2-(2-butoxyethoxy)ethyl 6-propylpiperonyl ether

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 82-4 (90-Day Inhalation Toxicity)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-butoxyethoxy)ethyl 6-propylpiperonyl ether

EC Number:

200-076-7

EC Name:

2-(2-butoxyethoxy)ethyl 6-propylpiperonyl ether

Cas Number:

51-03-6

Molecular formula:

C19H30O5

IUPAC Name:

2-(2-butoxyethoxy)ethyl 6-propylpiperonyl ether

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York, USA.
- Age at study initiation:Approx. 9 weeks .
- Weight at study initiation: 311-357 g (males) and 202-262 g (females).
- Number of animals per group: 15 per sex.
- Control animals: 15 per sex.
- Housing: During the non-exposure periods the animals were doubly housed in suspended stainless stell wire mesh cages during the first week of the equilibration period and individually housed during the remainder of the equilibration period and all other non-exposure pperiods. During the exposure periods the animals were individually housed in wire mesh, stainless stell cages within a 1000 liter glass and stainless steel exposure chamber.
- Diet: During the non-exposure periods ad libitum. During the exposure periods none. (See the diagram "EXPOSURE CHAMBER" attached in background material)
- Water: During the non-exposure periods ad libitum (by automated watering system). During the exposure periods none.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): During the non-exposure periods 16-24 °C. During the exposure periods 20-24 °C.
- Humidity (%): During the non-exposure periods 10-86. During the exposure periods 26-74%.
- Air changes (per hr): 12,4 (approximatly every 4.8 minutes)

IN-LIFE DATES: From 09 September 1991 (receipt of Animals) To : 19 December 1991

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: MMAD (mass median aerodynamic diameter) (+ GSD (geometric standard deviation) [µm]
Mean 2.7 +/- 1.7 µm. (See the table "DAILY MEAN EXPOSURE LEVELS" reported in "Any other information on materials and methods incl. tables)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure procedure: For groups II and III the appropriate amounts of PBO were placed in a glass syringe covered in foil and mounted on a syringe infusion pump. For the groups IV and V the appropriate amounts of PBO were placed into an Erlenmeyer flask covered in foil and connected to an FMI fluid metering pump.
For groups from II to V the test substance was fed via 1/8’’ Teflon from the pump tubing, directly into the liquid inlet of an air atomizing nozzle. House-line air was delivered through a regular and backpressure gauge via ¼” plastic tubing to a metering valve, flow meter and backpressure gauge into the air inlet of the atomizer to generate the aerosol.
The test atmosphere was directed into the inlet turret of the exposure chamber which housed the animals. he animals remained in the chamber for 30 minutes following the exposure to allow the chamber to clear, using clean air at the same airflow rate used during exposure.
Group I is for control
- Temperature, humidity, pressure in air chamber: Temperature (°C): During the non-exposure periods 16-24 °C. During the exposure periods 20-24 °C. Humidity (%): During the non-exposure periods 10-86. During the exposure periods 26-74%.
- Air change rate: Air changes (per hr) = 12,4 (approximatly every 4.8 minutes)
- Method of particle size determination. For group I: Particle size distribution measurements were performed once during each exposure for chamber and room air using a TSI Aerodynamic Particle Sizer. Sample were withdrawn at rate of 5 liters per minute for 20 seconds. The particle size were calculated by mean of a computer system. For groups to II to V : Particle size distribution measurements were performed once during each exposure for chamber and room air using a TSI Aerodynamic Particle Sizer. Sample were withdrawn at rate of 5 liters per minute for 20 seconds. The particle size were calculated by mean of a computer system. Samples for particle size distribution assessment were drawn once during each exposure using a Delron DCI-6 cascade impactor. The mass median aerodynamic diameter (MMAD), geometric standard deviation and percent of particles ≤1.0 and ≤ 10 microns were calculated based on the amount of material collected on the impactor stages (stainless steel slides and final filter) using a graphical analysis of an assumed lognormal distribution. (see the table "DAILY MEAN EXPOSURE LEVELS" reported in "Any other information on materials and methods incl. tables")

TEST ATMOSPHERE
- Samples taken from breathing zone: yes.
- Brief description of analytical method used: Sample for gravimetric determination of the PBO exposure levels were withdrawn from the breathing zone in the exposure chamber through glass fiber filter paper mounted open-face in a filter holder. Samples were withdrawn approximately every 90 minutes from the sampling portals. The filter papers were weighed before and after sample collection and the gravimetric concentration in mg/mc was calculated by dividing the weight difference in milligrams by the volume of air sampled.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Nominal concentration of the test substance: The nominal concentration (mg/mc) was determined by measuring the flow of air (lpm) through the chamber and the volumetric flow (µl/min) of test substance into the chamber (flowrate times total exposure time) to give a nominal concentration.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week (whole-body exposure) per 13 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 (control), 15, 74, 155 and 512 mg/m3
Basis:
analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0 (control), 38, 68, 230, 827 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

15/male/0; 15/male/15; 15/male/74; 15/male/155 and 15/male/512 [mg/m³];
15/female/0; 15/female/15; 15/female/74; 15/female/155 and 15/female/512 [mg/m³]

Control animals:

yes

Details on study design:

- Dose selection rationale: The exposure concentrations were selected on the basis of results of an acute inhalation toxicity study in rats in which no mortality or signs of systemic toxicity were observed at PBO concentration as high as 5.9 mg/l.
- Route: Inhalation, administered into the breathing zone of the animals as a respirable aerosol during whole-body exposures.
- Justification of route of administration: whole-body inhalation exposure was chosen as the route of administration since it is a potential route of human exposure to this test substance.
- Number of exposures: all animals received at least 65 exposures. In order to sacrifice the animals the day after the exposure and because of staggered sacrifice intervals, over three days, some animals received more exposures.

Examinations

Observations and examinations performed and frequency:

CLINICAL SIGS: Observations in mortality and clinical signs were observed daily commencing on exposure day 35. Body weights and food consumption were also examined. In addition haematology, clinical chemistry, gross pathology and histopathology were examined.
Mortality: Yes
BODY WEIGHT: Individual body weights were determined three times pre-test, weekly during treatment and terminally (after fasting).
OPHTHALMOSCOPIC EXAMINATION: Ophthalmoscopic examinations were performed pretest and prior to the terminal sacrifice.
HAEMATOLOGY: Clinical laboratory studies were conducted on 15 animals at study termination.
CLINICAL CHEMISTRY: Hematology studies were conducted on 15 animals at study termination.
URINALYSIS: Analysis of urine was not performed.
FOOD CONSUMPTION: Individual food consumption was measured weekly beginning one week prior to treatment. None during the test periods
FOOD EFFICIENCY: no data
WATER CONSUMPTION: none during the test periods

Sacrifice and pathology:

GROSS PATHOLOGY and HISTOPATHOLOGY: Examinations were performed on all animals which died during the study as well as all survivors at study termination. All animals/sex/dose group received a complete post-mortem examination under the direct supervision of a pathologist after exsanguination under carbon dioxide anaesthesia.
Histopathological examination of controls and high dose level groups.
Lungs, liver and larynx were examined in all groups.

Other examinations:

ORGAN WEIGHTS: liver, kidneys, adrenals, testes, epididymides, ovaries, , brain, heart, lungs.

Statistics:

Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure if needed. For the comparison of variance between groups Bartlett’s test was used. If the variance of the test groups were equal, parametric procedures like one way ANOVA was used using the F distribution to assess significance. Differences from the control were determined by using Dunnett’s test. If a nonparametric test was needed, the Kruskal-Wallis Test was used.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No animals died as a result of exposure to PBO.

Mortality:

mortality observed, treatment-related

Description (incidence):

No animals died as a result of exposure to PBO.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No food during the test period

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No food during the test period

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

no effects observed

Description (incidence and severity):

No water during the test period

Ophthalmological findings:

no effects observed

Description (incidence and severity):

See "Details on results"

Haematological findings:

no effects observed

Description (incidence and severity):

See "Details on results"

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results"

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results"

Gross pathological findings:

no effects observed

Description (incidence and severity):

See "Details on results"

Details on results:

CLINICAL SIGNS AND MORTALITY: Dose related increases in clinical signs included secretory signs in both males and females such as nasal discharge and dried material in the facial area ad extremities and anogenital staining in the two highest dose groups. No animals died as a result of exposure to PBO.

BODY WEIGHT AND WEIGHT GAIN: The absolute body weights and body weight gains were not affected

FOOD CONSUMPTION: The mean food consumption values were not affected

FOOD EFFICIENCY

WATER CONSUMPTION

OPHTHALMOSCOPIC EXAMINATION: There were no signs of any Piperonyl Butoxide related ocular effects.

HAEMATOLOGY: There was no indication of Piperonyl Butoxide related hematologic changes

CLINICAL CHEMISTRY: Significant differences in clinical chemistry parameters were seen in group V: Serum levels of SGOT, SGPT and glucose were decreased while BUN, total protein and albumin were increased. The differences were not always statistically significant in both sexes. (See the table "CLINICAL CHEMISTRY-SELECTED PARAMETERS" reported in "Any other information on results incl. tables")

URINALYSIS: not done

NEUROBEHAVIOUR

ORGAN WEIGHTS: Significant increases in organ weights or ratios were observed for liver and kidneys of group V males and females. Relative liver weights were also increased for group IV males and females. (See the table "SELECTED ORGAN WEIGHTS AND PATHOLOGIES" reported in "Any other information on results incl. tables")

GROSS AND HISTOPATHOLOGY: Changes noted in the larynx were indicative of local irritation rather than systemic and were minimal to slight in severity and were seen in control animals as well.
In the liver, vesiculation/vacuolation of hepatocellular cytoplasm (minimal to slight) was seen in animals of all groups but was slightly more pronounced in the highest dose group males.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

155 mg/m³ air

Sex:

male/female

Basis for effect level:

other: under the conditions of this study

2

Key result

Dose descriptor:

LOAEL

Effect level:

>= 512 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Based upon the hepatotoxicity (decreased serum enzymes activity, increased liver weight and increased kidney weight), together with a range of qualitatively different minimal to slight laryngeal lesions.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The tables reported below have been extracted by the full study report Project number 91-8333

Clinical Chemistry-Selected Parameters
Parameter	n = 15/sex	0 control	15 (mg/m^3)	74 (mg/m^3)	155 (mg/m^3)	512 (mg/m^3)
AST (SGOT) (IU/l)	M	62	60	57	53	51*
	F	53	53	48	60	47
ALT (SGPT) (IU/l)	M	29	31	28	27	25*
	F	29	28	27	33	21*
Total protein (g/dL)	M	6.5	6.6	6.6	6.7	7.1**
	F	7.2	7.0	7.2	7.4	7.6
Globulin (g/dL)	M	2.5	2.5	2.6	2.5	2.7
	F	2.5	2.4	2.5	2.6	2.6
Albumin (mg/dL)	M	4.0	4.1	4.0	4.2	4.4**
	F	4.8	4.6	4.7	4.7	5.0
Glucose (mg/dL)	M	165	166	160	146	149
	F	162	158	159	151	133*
BUN (mg/dL)	M	13.0	12.4	12.9	13.2	14.9**
	F	13.3	13.8	14.8	13.2	14.6

AST- Aspartate Amino Transferase (≙SGOT = Serum Glutamic Oxaloacetic Transaminase)

ALT - Alanine Amino Transferase (≙SGPT = Serum Glutamic Pyruvic Transaminase)

^*significantly different from the control group: p < 0.05

^**significantly different from the control group: p < 0.01

Selected Organ Weights and Pathologies
mg/m^3	Sex (n = 15)	Body weight (g)	Liver weight (g)	Relative liver weight (%)	Kidney weight (g)	Relative kidney weight (%)
0	m	563	14.83	2.63	5.00	0.73
	f	330	9.12	2.76	2.37	0.72
15	m	555	14.84	2.66	4.09	0.74
	f	334	9.08	2.73	2.49	0.76
74	m	541	14.76	2.73	4.03	0.75
	f	331	9.23	2.79	2.38	0.72
155	m	558	15.78	2.83*	4.11	0.74
	f	319	9.58	3.01*	2.41	0.76
512	m	538	18.20**	3.39**	4.40	0.82*
	f	319	10.90**	3.43**	2.52	0.80*

^*significantly different from the control group: p < 0.05

^** significantly different from the control group: p < 0.01

TOXIC RESPONSE

At 512 mg/m³ (males/females) differences were noted from control in clinical Chemistry parameters. Serum levels of SGOT, SGPT and glucose were decreased, while BUN, total protein and albumin were increased. These differences were not always statistically significant in both sexes. However, a similar trend was evident in both sexes. Therefore, a relationship to test substance exposure could not be excluded. Statistically significant increases in absolute or relative organ weights were seen in the liver and kidneys. The relative kidney weights were increased by about 11%. Absolute and relative liver weights were both increased by about 25%. In the liver, vesiculation/vacuolation of hepatocellular cytoplasm was slightlymore pronounced than in the other dose groups, especially for the males.

Applicant's summary and conclusion

Conclusions:

MATERIALS AND METHODS
Chronic inhalation toxicity study according to US EPA Pesticide Assessment Guideline, Subdivision F, 82-4.
Groups of 15 Sprague Dawley rats of each sex were whole body exposed to analytical concentrations of 0, 15, 74, 155, and 512 mg Piperonyl Butoxide /m³ for 13 weeks, 6 hours/day, generally 5 days/week and a minimum of 65 exposures in total. Determinations of size distribution showed an overall mass median aerodynamic diameter of 1.7 µm.

RESULTS AND DISCUSSION
No animals died. Clinical signs were observed from 74 mg/m³ onwards and included secretory signs.
There were no ocular effects.
At the highest dose (512 mg/m³) significant differences in clinical chemistry parameters were seen, however, the differences were not consistent between sexes.
Significant increases in liver weights and relative organ were observed for liver and kidneys.
At a dose level of 155 mg/m³ minimal increase was noted for the relative liver weights.
Morphological abnormalities in the larynx, observed by light microscopy were considered to be localized responses indicative of a treatment-related effect.

CONCLUSION
Based upon hepatotoxicity at 512 mg/m³, a NOAEL of 155 mg/m³ is considered for systemic toxicity under the conditions of this study.

The rat is highly susceptible to develop squamous laryngeal metaplasia due to its anatomy, airflow and epithelial pattern . There were no severe grade effects in the larynx of high dose level rats. In fact, all the findings of squamous metaplasia were essentially graded minimal to slight in all treated groups. The occurrence of additional qualitative changes at the high dose indicated 0.512mg/l to be the LOAEL. The weight of evidence, including current pathological diagnostic criteria do not classify minimal or slight effects as adverse.


LO(A)EL: 512 mg/m³. Based upon the hepatotoxicity (decreased serum enzymes activity, increased liver weight and increased kidney weight), together with a range of qualitatively different minimal to slight laryngeal lesions.

NO(A)EL: 155 mg/m³ is considered to be the NOAEL for systemic and local toxicity under the conditions of this study.

Reliability: 1
Deficiencies: No