Reaction mass of diisobutyl adipate and diisobutyl glutarate and diisobutyl succinate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

December 28, 1999. to December 06, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
The members of the category are all alcohol esters of dicarboxylic acids. All category members are manufactured by reacting an alcohol (methanol, butanol or isobutanol) with single dicarboxylic acids, succinic, glutaric or adipic acids or mixtures of these acids. The ester bonds are effectively metabolised by the body releasing the component alcohols and acids. The difference between members involves 3 parameters: 1) the alcohol used to esterify the acids, 2) the length of the acid molecule (4C, 5C or 6C) and 3) the presence of individual esters or mixtures thereof.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
The toxicity profile of the members (ecotoxicity and human health toxicity and the environmental fate) is consistent. All have low acute toxicity potential, are not sensitising, are mildly irritating to eyes and upper respiratory tract (where vapour pressure allows exposure), are not genotoxic or clastogenic (in vivo) and have minimal systemic toxicity. Data are available predominantly for the methyl esters (individual and mixture), dibutyl adipate and diisobutyl esters (mixture). Within the category, read across is used to cover the higher tier human health toxicity studies predominantly.

See attached document with the justification for the category/read-across approach.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl glutarate

EC Number:

214-277-2

EC Name:

Dimethyl glutarate

Cas Number:

1119-40-0

Molecular formula:

C7H12O4

IUPAC Name:

dimethyl glutarate

Constituent 2

Reference substance name:

Dimethyl succinate

EC Number:

203-419-9

EC Name:

Dimethyl succinate

Cas Number:

106-65-0

Molecular formula:

C6H10O4

IUPAC Name:

dimethyl succinate

Constituent 3

Reference substance name:

Dimethyl adipate

EC Number:

211-020-6

EC Name:

Dimethyl adipate

Cas Number:

627-93-0

Molecular formula:

C8H14O4

IUPAC Name:

dimethyl adipate

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were from Charles River, Raleigh, North Carolina. The rats were approximately 3 weeks old on the day of arrival.

Rats were quarantined for approximately 8 days, and in pretest for approximately 3 weeks prior to test initiation. Rats were weighed and observed for clinical signs of disease 3 times during the quarantine period and at least weekly during the pretest period.

Rats were housed in a total of 4 Bioclean® rooms (laminar flow air circulation pattern) in close proximity to the inhalation chambers. Groups exposed to 400 mg/m3 DMG, DMS, or DMA were housed in separate rooms. The control rats and groups exposed to 10 or 50 mg/m3 DMG were
housed in the remaining room. Except during exposures, rats were housed in stainless steel, wire-mesh cages suspended above cage boards. Male and female rats were housed on separate cage racks. The position of cages on
racks was rotated every 2 weeks. During quarantine and the majority of the pretest, rats were housed 3 per cage. Upon grouping and during the test period, rats were housed individually.

Acceptable ranges for animal room relative humidity and temperature were 50 ± 20% and 22 ± 3°C, respectively. Rooms were artificially illuminated (fluorescent light) on a 12 hour light/12 hour dark cycle.


Animal Selection and Identification: During the pretest period, rats were assigned to control and treatment groups of 36 rats per sex. Rats were divided into groups with the aid of a computerized, stratified, randomization program, so that there were no significant differences in the pretest group mean body weights. Rats were grouped based on body weights collected approximately 10 days prior to the first exposure. In general, at the commencement of the study, the weight variation of animals used did
not exceed ± 20% of the mean weight for each sex. Upon arrival, each rat was assigned a unique 6-digit animal number. Rat tails and cage cards
were color-coded with water-insoluble markers so rats could be identified. Following release from quarantine and prior to grouping, the last 3 digits of the animal number were tattooed onto the tail of each rat. Tattoo numbers and corresponding 6-digit animal numbers were recorded in the study records and on cards affixed to the cages.

Male rats were approximately 7 weeks old and weighed from 197.9 to 286.6 grams at the start of the exposures. Female rats were approximately 7 weeks old and weighed between 149.1 and 231.9 grams at the start of the exposures.


Feed and Water

Except during exposures, PMI Nutrition International, Inc. Certified Rodent LabDiet® 5002 and tap water was available ad libitum. During the urine collection period or fasting period prior to sacrifice, rats were fasted overnight for 12 to 20 hours; water, however, was available ad libitum.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: 5.7-6.9 µm

Details on inhalation exposure:

Filtered, high-pressure air and filtered, conditioned air were passed through the control chambers at rates to achieve at least 10 air changes per hour.
Control chamber atmospheres were exhausted into the main plenum exhaust system and emitted into the atmosphere.


10 or 50 mg/m3 DMG

Chamber atmospheres were generated by atomization of the test substances in air with a Spraying Systems nebulizer. Test substances were metered into the nebulizer with a Harvard Apparatus Syringe Infusion pump. Filtered, high-pressure air introduced at the nebulizer atomized the test substance and aerosol was delivered directly into the top of the exposure chamber. Filtered,
conditioned air was added to the exposure chamber to achieve at least 10 air changes per hour in the chamber. Chamber concentrations of DMG were controlled by varying the test substance feed rate to the nebulizer. Chamber atmospheres were exhausted into the main plenum exhaust system, through a scrubbing unit containing water, and emitted into the atmosphere.

400 mg/m3 DMG, DMS, or DMA

Chamber atmospheres were generated by atomization of the test substances in air with a Spraying Systems nebulizer. Test substances were metered into the nebulizer with a Masterflex® Console Drive pump. Filtered, high pressure air introduced at the nebulizer atomized the test substance and aerosol was delivered directly into the top of the exposure chamber. Filtered, conditioned air was added to the exposure chamber to achieve at least 10 air changes per hour in the chamber. Chamber concentrations of DMG, DMS, or DMA were controlled by varying the test substance feed rate to the nebulizer.

Exposure Mode

During exposure, rats were placed within wire-mesh cages and exposed whole-body inside the exposure chamber. The chamber volume was chosen so that the total body volume of the test animals did not exceed 5% of the chamber volume.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test Substance Sampling and Analysis

The atmospheric concentration of DMG, DMS, or DMA was determined at approximately 70-minute intervals (5 times during each 6-hour exposure) by gas chromatography and gravimetric analysis for the vapor and aerosol components, respectively. The control chambers were sampled once during each exposure. Known volumes of chamber atmosphere were drawn
from the breathing zone of the rats through a sampling train consisting of a 25 mm filter cassette containing a preweighed Gelman glass fiber (Type AlE) filter followed by a midget glass impinger containing acetone as the collection medium. For each exposure, the aerosol concentration was added to the vapor concentration to calculate the total chamber concentration
of each test substance. The filters were weighed on a Cahn model C-31 Microbalance®. The atmospheric concentration of the DMG, DMS, or DMA aerosol component was calculated from the difference in the pre- and post-sampling filter weights divided by the volume of chamber atmosphere sampled.

Aliquots of impinger solutions were injected on a Hewlett Packard model 6890 Series Gas Chromatograph equipped with a flame ionization detector. All samples were chromatographed isothermally at 165°C on an Alltech Carbowax 20M fused silica glass column. The atmospheric concentration of the DMG, DMS, or DMA vapor component was determined by comparing the detector response of the impinger samples with a standard curve calculated prior to exposure.

Liquid standards were prepared weekly by the quantitative dilution of DMG, DMS, or DMA in acetone. Samples to determine particle size distribution (mass median aerodynamic diameter and percent particles less than 1,3, and 10 µm diameter) were taken from the DMA chamber 5 times during the study with a Sierra® Series 210 Cyclone Preseparator/Cascade Impactor and Sierra® Series Detector.

Duration of treatment / exposure:

6 hours of exposure per day;

Frequency of treatment:

5 days per week, over a 90-day period;

Doses / concentrations

Remarks:

Doses / Concentrations:
0.0, 10, 50, or 400 mg/m3 for DMG, 400 mg/m3 for DMS, and 400 mg/m3 for DMA
Basis:
nominal conc.

No. of animals per sex per dose:

no data

Control animals:

yes, concurrent no treatment

Details on study design:

Six groups of male and 6 groups of female Crl:CD®(SD)IGS BR rats each were exposed via inhalation to 0,10,50, or 400 mg/m3 DMG, 400 mg/m3 DMS, or 400 mg/m3 DMA 6 hours per day, 5 days a week over a 90-day period. The exposure period was followed by an approximately 1-month
recovery period. Rats were weighed once per week and clinical signs were taken prior to exposure on days the rats were weighed. Group clinical signs were observed during each exposure, and the alerting response to an auditory stimulus was checked during each exposure in rats visible from the front of the chamber. After each exposure, rats were checked for the
alerting response to an auditory stimulus prior to removal from chambers and were observed for clinical signs immediately as they were returned to their cages. Food consumption was determined on a weekly basis.

Samples for hepatic, lung, and nasal (levels II and III) cell proliferation (CP) were collected from 5 rats/ sex/group approximately 2 weeks after initiation of the study and approximately 90 days after study initiation.

A clinical pathology evaluation was conducted on 10 rats/sex/group designated for subchronic toxicity evaluation approximately 45 and 90 days after initiation of the study. Approximately 90 days after study initiation, rats
designated for the clinical pathology evaluation were sacrificed for pathological examination and evaluation of male reproductive endpoints, including sperm motility, sperm number, and sperm morphology.

A neurobehavioral test battery, consisting of functional observational battery
assessments and motor activity, was conducted on 10 rats/sex/group designated for behavioral evaluation and recovery prior to test substance administration to obtain baseline measurements, and during test weeks 4, 8, 13, and 18 (recovery). Approximately 90 days after study initiation, 6 rats/sex/group designated for neuropathology were sacrificed and evaluated. After approximately 1 month of recovery, rats designated for neuropathological evaluation at this timepoint were sacrificed for evaluation of this endpoint.

The estrous cycle of female rats was determined for the last 21 days of exposure in rats designated for subchronic toxicity evaluation. Following 90 days of exposure, blood was collected via the tail vein from 10 rats/sex/group and serum was subjected to hormonal analyses. In male rats, serum luteinizing hormone, follicle stimulating hormone, and testosterone concentrations were measured. In female rats, serum estradiol and progesterone concentrations were measured.

Positive control:

No data available

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Effect levels

1

Dose descriptor:

NOAEC

Remarks:

Systemic

Effect level:

400 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: MInimal treatment related findings in this dose group suggestive of systemic toxicity

2

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

50 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on irritation of the upper respiratory tract and cell proliferation indicative of regeneration of epithelia.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Results

Chamber Concentrations of DMG, DMS, and DMA

For each exposure, the mean aerosol concentration was added to the mean vapor concentration to calculate the total chamber atmospheric concentration of test substance.

 

The analytically determined overall mean concentrations ± S.D. of DMG in the exposure chambers targeted to 10, 50, or 400 mg/m³were 10 ± 1.5, 49 ± 7.3, and 410 ± 41 mg/m³, respectively.

 

The analytically determined overall mean concentrations ± S.D. of DMS and DMA in the exposure chambers targeted to 400 mg/m³were 400 ± 34 and 390 ± 49 mg/m³, respectively.

 

In general, daily mean concentrations were consistent from day-to-day throughout the study and were within 20% of the targeted concentration for each respective chamber. The analytically determined concentrations were considered acceptable for evaluating the toxicity of the test substances at the selected targeted concentrations. The exposure concentrations described in this report will be referenced to the targeted (nominal) concentrations of DMG, DMS, and DMA.

 

A sample to determine particle size distribution was taken from the DMA chamber during exposures number 3, 20, 35, 50, and 65.  The mass median aerodynamic diameter (MMAD) determined from 5 samples ranged from 5.7-6.9 µm. The test atmosphere was considered respirable in rats. No samples were taken from the DMG or DMS chambers since appreciable aerosol concentration was not present.

 

Mean Body Weights and Body Weight Gains

 

Body weights were collected weekly during the study. No statistically significant effects on body weight were observed in male or female rats during the exposure phase of the study.

 

DMG:

Male rats in the 50 or 400 mg/m³DMG exposure groups had significantly lower mean body weights during the recovery period. The significantly lower weights in male rats from the 50 mg/m3 group are considered equivocal since no adverse effects on body weight were observed during the exposure period. No statistically significant effects on bodyweight were observed in female rats during recovery.

 

Mean body weight gains of male rats in the 10 and 400 mg/m³DMG groups were significantly lower than controls during various times during the study. The effects on body weight gain were isolated in nature and had resolved by the following week. Although statistically significant, these transient changes in body weight gain are considered not to be adverse.

 

For the overall exposure period (days 0-84), male rats in the 400 mg/m³DMG group had significantly lower mean body weight gains. These changes are considered test substance related.

 

Mean body weight gains of female rats in the 400 mg/m³DMG group were significantly lower during the period day 7-14 of the exposure period. Mean body weight gains in female rats in the 50 mg/m³DMG group were significantly higher during the period day 28-35 of the exposure period. These findings were isolated, had resolved by the following week, and are considered not to be test substance related. No additional significant effects on body weight gains were observed in female rats during the exposure or recovery phases of the study.

 

DMA

Male rats in 400 mg/m³DMA exposure group had significantly lower mean body weights during the recovery period. No statistically significant effects on bodyweight were observed in female rats during recovery.

 

Mean body weight gains of male rats 400 mg/m³DMA group were significantly lower than controls during various times during the study. The effects on body weight gain were isolated in nature and had resolved by the following week. Although statistically significant, these transient changes in body weight gain are considered not to be adverse.

 

For the overall exposure period (days 0-84), male rats in the 400 mg/m³DMG group had significantly lower mean body weight gains. These changes are considered test substance related.

 

DMS

Mean body weight gains of male rats 400 mg/m³DMS group, and 400 mg/m³DMA group were significantly lower than controls during various times during the study. The effects on body weight gain were isolated in nature and had resolved by the following week. Although statistically significant, these transient changes in body weight gain are considered not to be adverse.

 

 

Food Consumption and Food Efficiency

 

DMG 

Food consumption by male rats in the 400 mg/m³DMG group was significantly lower compared to controls beginning on day 49 and continuing throughout the remainder of the exposure and recovery periods. In addition, food consumption in this group was significantly lower during the overall exposure period (days 0-84). These findings are considered indirectly test substance related.

 

Food consumption by female rats in the 400 mg/m³DMG group was significantly lower compared to controls on day 14 and days 35-84. The mean daily food consumption was also significantly lower in female rats in the 400 mg/m³DMG group during the overall exposure period (days 0-84). No additional significant findings on food consumption were seen in female rats during the study.

 

At various intervals during the study, mean daily food efficiency was observed to be significantly lower in all test groups or higher in the 400 mg/m³DMG group on day 7-14 and day 56-63 compared to controls. The mean daily food efficiency was also significantly lower in male rats in the 50 mg/m³DMG group and 400 mg/m³DMA group during the overall exposure period (days 0-84). The effect on food efficiency observed at 50 mg/m³DMG is considered not to be test substance related since the results observed were not part of a dose-response relationship.

 

Food efficiency of female rats in the 400 mg/m³DMG group was significantly lower compared to controls during the interval day 7 -14. These findings were isolated, had resolved by the following week, and are considered not to be test substance related. No additional significant effects on food efficiency were observed in female rats during the exposure or recovery phases of the study.

 

DMA

The mean daily food efficiency was also significantly lower in male rats in the 400 mg/m³DMA group during the overall exposure period (days 0-84).

 

Food efficiency of female rats in the 400 mg/m³DMA group was significantly lower compared to controls during the interval day 7 -14. These findings were isolated, had resolved by the following week, and are considered not to be test substance related. No additional significant effects on food efficiency were observed in female rats during the exposure or recovery phases of the study.

 

DMS

No adverse effects on food consumption identified.

 

 

Mortality and Clinical Observations

 

There were no test substance-related deaths in the study. There were 2 early deaths (found dead) in 400 mg/m³DMG male rats, whose cause of death was undetermined. All remaining rats survived until their scheduled termination. During the clinical observation period prior to exposure on the days when rats were weighed, there were no clinical signs of toxicity in male or female rats that could be attributed to DMG, DMS, or DMA. Clinical signs observed, including ocular discharge, hair loss, dehydration, wounds, misshapen tail, and missing end of tail, were considered incidental findings.

 

No treatment related clinical signs of toxicity attributable to DMG, DMS, or DMA were evident during the study. Clinical signs observed, including ocular discharge, dehydration, wounds, and missing end of tail, were considered incidental findings.

 

Ophthalmological Evaluations

 

No test substance-related abnormalities were identified in rats examined at the end of the exposure period. The abnormalities identified were attributed to trauma to the globe and/or orbit during previous orbital bleeding or were considered incidental findings.

 

Clinical Pathology Evaluations

 

A. Hematology/Coagulation

 

There were no toxicologically significant changes in male or female rats for hematologic or coagulation parameters.

 

B. Serum Chemistry

 

There were no toxicologically significant changes in male or female rats for clinical chemistry parameters. All statistically significant changes were considered not toxicologically significant (non-adverse) or not related to compound.

 

C. Urinalysis

 

There were no toxicologically or statistically significant changes in urinalysis parameters for male or female rats.

 

D. Conclusions on Clinical pathology

 

There were no toxicologically significant changes in hematology, clinical chemistry, or urinalysis parameters. Therefore, under the conditions of this study and for the clinical pathology parameters measured, the no-observed-effect level (NOEL) was greater than 400 mg/m³for DMG, DMS, and DMA for male and female rats.

 

 

Neurobehavioral Evaluations

A. Functional Observational

1. Forelimb Grip Strength

 

There were no test substance-related effects on forelimb grip strength in males or females for any exposure concentration of DMG, DMS, or DMA.

 

2. Hindlimb Grip Strength

 

There were no test substance-related effects on hindlimb grip strength in males or females for any exposure concentration of DMG, DMS, or DMA.

 

3. Hindlimb Foot Splay

 

There were no test substance-related effects on hindlimb foot splay in males or females for any exposure concentration of DMG, DMS, or DMA.

 

4. Other Functional ObservationalEndpoints

 

There were no test substance-related, toxicologically significant effects on the 37 neurobehavioral parameters evaluated in either males or females for any exposure concentration of DMG, DMS, or DMA.

 

B. Motor Activity

 

There were no test substance-related, toxicologically significant effects on duration of movement or number of movements for either males or females for any exposure concentration of DMG, DMS, or DMA.  

 

C. Conclusions

 

There were no test substance-related effects on forelimb grip strength, hindlimb grip strength, foot splay, functional observation parameters, or motor activity for either males or females exposed to DMG, DMS, or DMA.

 

Organ Weight Data

 

There were no test substance-related changes in mean organ weights.

 

There were statistically significant increases in the mean organ weight relative to body weights of the liver (10 and 50 mg/m³DMG) , epididymides (50 mg/m³DMG, 400 mg/m³DMA) , and spleen (400 mg/m³DMA). The mean weight of the epididymides, as a percent of brain weight, was also statistically increased (50 mg/m³DMG). These organ weight changes were not associated with test substance-related microscopic lesions, and/or did not exhibit a dose relationship. Thus, these increases were considered to be spurious or related to the slight decreases (not statistically significant) in mean body weights in the respective groups. There were no significant organ weight changes in females.

 

Gross Observations

 

There were no test substance-related gross observations. The few gross observations in this study were typical of spontaneously occurring lesions in this strain of rat.

 

Microscopic Findings

 

Test substance-related effects were observed in the noses of 3 month male and female rats exposed to 400 mg/m³of DMG, DMS, and DMA. These effects consisted primarily of degeneration/atrophy of the olfactory mucosa of the dorsal meatus and of the dorsomedial aspect of the dorsal endoturbinate. Less commonly, focal respiratory metaplasia of the olfactory mucosa of the dorsal meatus was also present. Lesions were minimal to mild in severity and occurred in higher incidences in the DMG groups.

 

Degeneration/atrophy of the olfactory mucosa occurred in recovery animals in the same locations as was apparent at the 90-day sacrifice in animals exposed to DMG, DMS, and DMA. The lesions were usually focal and minimal in severity. The incidence of lesions in female recovery groups was higher in the DMG and DMS groups compared to DMA group, while in male recovery groups, incidences were somewhat higher for the DMA group.

 

Conclusions

 

The no-observed-effect level (NOEL) for this study is defined as the highest dose at which toxicologically important effects attributable to the test substance were not detected. Under the conditions of this study, the NOEL for pathology was 50 mg/m³DMG in both males and females.

NOELs for DMS and DMA were not established, as there were effects in the only concentration of these materials tested. There were some minimal test substance-related effects in noses of recovery animals from the DMG, DMS, and DMA groups. Thus, complete reversibility of lesions was not demonstrated over the recovery period evaluated.

 

Neuropathology Evaluations

 

A. Gross Observations

There were no test substance-related gross lesions observed in this study.

B.  Microscopic Findings

There were no test substance-related microscopic observations in this study.

Reproductive Evaluations

 

A.   

 

DMG

In male rats exposed to DMG, serum testosterone concentrations were statistically significantly decreased at concentrations of 50 and 400 mg/m³(59 and 50% of control, respectively).

Similarly, serum LH concentrations were decreased in a dose-dependent manner and were statistically significantly decreased at 400 mg/m³(71 % of control). Serum concentrations of FSH were not affected by DMG treatment. The decreases in serum testosterone and LH were not accompanied by alterations in organ weights or histopathology of the male reproductive organs. Therefore, the biological significance of the hormonal alterations is unclear.

 

In female rats, DMG exposure did not alter serum estradiol or progesterone concentrations in a dose-responsive or statistically significant manner.

 

DMS

DMS caused a statistically significant decrease in serum estradiol concentrations (43% of control); serum progesterone concentrations were not affected. However interpretation of the female data was confounded due to differences in the stage of the estrous cycle for each of the females at the time of blood collection. Due to the small number of animals (n= 10) , it was not possible to analyze the data on the basis of stage of estrous cycle as would be recommended when analyzing serum hormone data from female rats. Therefore, only combined (i.e., rats in all stages of estrus) serum hormone concentrations were evaluated statistically. Due to the variability associated with the female data, a conclusion that the alterations in serum estradiol were compound-related is not possible.

 

In male rats exposed to DMS or DMA or in female rats exposed to DMG or DMA, no significant alterations in serum hormone concentration were observed.

 

B. Sperm Parameters

 

No compound-related effects were observed on sperm motility, sperm morphology or testicular spermatid counts (per testis and per gram testis) following inhalation exposure to DMG, DMS or DMA.

 

A treatment-related increase was detected in epididymal sperm counts (per cauda epididymis and per gram cauda epididymis) following exposure to DMG and the number of sperm per cauda and per gram cauda epididymis was significantly increased at 50 and 400 mg/m³(124-131 % of control). Epididymal sperm counts were similar to control at 10 mg/m³DMG. In male rats exposed to DMS, epididymal sperm counts (per cauda epididymis and per gram cauda epididymis) were significantly increased (153 and 141 % of control, respectively). Although not statistically significant, a similar trend of increased epididymal sperm counts was observed in the DMA group (124 and 114% of control for sperm per cauda and per gram cauda epididymis, respectively) that was considered compound-related.

 

Anatomic pathology evaluations indicated there were statistically significant increases in the mean organ weight relative to body weights of the epididymides at 50 mg/m³DMG and 400 mg/m³DMA. No statistically significant increases were observed at 10 or 400 mg/m³DMG or 400 mg/m³DMS. The mean weight of the epididymides, as a percent of brain weight, was also statistically increased at 50 mg/m3 DMG, but not at other concentrations.

 

No statistically significant differences were observed in absolute epididymidal weight at any concentration. These organ weight changes were not associated with test substance-related microscopic lesions. The statistically significant increases in relative epididymidal weight were considered not to be related to the changes seen in epididymal sperm count since no statistically significant increases in relative organ weight were observed at 400 mg/m³DMG or 400 mg/m3 DMS and no statistically significant increases in absolute epididymidal weight were observed at any \concentration. Rather, the changes were considered to be spurious or related to the slight decreases (not statistically significant) in mean body weights in the respective groups.

 

C. Estrous Cycling

 

There were no compound-related effects on estrous cycling.

 

Cell Proliferation Evaluations

A. Labeling Indices

              

1. Liver

 

Male rats exposed to 400 mg/m³DMS or DMA showed significantly increased CP in the liver at day 14 compared to controls (labeling indices of 6.64 for DMS, 4.52 for DMA, and 1.78 for controls). No significant effects were observed in the liver from males evaluated at 87 days or from females evaluated at either time point, compared to controls. No pathological evaluations were conducted on rats sacrificed at day 14. The biological significance of the CP data for liver is unclear due to a lack of pathological findings in this tissue at 90 days.

 

2. Lung

Female rats exposed to 400 mg/m³DMA had significantly greater CP in the lung relative to controls at days 14 and 87 (labeling indices of 11.34 and 3.28 for DMA at days 14 and 87,  respectively, versus 5.88 and 1.06 for controls). No additional significant effects on CP were observed in the remaining groups of females or males. No pathological evaluations were conducted on rats sacrificed at day 14. The biological significance of the CP data for lung is unclear due to a lack of pathological findings in this tissue at 90 days.

 

3. Nose Level II

Male rats exposed to 400 mg/m³DMG and DMA showed significantly greater CP in the nose level II compared to controls at day 87 (labeling indices of 19.62 for DMG, 21.00 for DMA, and 9.06 for controls). Female rats exposed to 400 mg/m3 DMG had significantly greater CP in the nose level II at day 14 (21.58 versus 10.92). No other significant findings were observed in male or female rats. The increased CP in the nose level II of rats evaluated at day 87 was expected based on histopathology findings observed in rats sacrificed at the end of the exposure period. No pathological evaluations were conducted on rats sacrificed at day 14.

              

4. Nose Level III

Compared to controls, male rats exposed to 10 or 50 mg/m3 DMG, 400 mg/m3 DMS, or 400 mg/m3 DMA had significantly lower CP in the nose on day 14. Labeling indices for these groups were 6.60, 9.08, 6.42, and 4.16, respectively, versus 16.30 (controls). Male rats exposed to 400 mg/m3 DMG had significantly greater CP in the nose level III at day 87 (23.56 versus 13.74). CP in the nose level III of female rats exposed to 400 mg/m3 DMG was significantly greater than controls on day 14 (16.58 versus 7.70). Female rats exposed to 400 mg/m3 DMS had significantly greater CP in the nose level III compared to controls on day 87 (13.38 versus 5.74).

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the systemic no-observed-effect level (NOAEL) for repeated exposure to DMG, DMA and DMS was 400 mg/m3, based on the minimal treatment related findings at this dose. the Local NOEL for irritation of the upper respiratory tract is 50 mg/m3 for DMG.