- and low-exposure groups on Day 42 and
from the mid-exposure group on Day 48, while not statistically significantly reduced, continued
to be lower than those of the control group. Body weight gain was reduced (p !S 0.05) in an
exposure dependent manner between Days 0 and 7 for all male test substance exposure groups
and was reduced between Days 7 and 14 and Days 0 and 48 for the male high-exposure group
when compared to the control group. During the study, the male high-, mid-, and low-exposure
groups gained 29, 11, and 11 % less weight then the control group, respectively.
For female rats from the main study, the reduction in mean body weights was statistically
significant (p s 0.05) for all test substance exposure groups on Days 7 and 14 of the pre-mating
phase, Days 0 to 20 of the gestation phase, and on Day 0 of the lactation phase when compare.d
to the control group. Mean body weights for female rats from all test substance exposure groups
on Day 4 of the lactation phase were not statistically significantly reduced when compared to the
control group, but continued to be lower than those of the control group. Body weight gain,
during the gestation and post-partum phases, was comparable between the female test substance
exposure groups and the control group.
The body weight gain observed for the unexposed female rats from the male high-exposure
group second mating exceeded the body weight gain of all test substance exposure groups, and
the control group from the main study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For male rats, mean feed consumption was reduced (p s 0.05) in an exposure-related manner
for all test substance exposure groups on Day 7 and for the high- and mid-exposure groups on
Day 14 when compared to the control group. For the remainder of the study, mean feed
consumption was comparable between the male test substance exposure groups and the control
group.
For female rats, mean feed consumption was reduced (p s 0.05) in an exposure-related manner
for all test substance exposure groups on Day 7 and 14 of the pre-mating phase and for the midand
high-exposure groups on Day 7 of the gestation phase when compared to the control group.
Feed consumption for all test substance exposure groups remained slightly lower than was
observed for the control group through Day 20 of gestation; however, .this reduction was not
statistically significant. Feed consumption during the lactation phase for all test substance
exposure groups was statistically comparable to, though slightly higher than, the control group
consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

All hematologic parameters for male rats from all test substance exposure groups were
comparable to the control groups.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Mean creatinine levels were reduced (p ~ 0.05) in an exposure-related manner for all male test
substance exposure groups when compared to the control group.
The serum of two to seven male rats per group, including the control group, was slightly to
markedly hemolyzed. This hemolysis was believed to be an artifact of bleeding.
All other clinical chemistry parameters for male rats from all test substance exposure groups
were comparable to the control groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

All urinalysis parameters for male rats from all test substance exposure groups were comparable
to the control groups.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean absolute and relative testes and epididymides weights for all male test substance
exposure groups were statistically comparable to the control group. However, when Rat 643
(a high-exposure male rat observed to have small testes at the time of ne.cropsy) was eliminated
from the analysis, the mean relative testes, for the high-exposure group, and mean relative
epididymides weights, for the mid- and high-exposure groups, became significantly higher
(p < 0.05) than the mean control weights. This difference was probably the result of a
de.creased body weight for the mid- and high-exposure male rats rather then an effect on the
testes or epididymides. All other organ weight me.asurements for male test substance exposure
groups and all organ weight me.asurements for all female test substance exposure groups were
comparable to the control group organ weights.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY
No mortality occurred during the study.

CLINICAL SIGNS
During exposure, effects of the test substance consisted of a transient reduction in activity for the high- and mid-exposure groups, and partially closed eyes for the high-exposure group. An increased incidence of post-exposure porphyrin nasal discharge was observed for all test substance exposure groups with the incidence being slightly higher for the female rats when compared to the male rats. Additionally, post-exposure sialorrhea was observed for 3 of 12 high-exposure male rats. These clinicat signs were indicative of the irritating nature of the vaporized test substance.

BODY WEIGHT AND WEIGHT GAIN
An exposure-dependent reduction in mean body weight was observed for all male and female test substance-exposed groups from the main study.

For male rats, the reduction in mean body weights was statistically significant (p <= 0.05) on Days 7 to 35 for all test substance exposure groups, on Day 42 for the high-exposure group only, and on Day 48 for the high- and low-exposure groups when compared to the control group. Mean body weights for male rats from the mid- and low-exposure groups on Day 42 and from the mid-exposure group on Day 48, while not statistically significantly reduced, continued to be lower than those of the control group. Body weight gain was reduced (p <= 0.05) in an exposure dependent manner between Days 0 and 7 for all male test substance exposure groups and was reduced between Days 7 and 14 and Days 0 and 48 for the male high-exposure group when compared to the control group. During the study, the male high-, mid-, and low-exposure groups gained 29, 11, and 11 % less weight then the control group, respectively.

For female rats from the main study, the reduction in mean body weights was statistically significant (p <= 0.05) for all test substance exposure groups on Days 7 and 14 of the pre-mating phase, Days 0 to 20 of the gestation phase, and on Day 0 of the lactation phase when compared to the control group. Mean body weights for female rats from all test substance exposure groups on Day 4 of the lactation phase were not statistically significantly reduced when compared to the control group, but continued to be lower than those of the control group. Body weight gain, during the gestation and post-partum phases, was comparable between the female test substance exposure groups and the control group.

The body weight gain observed for the unexposed female rats from the male high-exposure group second mating exceeded the body weight gain of all test substance exposure groups, and the control group from the main study.

FOOD CONSUMPTION
For male rats, mean feed consumption was reduced (p <= 0.05) in an exposure-related manner for all test substance exposure groups on Day 7 and for the high- and mid-exposure groups on Day 14 when compared to the control group. For the remainder of the study, mean feed consumption was comparable between the male test substance exposure groups and the control group.

For female rats, mean feed consumption was reduced (p <= 0.05) in an exposure-related manner for all test substance exposure groups on Day 7 and 14 of the pre-mating phase and for the mid¬ and high-exposure groups on Day 7 of the gestation phase when compared to the control group. Feed consumption for all test substance exposure groups remained slightly lower than was observed for the control group through Day 20 of gestation; however, this reduction was not statistically significant. Feed consumption during the lactation phase for all test substance exposure groups was statistically comparable to, though slightly higher than, the control group consumption.

HAEMATOLOGY
All hematologic parameters for male rats from all test substance exposure groups were comparable to the control groups.

CLINICAL CHEMISTRY
A slight reduction in mean serum creatinine levels for all male exposure groups was not considered biologically significant since organ toxicity is associated with increases rather than decreases in serum creatinine level. All other clinical chemistry parameters for male rats from all test substance exposure groups were comparable to the control groups.

URINALYSIS
All urinalysis parameters for male rats from all test substance exposure groups were comparable to the control groups.

ORGAN WEIGHTS
The mean absolute and relative testes and epididymides weights for all male test substance exposure groups were statistically comparable to the control group. However, when Rat 643 (a high-exposure male rat observed to have small testes at the time of necropsy) was eliminated from the analysis, the mean relative testes, for the high-exposure group, and mean relative epididymides weights, for the mid- and high-exposure groups, became significantly higher (p 0.05) than the mean control weights. This difference was probably the result of a decreased body weight for the mid- and high-exposure male rats rather then an effect on the testes or epididymides. All other organ weight measurements for male test substance exposure groups and all organ weight measurements for all female test substance exposure groups were comparable to the control group organ weights.

GROSS PATHOLOGY
No exposure-related changes were detected on necropsy examinations.

HISTOPATHOLOGY: NON-NEOPLASTIC
The incidence of exposure-related changes observed in the olfactory and respiratory epithelium of the nasal passages are listed in Table below.

Table: Incidence of Exposure-related Changes Observed in the Nasal Passages
Location Male Croups Female Croups
Abnormality within the
Nasal Passages Severity Low Mid High Low Mid High
Olfactory Epithelium
Sero-cellular Exudate Minimal 3 9 9 7 9 3
Sero-cellular Exudate Minor 3
Squamous Metaplasia Minimal 1 1
Respiratory Metaplasia Minimal 2
Respiratory Epithelium
Sero-cellular Exudate Minimal 1 1
Sero-cellular Exudate Minor 2
Squa nous Metaplasia Minimal 3 1 3
Chronic Focal Inflammation Minimal 1
Chronic Focal Inflammation Minor 1

Histopathological examination of nasal passages from the control animals did not exhibit any of these changes.
The sero-cellular exudate, was composed of a proteinaceous serum-like component and a cellular component which contained a small number of polymorphonuclear leukocytes.

No other exposure-related changes were observed during the histopathology examinations.

A single high-exposure male (Rat 643) exhibited decreased spermatozoa and spermatids in the testes and decreased spermatozoa and degeneration of spermatids in the epididymides.

Key result

Dose descriptor:

LOAEC

Effect level:

31 ppm

Based on:

other: vapor concentration

Sex:

male/female

Basis for effect level:

other: effects on feed consumption, body weights, body weight gain, and nasal passage histopathology

Key result

Critical effects observed:

not specified

Conclusions:

Exposure to 302, 103, or 31 ppm of the test substance was associated with a concentration-dependent lower feed consumption, body weight, and body weight gain; clinical abnormalities and nasal passage pathology in the test groups were consistent with exposure to an irritating vapor. Thus, a lowest-observed-adverse-effect level (LOAEL) for toxicity was determined to be 31 ppm based on effects on feed consumption, body weights, body weight gain, and nasal passage histopathology.

Executive summary:

The potential toxicity of the mesityl oxide was evaluated using a combined repeated dose and reproductive/developmental toxicity screening test. The test consisted of four phases: pre-mating (14 days), mating (1-14 days), gestation (21-22 days), and early lactation (4 days). Male and female Sprague-Dawley rats were exposed to mean chamber vapor concentrationsof 0, 31, 103, or 302 ppm of the test substance (target concentrations of 0, 30, 100, or 300 ppm) for six hrs/day, 7 days/week for a total of 36 to 49 exposures for female rats (through Day 20 of gestation) and 49 exposures for male rats. Parameters examined consisted of body weight, body weight change, feed consumption, organ weights, gross pathology, histopathology, hematology, clinical chemistry, reproductive performance, and litter data. No mortality of adult animals was observed during the study.

An exposure-dependent reduction (p =< 0.05) in feed consumption, and a corresponding reduction in mean body weight were observed during the pre-mating phase for male and female rats from all testsubstance exposure groups, and during the first week of the gestation phase for the high-and mid-exposure female rats. During exposure, effects of the test substance consisted of a transient reduction in activity for the high- and mid-exposure groups, and partially closed eyes for the high-exposure group. An increased incidence of post-exposure porphyrin nasal discharge was observed for all test substance exposure groups with the incidence-being slightly higer for the female rats when compared to the male rats. Additionally, post-exposure sialorrhea vas observed for 3 of 12 high-exposure male rats. These clinical signs were indicative of the irritating nature of the vaporized test substanse. A slight reduction in mean serum creatinine levels for all male exposure groups was not considered biologically significant since organ toxicity is associated with increases rather than decrease in serum creatinine level.

No exposure-related changes were seen at necropsy for male or female rats. Histopathology revealed exposure-dependent changes in the olfactory and respiratory epithelium of the nasal passages which were characterized by the presence of sero-cellular exudates, chronic focal inflammation, and focal metaplasia of the respiratory and olfactory epithelium of the nasal passages. These changes are a common response to an irritating vapor. No other exposure-related changes were observed during histopathology examinations.

In summary, exposure to 302, 103, or 31 ppm of the test substance was associated with a concentration-dependent lower feed consumption, body weight, and body weight gain; clinical abnormalities and nasal passage pathology in the test groups were consistent with exposure to an irritating vapor. Thus, a lowest-observed-adverse-effect level (LOAEL) for toxicity was determined to be 31 ppm based on effects on feed consumption, body weights, body weight gain, and nasal passage histopathology.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

124 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Neither deviation had a detrimental effect on the outcome of the study

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Kingston, NY)
- Age at study initiation: The male and female rats were 66 days of age at the beginning of the pre-mating phase and 80 days of age at the beginning of the mating phase
- Weight at study initiation: The male and female rats weighed 330.3 ± 10.6 or 218.5 ± 9.2 grams (mean ± SD), respectively, at the beginning of the pre-mating phase.
- Fasting period before study: no
- Housing: During the acclimation period, animals were housed by sex in groups of five in suspended, stainless-steel, wire-mesh cages. During exposure periods, the main study animals were singly housed in multicompartmented, stainless-steel, wire-mesh cages. During nonexposure period, the rats were housed in suspended, stainless-steel, wire-mesh cages in a room separate from the exposure room. Starting at study initiation and continuing through the premating period, and following the mating period, male and female animals were singly housed during nonexposure periods. During the mating period, the animals were housed one male with one female during nonexposure periods. On the 20th day of gestation, solid bottom pans containing bedding material for nesting were put in the nonexposure cages of the female rats.
- Diet: Agwaya Prolab' Animal Diet (RMH 3200 ad libitum
- Water: municipal tap water ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70-76
- Humidity (%): 36-58
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: October 7, 1991 to May 7, 1992

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 420 L stainless steel and glass inhalation chambers
- Method of holding animals in test chamber: male and female rats were singly housed and exposed simultaneously
- Air flow rate and air change rate:
Exposure Level Compressed AirFlow (Lpm) Dilution Air Flow (Lpm) Total Air Flow (Lpm) Air Changes Per
Hour
High 40 50-62 89-102 13-15
Mid 25 65-93 90-118 13-17
Low 25 62-68 89-95 12-13
Control 25 65 90 13
- Method of conditioning air: Compressed air (Kodak Park Utilities Division) was passed through silices gel (EM Science, Cherry Hill, NJ) and an oil filter (Deltech Engineering Inc., New Castle, DE).
- System of generating vapors: The test substance was metered by a Sage peristaltic pump Model No. 375A (Orion Research Inc., Cambridge, MA) [high-exposure group] from a graduated cylinder reservoir or by a Harvard syringe pump Model No. 2274 (Harvard Apparatus, South Natick, MA) [mid- and low¬exposure groups] from a 25 mL syringe into a glass distillation column (1.5" o.d. x 17" [high¬exposure group] or 1.0" o.d. x 20.5" [mid- and low-exposure groups] Ace Glass Inc., Vinland, NJ). The liquid trickled over the surface of tightly packed glass beads (4 mm diameter) within the distillation column. The beads were used to increase the surface area and enhance vaporization.
- Temperature, humidity, pressure in air chamber: Chamber temperatures for the high-, mid-, and low-exposure groups and the control group were 23.5 ± 0.6, 24.0 ± 0.6, 23.7 ± 0.7, and 23.5 ± 0.8-C (mean ± SD), respectively, and chamber relative humidity were 49.7 ± 6.9, 50.4 ± 4.4, 52.4 ± 3.7, and 43.6 ± 4.1%, respectively.
- Method of particle size determination: Nongaseous airborne material within the exposure inhalation chambers was measured using a Micro Laser Particle Counter model µLCP-301 at a sampling rate of 0.1 cmf for 10 sec. (Particle Measuring Systems, Inc., Boulder, CO). Airborne particles greater than 0.3 mm. were counted. Samples were collected from a fixed reference position within each chamber at least six days per week during the exposure. The samples collected from the test substance exposure chambers were compared to samples collected from the control chamber. The results indicated the absence of a test substance aerosol.
- Treatment of exhaust air: The effluent from all Chambers was filtered through a coarse prefilter, a HF-PA filter, and a charcoal filter.

TEST ATMOSPHERE
- Brief description of analytical method used:
Chamber vapor concentrations were monitored with an automated multipositional air sampling and analysis system. The system consisted of a MIRAN IA infrared gas analyzer (Wilks Foxboro Analytical, South Norwalk, Cl.), a Perkin-Elmer SIGMA 15 data station, and a four-port multipositional environmental sampling valve (Valco Instruments, Houston, TX). Chamber vapor samples were continuously collected, from a fixed reference position within each chamber, through the four port valves using TEFLON tubing (3/16" i.d.). On Day 10 of the study, the microprocessor of the Pekin-Elmer SIGMA 15 data station (which controls the automatic sampling, analyses, and recording of the chamber concentrations) began to malfunction, and it failed on Day 11. This did not affect the performance of the Miran, but did make it necessary to manually sample and record the concentrations for the duration of the study.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see "Details on inhalation exposure"

Duration of treatment / exposure:

pre-mating (14 days), mating (1-14 days), gestation (21-22 days), and early lactation (4 days) (36 to 49 exposures (females) and 49 exposures (males))

Frequency of treatment:

six hours per day, seven days per week

Remarks:

Doses / Concentrations:
31, 103 and 302 ppm (124, 413 and 1211 mg/m3)
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
35, 10 and 388 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
30, 100 and 300 ppm
Basis:
other: target conc.

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
A range-finding inhalation study in pregnant female rats was conducted 1) to evaluate maternal toxic effects of the test substance following repeated inhalation exposures, and 2) to establish exposure levels for this study.
- Rationale for animal assignment (if not random):
All culling and randomization of the animals was done by computer-generated lists using the Automated Animal Toxicology System (AATS). The male and female rats were assigned to the study groups on the basis of body weights so that the mean body weights were similar among the groups at the start of the study. Within an exposure group, male and female rats were randomly paired for mating. For the second week of mating, the uninseminated female rats were randomly paired with a male from the same exposure group that had inseminated a female the previous week.
- Post-exposure recovery period in satellite groups: none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
For adult animals, clinical observations were made at least once daily. Observations included, but were not limited to, examination of the hair, skin, eyes, mucous membranes, behavior patterns, respiratory patterns, feces, urine, and general activity. Post-partum neonatal observations were conducted similarly. Rats visible through chamber windows were observed for clinical signs during exposure. The dams were observed for the beginning of parturition and this date was used to calculate the gestation period. The day on which the dam had completely finished delivering was considered Day 0 post-partum.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes The male rats were weighed on Days 0, 7, and weekly thereafter. Male rats were also weighed the day before euthanasia. Body weight change was calculated for Days 0-7, 7-14, 14-21, 21-28, 28-35, 35-42, 42-48, and 0-48.
The female rats were weighed on Days 0, 7, 14 and 21 of the premating/mating phase, Days 0, 7, 14, and 20 of the gestation phase, and Days 0 and 4 post-partum. Body weight change was calculated for Days 0-7, 7-14, 14-20, and 0-20 of the gestation phase and for Days 0-4 post-partum.
The unexposed female rats used in the second mating of the male high-exposure group were weighed on Days 0 and 20 of gestation. Body weight change between Days 0-20 of gestation was calculated.

FOOD CONSUMPTION: Yes
For male rats, feed consumption was determined between Days 0-7, 7-14, 28-35, 35-41, 41-42, and 42-48. Feed consumption was not determined during the mating period. The male high-exposure group did not have feed consumption determined between Days 42-48 because of the second mating period. Male rats were fasted the day before euthanasia.
For female rats from the main study, feed consumption was determined between Days 0-7, and 7-14 of the pre-mating phase, Days 0-7, 7-14, and 14-20 of the gestation phase, and Days 0-4 post-partum. Feed consumption was not determined during the mating period.
Feed consumption was not determined for the unexposed female rats from the male high¬exposure group second mating.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy, blood was collected from the posterior vena cava of all male animals while the rats were under CO2 anesthesia.
- Animals fasted: Yes
- How many animals: all
- Parameters checked : hematocrit, hemoglobin concentration, red blood cell count, and white blood cell count. Fixed blood smears were prepared and stored.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy, blood was collected from the posterior vena cava of all male animals while the rats were under CO2 anesthesia.
- Animals fasted: Yes
- How many animals:
- Parameters checked: aspartate aminotransferase (AST1.), alanine aminotransferase (ALT), glucose, urea nitrogen (UN), gamma glutamyl transpeptidase (GGTP), total bilirubin, creatinine, total protein, albumin, albumin/globulin ratio (A/G ratio), potassium, chloride, calcium, sodium, and phosphorus.

URINALYSIS: Yes
- Time schedule for collection of urine: On Day 41, male rats were placed in metabolism cages for approximately 16 hours for urine collection on Day 42
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: The osmolality and volume of the urine was measured and the urine was evaluated for color and appearance. The urine was tested using Multistix® for nitrite, urobilinogen, protein, pH, blood, ketones, bilirubin, and glucose. Microscopic examination was conducted on urinary sediment for the presence of red blood cells, white blood cells, epithelial cells, triple phosphate crystals, amorphous sediment, sperm, and bacteria.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
Wet weights of the liver, kidneys, thymus, lungs, testes, and epididymides were collected at necropsy. Paired organs were weighed together. Org/body weight ratios were calculated.

HISTOPATHOLOGY: Yes
The following tissues were collected from adult animals and were fixed in 10% buffered formalin: liver (2 sections), kidneys (1 section/kidney), adrenal glands, brain (3 sections including medulla oblongata, pons, cerebellar cortex, and cerebral cortex), heart, spleen, thymus, trachea, lungs, nasal passages, ovaries, and gross lesions. The testes and epididymides were preserved in Bouin's fixative.
Histological evaluation was conducted on the tissues listed above from control and high-exposure adult male and female animals. If abnormalities or equivocal results were seen in any of these tissues, the same tissues from lower exposure groups were examined. Histopathology was performed on significant gross lesions for all adult animals. Ovaries from any female which failed to complete its pregnancy were examined.

Statistics:

Mean values were calculated for test substance concentration, chamber temperature, chamber relative humidity, body weight, body weight change, feed consumption, organ weights, hematology, and clinical chemistries. Mean body weight, feed consumption, organ weight, hematology, and clinical chemistry data were evaluated using the following computer-generated statistical, tests: Bartlett's test (p <= 0.01), one-way analysis of variance (ANOVA) (p <= 0.05), and Duncan's multiple range test (p <= 0.05) to indicate statistical significance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During-exposure, male and female rats from the high-exposure group exhibited reduced general
activity on Days Oto 13 (Day 0 - minor, Days 1 to 13 - minimal) and partially closed eyes on
Days Oto 3 (Day 0 - minor, Days 1 to 3 - minimal). No clinical abnormalities were observe,d
during exposure for the high-exposure group from Day 14 ·to study termination. During
exposure clinical abnormalities for male and female rats from the-~ mid-exposure group were
restricted to a minimal reduction of general activity on Day 0 only. Reduced activity was not
observed for the low-exposure group.
A total of nine incidences of post-exposure sialorrhea were observed for three high-exposure
male rats. Sialorrhea was not observed for any other test substance exposed or control rats.
The incidence of post-exposure porphyrin nasal discharge was higher for the test substance
exposed male and female rats when compared to the control rats (

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

An exposure-dependent reduction in mean body weight was observed for all male and female
test substance-exposed groups from the main study.
For male rats, the reduction in mean body weights was statistically significant (p < 0.05) on
Days 7 to 35 for all test substance exposure groups, on Day 42 for the high-exposure group
only, and on Day 48 for the high- and low-exposure groups when compared to the control
group. Mean body weights for male rats from the mid- and low-exposure groups on Day 42 and
from the mid-exposure group on Day 48, while not statistically significantly reduced, continued
to be lower than those of the control group. Body weight gain was reduced (p !S 0.05) in an
exposure dependent manner between Days 0 and 7 for all male test substance exposure groups
and was reduced between Days 7 and 14 and Days 0 and 48 for the male high-exposure group
when compared to the control group. During the study, the male high-, mid-, and low-exposure
groups gained 29, 11, and 11 % less weight then the control group, respectively.
For female rats from the main study, the reduction in mean body weights was statistically
significant (p s 0.05) for all test substance exposure groups on Days 7 and 14 of the pre-mating
phase, Days 0 to 20 of the gestation phase, and on Day 0 of the lactation phase when compare.d
to the control group. Mean body weights for female rats from all test substance exposure groups
on Day 4 of the lactation phase were not statistically significantly reduced when compared to the
control group, but continued to be lower than those of the control group. Body weight gain,
during the gestation and post-partum phases, was comparable between the female test substance
exposure groups and the control group.
The body weight gain observed for the unexposed female rats from the male high-exposure
group second mating exceeded the body weight gain of all test substance exposure groups, and
the control group from the main study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For male rats, mean feed consumption was reduced (p s 0.05) in an exposure-related manner
for all test substance exposure groups on Day 7 and for the high- and mid-exposure groups on
Day 14 when compared to the control group. For the remainder of the study, mean feed
consumption was comparable between the male test substance exposure groups and the control
group.
For female rats, mean feed consumption was reduced (p s 0.05) in an exposure-related manner
for all test substance exposure groups on Day 7 and 14 of the pre-mating phase and for the midand
high-exposure groups on Day 7 of the gestation phase when compared to the control group.
Feed consumption for all test substance exposure groups remained slightly lower than was
observed for the control group through Day 20 of gestation; however, .this reduction was not
statistically significant. Feed consumption during the lactation phase for all test substance
exposure groups was statistically comparable to, though slightly higher than, the control group
consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

All hematologic parameters for male rats from all test substance exposure groups were
comparable to the control groups.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Mean creatinine levels were reduced (p ~ 0.05) in an exposure-related manner for all male test
substance exposure groups when compared to the control group.
The serum of two to seven male rats per group, including the control group, was slightly to
markedly hemolyzed. This hemolysis was believed to be an artifact of bleeding.
All other clinical chemistry parameters for male rats from all test substance exposure groups
were comparable to the control groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

All urinalysis parameters for male rats from all test substance exposure groups were comparable
to the control groups.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean absolute and relative testes and epididymides weights for all male test substance
exposure groups were statistically comparable to the control group. However, when Rat 643
(a high-exposure male rat observed to have small testes at the time of ne.cropsy) was eliminated
from the analysis, the mean relative testes, for the high-exposure group, and mean relative
epididymides weights, for the mid- and high-exposure groups, became significantly higher
(p < 0.05) than the mean control weights. This difference was probably the result of a
de.creased body weight for the mid- and high-exposure male rats rather then an effect on the
testes or epididymides. All other organ weight me.asurements for male test substance exposure
groups and all organ weight me.asurements for all female test substance exposure groups were
comparable to the control group organ weights.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY
No mortality occurred during the study.

CLINICAL SIGNS
During exposure, effects of the test substance consisted of a transient reduction in activity for the high- and mid-exposure groups, and partially closed eyes for the high-exposure group. An increased incidence of post-exposure porphyrin nasal discharge was observed for all test substance exposure groups with the incidence being slightly higher for the female rats when compared to the male rats. Additionally, post-exposure sialorrhea was observed for 3 of 12 high-exposure male rats. These clinicat signs were indicative of the irritating nature of the vaporized test substance.

BODY WEIGHT AND WEIGHT GAIN
An exposure-dependent reduction in mean body weight was observed for all male and female test substance-exposed groups from the main study.

For male rats, the reduction in mean body weights was statistically significant (p <= 0.05) on Days 7 to 35 for all test substance exposure groups, on Day 42 for the high-exposure group only, and on Day 48 for the high- and low-exposure groups when compared to the control group. Mean body weights for male rats from the mid- and low-exposure groups on Day 42 and from the mid-exposure group on Day 48, while not statistically significantly reduced, continued to be lower than those of the control group. Body weight gain was reduced (p <= 0.05) in an exposure dependent manner between Days 0 and 7 for all male test substance exposure groups and was reduced between Days 7 and 14 and Days 0 and 48 for the male high-exposure group when compared to the control group. During the study, the male high-, mid-, and low-exposure groups gained 29, 11, and 11 % less weight then the control group, respectively.

For female rats from the main study, the reduction in mean body weights was statistically significant (p <= 0.05) for all test substance exposure groups on Days 7 and 14 of the pre-mating phase, Days 0 to 20 of the gestation phase, and on Day 0 of the lactation phase when compared to the control group. Mean body weights for female rats from all test substance exposure groups on Day 4 of the lactation phase were not statistically significantly reduced when compared to the control group, but continued to be lower than those of the control group. Body weight gain, during the gestation and post-partum phases, was comparable between the female test substance exposure groups and the control group.

The body weight gain observed for the unexposed female rats from the male high-exposure group second mating exceeded the body weight gain of all test substance exposure groups, and the control group from the main study.

FOOD CONSUMPTION
For male rats, mean feed consumption was reduced (p <= 0.05) in an exposure-related manner for all test substance exposure groups on Day 7 and for the high- and mid-exposure groups on Day 14 when compared to the control group. For the remainder of the study, mean feed consumption was comparable between the male test substance exposure groups and the control group.

For female rats, mean feed consumption was reduced (p <= 0.05) in an exposure-related manner for all test substance exposure groups on Day 7 and 14 of the pre-mating phase and for the mid¬ and high-exposure groups on Day 7 of the gestation phase when compared to the control group. Feed consumption for all test substance exposure groups remained slightly lower than was observed for the control group through Day 20 of gestation; however, this reduction was not statistically significant. Feed consumption during the lactation phase for all test substance exposure groups was statistically comparable to, though slightly higher than, the control group consumption.

HAEMATOLOGY
All hematologic parameters for male rats from all test substance exposure groups were comparable to the control groups.

CLINICAL CHEMISTRY
A slight reduction in mean serum creatinine levels for all male exposure groups was not considered biologically significant since organ toxicity is associated with increases rather than decreases in serum creatinine level. All other clinical chemistry parameters for male rats from all test substance exposure groups were comparable to the control groups.

URINALYSIS
All urinalysis parameters for male rats from all test substance exposure groups were comparable to the control groups.

ORGAN WEIGHTS
The mean absolute and relative testes and epididymides weights for all male test substance exposure groups were statistically comparable to the control group. However, when Rat 643 (a high-exposure male rat observed to have small testes at the time of necropsy) was eliminated from the analysis, the mean relative testes, for the high-exposure group, and mean relative epididymides weights, for the mid- and high-exposure groups, became significantly higher (p 0.05) than the mean control weights. This difference was probably the result of a decreased body weight for the mid- and high-exposure male rats rather then an effect on the testes or epididymides. All other organ weight measurements for male test substance exposure groups and all organ weight measurements for all female test substance exposure groups were comparable to the control group organ weights.

GROSS PATHOLOGY
No exposure-related changes were detected on necropsy examinations.

HISTOPATHOLOGY: NON-NEOPLASTIC
The incidence of exposure-related changes observed in the olfactory and respiratory epithelium of the nasal passages are listed in Table below.

Table: Incidence of Exposure-related Changes Observed in the Nasal Passages
Location Male Croups Female Croups
Abnormality within the
Nasal Passages Severity Low Mid High Low Mid High
Olfactory Epithelium
Sero-cellular Exudate Minimal 3 9 9 7 9 3
Sero-cellular Exudate Minor 3
Squamous Metaplasia Minimal 1 1
Respiratory Metaplasia Minimal 2
Respiratory Epithelium
Sero-cellular Exudate Minimal 1 1
Sero-cellular Exudate Minor 2
Squa nous Metaplasia Minimal 3 1 3
Chronic Focal Inflammation Minimal 1
Chronic Focal Inflammation Minor 1

Histopathological examination of nasal passages from the control animals did not exhibit any of these changes.
The sero-cellular exudate, was composed of a proteinaceous serum-like component and a cellular component which contained a small number of polymorphonuclear leukocytes.

No other exposure-related changes were observed during the histopathology examinations.

A single high-exposure male (Rat 643) exhibited decreased spermatozoa and spermatids in the testes and decreased spermatozoa and degeneration of spermatids in the epididymides.

Key result

Dose descriptor:

LOAEC

Effect level:

31 ppm

Based on:

other: vapor concentration

Sex:

male/female

Basis for effect level:

other: effects on feed consumption, body weights, body weight gain, and nasal passage histopathology

Key result

Critical effects observed:

not specified

Conclusions:

Exposure to 302, 103, or 31 ppm of the test substance was associated with a concentration-dependent lower feed consumption, body weight, and body weight gain; clinical abnormalities and nasal passage pathology in the test groups were consistent with exposure to an irritating vapor. Thus, a lowest-observed-adverse-effect level (LOAEL) for toxicity was determined to be 31 ppm based on effects on feed consumption, body weights, body weight gain, and nasal passage histopathology.

Executive summary:

The potential toxicity of the mesityl oxide was evaluated using a combined repeated dose and reproductive/developmental toxicity screening test. The test consisted of four phases: pre-mating (14 days), mating (1-14 days), gestation (21-22 days), and early lactation (4 days). Male and female Sprague-Dawley rats were exposed to mean chamber vapor concentrationsof 0, 31, 103, or 302 ppm of the test substance (target concentrations of 0, 30, 100, or 300 ppm) for six hrs/day, 7 days/week for a total of 36 to 49 exposures for female rats (through Day 20 of gestation) and 49 exposures for male rats. Parameters examined consisted of body weight, body weight change, feed consumption, organ weights, gross pathology, histopathology, hematology, clinical chemistry, reproductive performance, and litter data. No mortality of adult animals was observed during the study.

An exposure-dependent reduction (p =< 0.05) in feed consumption, and a corresponding reduction in mean body weight were observed during the pre-mating phase for male and female rats from all testsubstance exposure groups, and during the first week of the gestation phase for the high-and mid-exposure female rats. During exposure, effects of the test substance consisted of a transient reduction in activity for the high- and mid-exposure groups, and partially closed eyes for the high-exposure group. An increased incidence of post-exposure porphyrin nasal discharge was observed for all test substance exposure groups with the incidence-being slightly higer for the female rats when compared to the male rats. Additionally, post-exposure sialorrhea vas observed for 3 of 12 high-exposure male rats. These clinical signs were indicative of the irritating nature of the vaporized test substanse. A slight reduction in mean serum creatinine levels for all male exposure groups was not considered biologically significant since organ toxicity is associated with increases rather than decrease in serum creatinine level.

No exposure-related changes were seen at necropsy for male or female rats. Histopathology revealed exposure-dependent changes in the olfactory and respiratory epithelium of the nasal passages which were characterized by the presence of sero-cellular exudates, chronic focal inflammation, and focal metaplasia of the respiratory and olfactory epithelium of the nasal passages. These changes are a common response to an irritating vapor. No other exposure-related changes were observed during histopathology examinations.

In summary, exposure to 302, 103, or 31 ppm of the test substance was associated with a concentration-dependent lower feed consumption, body weight, and body weight gain; clinical abnormalities and nasal passage pathology in the test groups were consistent with exposure to an irritating vapor. Thus, a lowest-observed-adverse-effect level (LOAEL) for toxicity was determined to be 31 ppm based on effects on feed consumption, body weights, body weight gain, and nasal passage histopathology.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

124 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Key study

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

The potential toxicity of the mesityl oxide was evaluated using a combined repeated dose and reproductive/developmental toxicity screening test (Bernard and Faber, 1992). The test consisted of four phases: pre-mating (14 days), mating (1-14 days), gestation (21-22 days), and early lactation (4 days). Male and female Sprague-Dawley rats were exposed to mean chamber vapor concentrations of 0, 31,103, or 302 ppm of the test substance (target concentrations of 0, 30, 100, or 300 ppm) for six hrs/day, 7 days/week for a total of 36 to 49 exposures for female rats (through Day 20 of gestation) and 49 exposures for male rats. Parameters examined consisted of body weight, body weight change, feed consumption, organ weights, gross pathology, histopathology, hematology, clinical chemistry, reproductive performance, and litter data. No mortality of adult animals was observed during the study.

An exposure-dependent reduction (p =< 0.05) in feed consumption, and a corresponding reduction in mean body weight were observed during the pre-mating phase for male and female rats from all test substance exposure groups, and during the first week of the gestation phase for the high-and mid-exposure female rats. During exposure, effects of the test substance consisted of a transient reduction in activity for the high- and mid-exposure groups, and partially closed eyes for the high-exposure group. An increased incidence of post-exposure porphyrin nasal discharge was observed for all test substance exposure groups with the incidence-being slightly higher for the female rats when compared to the male rats. Additionally, post-exposure sialorrhea vas observed for 3 of 12 high-exposure male rats. These clinical signs were indicative of the irritating nature of the vaporized test substance. A slight reduction in mean serum creatinine levels for all male exposure groups was not considered biologically significant since organ toxicity is associated with increases rather than decrease in serum creatinine level.

No exposure-related changes were seen at necropsy for male or female rats. Histopathology revealed exposure-dependent changes in the olfactory and respiratory epithelium of the nasal passages which were characterized by the presence of sero-cellular exudates, chronic focal inflammation, and focal metaplasia of the respiratory and olfactory epithelium of the nasal passages. These changes are a common response to an irritating vapor. No other exposure-related changes were observed during histopathology examinations.

In summary, exposure to 302, 103, or 31 ppm of the test substance was associated with a concentration-dependent lower feed consumption, body weight, and body weight gain; clinical abnormalities and nasal passage pathology in the test groups were consistent with exposure to an irritating vapor. Thus, a lowest-observed-adverse-effect concentration (LOAEC) for toxicity was determined to be 31 ppm (124 mg/m³) based on effects on feed consumption, body weights, body weight gain, and nasal passage histopathology.

 

In a repeat dose inhalation study, groups of 10 male Wistar rats and 10 guinea pigs of both sexes were exposed to mesityl oxide at concentrations of 0, 50, 100, 250 or 500 ppm (equivalent to 0, 0.2, 0.4, and 1.0 mg/L) for 8 hr/day, 5 days/week for 6 weeks (Smyth et al., 1942). The 500 ppm group was terminated after 10 days due to high mortality (65 percent). Observations were recorded on the growth, blood count, urine, and microscopic pathology. Effects were seen at all concentrations except 50 ppm. Chronic conjunctivitis and nasal irritation were observed at 250 and 500 ppm. Poor growth was observed in animals exposed to 250 ppm. No blood cell changes were noted at any concentration. Animals exposed to 250 and 500 ppm had mild albuminuria. In addition, exposure to mesityl oxide resulted in vascular congestion, primarily in the kidneys. The liver and lungs were affected to a lesser degree. Liver pathology was observed in 2 of 16, 2 of 4, and 3 of 5 animals exposed to 100, 250 and 500 ppm, respectively. Kidney pathology was observed in 5 of 16 animals exposed to 100 ppm and was observed in all of the animals exposed to 250 and 500 ppm. Mortality only occurred at 500 ppm, with a death rate of 65 percent. Death was attributed to mesityl oxide's narcotic action.

 

Hart et al. (1939) exposed 10 mice daily to concentrations of 4-methylpent-3-en-2-one of 13,000 ppm (53,170 mg/m3), for 15 minutes. All animals survived 5 exposures, but after 11 exposures, 3 had died. When increasing exposure duration to 30 minutes/day, all 10 mice died within 6 days. Post-mortem examinations of the exposure-related deaths showed hepatic necrosis, pulmonary haemorrhage and oedema, renal tubular degeneration, and gastrointestinal tract distension. When 6 rabbits were exposed to this concentration, 30 minutes/day for 15 days, there was slight eye and nose irritation. Extension of exposure periods to 60 minutes/day resulted in spastic paralysis, within 10 days, and death, within 7-11 days after the onset of paralysis. Post-mortem examinations showed organ lesions similar to those found in mice.

Justification for classification or non-classification

No classification is warranted for repeated dose toxicity according to Regulation no. 1272-2008 and Directive 67/548/EEC.