Registration Dossier

Administrative data

Description of key information

- Oral route (14 days, rat): NOEL = 10000 ppm (equivalent to 980 mg/kg bw)

- Dermal route (14 days, rat): NOEL (systemic toxicity) = 1000 mg/kg bw

- Inhalation (90 days, rat):       NOEC (respiratory local toxicity) = 50 mg/m3

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
1981
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Documentation insufficient for assessment.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 10 rats per concentration received the test item in feed for 14 consecutive days. An additional group of 10 rats served as controls and was fed standard chow only. All rats were weighed daily (weekends excluded) during the test period and observations for clinical signs were made at the same time. Food consumption for the test period was recorded. On day 14, 5 rats from each group were sacrificed. The remaining half of each group were weighed every other day and observed for a 14-day recovery period on standard feed without test item. All rats were examined grossly, selected tissues were weighed, and selected tissues and organs evaluated histologically.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male
Details on test animals and environmental conditions:
Young adult animals were used for this study
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
The test material mixed with ground Purina Lab Chow (GPLC) was fed .
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not applicable
Duration of treatment / exposure:
14 days of treatment. Half of the animals were observed for further 14 days without treatment.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
10, 20, 50 g/kg feed
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
10000, 20000, 50000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10 male animals per sex and dose
Control animals:
yes, plain diet
Details on study design:
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
no positive control used
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: treatment phase: daily; recovery period: every other day

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 1)
HISTOPATHOLOGY: Yes (see table 1)
Other examinations:
None
Statistics:
Standard statistical methods were used
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
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
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths occured during the study period.
No clinical signs other than initial and sporadic weight loss were observed.

BODY WEIGHT AND WEIGHT GAIN
The rate of body weight gain was reduced in rats fed either 20 or 50 g/kg feed. This reflects, at least in part, lower food intake by these rats. Upon return to the ground lab chow (control diet), test rats in these 2 groups gained slightly more weight than did the controls although the rats in the 50 g/kg feed group weighed approximately 7% less than controls at the end of the recovery period.

GROSS PATHOLOGY AND HISTOPATHOLOGY
No gross or microscopic pathologic changes that could be attributed to exposure of DBE were detected.
Critical effects observed:
not specified

Table 2:Weight and Food Consumption Data

Dose [ppm]

Dose [g/kg feed]

Average initial weight [g]

Average weight, day 14 [g]

Average weight, after 14 day recovery [g]

Average food consumption during test period [g/day/rat]

Estimated test item uptake [mg/kg bw/day] a)

0 (control)

0 (control)

244

329

391

26

-

10000

10

245

325

401

24

980

20000

20

239

302

394

22

1841

50000

50

240

290

364

19

3958

a) Estimation based on average food consumption and average initial body weight

Conclusions:
Except for the observed slight weight gain effects, under the conditions of this test, Dibasic esters appeared to be devoid of general toxicity in this 14-day feeding study.
Executive summary:

Dibasic Esters (DBE) has been tested in a 14-day oral toxicity study on Crl:CD rats.

Groups of 10 male rats received feed containing DBE at concentrations of 0 (control), 10000, 20000, or 50000 ppm for 14 consecutive days. All rats were weighed daily during the test period and observations for clinical signs were made at the same time. Food consumption for the test period was recorded. On the 14th test day, half of the rats of each test group were sacrificed. The remaining half of each group were weighed every other day, observed for a 14-day recovery period, and sacrificed 14 days later. All rats were examined grossly, selected tissues were weighed, and selected tissues and organs evaluated histologically.

Oral administration of DBE to rats over 14 days produced no deaths. No gross or microscopic pathologic changes that could be attributed to exposure of DBE were detected. No clinical signs other than initial and sporadic weight loss were observed. The rate of body weight gain was reduced in rats of the mid- and high-dose group which is reflected, at least in part, by lower food intake by these rats. During the recovery period, test rats in these 2 groups gained slightly more weight than the controls although the high-dose rats weighed approximately 7% less at the end of the recovery period.

Except for the observed slight weight gain effects, under the conditions of this test, DBE appeared to be devoid of general toxicity in this 14-day feeding study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
980 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 28, 1999. to December 06, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study equivalent or similar to OECD Guideline 413; Study under EPA GLP Regulations 40 CFR 792;
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
other: Crl:CD (SD)IGS BR
Sex:
male/female
Details on test animals 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.
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
Remarks:
Doses / Concentrations:
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
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOEC
Remarks:
systemic toxicity
Effect level:
10 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Other: decreases in serum testosterone concentrations + increased epididymal sperm counts at 50 mg/m3 and above (as concluded in study report)
Dose descriptor:
NOEC
Remarks:
respiratory local toxicity
Effect level:
50 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Histopathology: olfactory mucosa degeneration/atrophy
Critical effects observed:
not specified

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 Observational Endpoints

 

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 on neurobehavioural evaluation

 

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 Weights

 

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 concentration (NOEC) 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 NOEC for pathology was 50 mg/m3DMG in both males and females.

NOECs 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

  • 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).

Conclusion on cell proliferation

Due to inconsistency between genders, between exposure timepoints (14 or 90 days), between individual esters at the same test concentration of 400 mg/m3, and provided that no functional disturbances (as illustrated by clinical pathology and histopathological findings) were observed for these organs, the cell proliferation findings in liver and lungs were considered not to bear any toxicological relevance. The cell proliferation findings in the nose usually correlated with the local irritation effects.

Conclusions:
Under the conditions of this study, the no-observed-effect level (NOELd) for repeated exposure to DMG was 10 mg/m3, based on the decreases in serum testosterone and serum LH concentrations and increased epididymal sperm counts at concentrations of 50 mg/m3 and above. NOELs for DMS and DMA were not established, as there were effects in the only concentration of these materials tested.

Executive summary:

A 90-day inhalation toxicity study on dimethyl succinate (DMS), dimethyl glutarate (DMG), and dimethyl adipate (DMA was performed in the rat under conditions equivalent to the OECD Guideline 413 and under EPA GLP Regulation.

No compound-related effects were observed on mortality, clinical signs, hematology, neurobehavioral endpoints, sperm motility or morphology, or estrous cycle parameters.

Male rats exposed to nominal 400 mg/m3 DMG or 400 mg/m3 DMA had lower mean body weights and mean body weight gains during the study. In addition, male and female rats exposed to 400 mg/m3 DMG had lower food consumption, and male rats exposed to 400 mg/m3DMA had lower food efficiency during the study.

Compound-related effects were observed in the noses of male and female rats exposed to 400 mg/m3 of DMG, DMS, or DMA for 90 days. 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 group. 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 the DMA group, while in male recovery groups, incidences were somewhat higher for the DMA group.

Male rats exposed to 400 mg/m3 DMS and DMA showed significant increased Cell Proliferation (CP) in the liver at day 14 compared to controls. No significant effects were observed in the liver from males evaluated at 90 days or from females evaluated at either time point. Female rats exposed to 400 mg/m3 DMA had significantly greater CP in the lung relative to controls at days 14 and 90. No effects on CP were observed in the lung from male rats. These liver and lung findings were considered not to bear any toxicological relevance.

Male rats exposed to 400 mg/m3 DMG and DMA showed significantly greater CP in the nose level II compared to controls at day 90. Female rats exposed at 400 mg/m3 DMG had significantly greater CP in the nose level II at day 14. Male rats exposed to 400 mg/m3 DMG had significantly greater CP in the nose level III at day 90. CP in the nose level III of female rats exposed to 400 mg/m3 DMG was significantly greater than controls on day 14. Female rats exposed at 400 mg/m3 DMS had significantly greater CP in the nose level III compared to controls on day 90.

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. In female rats, DMG exposure did not alter serum estradiol or progesterone concentrations. In male rats exposed to DMS or DMA, no significant alterations in serum hormone concentration were observed. In female rats, DMS caused a statistically significant decrease in serum estradiol concentrations (43% of control); serum progesterone concentrations were not affected. In female rats, DMA exposure did not alter serum estradiol or progesterone concentrations.

 

There was a treatment-related increase 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/m3 DMG. 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.

In conclusion the LOEL for systemic and local effects of DMA was the only dose tested (400 mg/m3). Based on the structural similarity to the other dibasic esters (DMG and DMS), it is considered that the NOEC for systemic toxicity of DMA would likely be similar to that of DMG, i.e. approximately 10 mg/m3. However it should be noted that the sperm and hormonal effects were not replicated in other repeated dose studies on these materials.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
50 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
February 24 to December 20, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to to OECD Guideline 410; Study under EPA GLP Regulation 40 CFR &92;
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
14 days exposure period, followed by 14 days recovery period; no separate limit test performed;
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
other: Crl:CD (SD)IGS BR
Sex:
male/female
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test articles were applied seven days per week for two weeks to the shaved intact dorsal skin of each rat. The application sites were covered with gauze, occluded with plastic wrap and secured with non-irritating tape for a period of six hours per exposure. Following the exposure period, the application sites were gently washed with a solution of non-
irritating soap and disposable paper towels to remove residual test article. Each test article group consisted of 10 males and 10 females. Dosage levels were 100, 300 and 1000 mg/kg/day. The test article was administered undiluted based on specific gravity at varying dosage volumes. A concurrent control group received deionized water on a comparable regimen. The animals were examined twice daily for mortality and moribundity.

Clinical and dermal observations were performed daily prior to and following exposure and once each day of the recovery period. Detailed physical examinations were performed weekly. Ophthalmological examinations were performed prior to dosing and during study weeks 1 and 3. Clinical pathology evaluations (hematology, serum chemistry and urinalysis) were performed prior to the scheduled primary necropsy. Complete necropsies were performed for all animals and selected organs weighed from animals at the primary and recovery necropsies. Selected tissues were examined microscopically.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Six (6) hours per day, seven (7) days per week for two (2) weeks;
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw;
Basis:
nominal per unit area
No. of animals per sex per dose:
10 male animals per dose;
10 female animals per dose;
excluding satelitte animals;
Control animals:
yes
Details on study design:
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
no data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, before and after exposure, once daily in recovery period;
DERMAL IRRITATION (if dermal study): Yes, mild;
- Time schedule for examinations: daily, and at necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, selected organs;
Other examinations:

Body weights

Individual body weights were recorded twice weekly, beginning
approximately 10 days prior to test article administration. Mean body weight
changes were calculated for each corresponding interval.

A final (fasted) body weight was collected for each animal on the day of
scheduled necropsy.

Food consumption

Individual food consumption was measured weekly, beginning
approximately 10 days prior to test article administration. Food intake was
calculated as g/animal/day for the corresponding body weight intervals.


Clinical pathology

Blood and urine samples for clinical pathology evaluation were collected
from all surviving animals at the time of the primary necropsy (study week 2). Blood was collected from a lateral tail vein or the retro-orbital sinus. Urine was collected overnight using metabolism cages. The animals were fasted overnight prior to collection of blood samples. Clinical pathology methods, procedures and references are presented in Appendix F of the attached complete study report, as is the list of the clinical parameters in hematology, serum chemistry, and urinanalysis, examined in this study (Study report DBE WIL 385001, (2000), pages 28 & 29).

Ophthalmological examinations

Ocular examinations were conducted on all animals prior to initiation of test
article administration (study week -1) and during study week 1. Even though no ocular changes attributed to test article administration were noted at study week 2, a recovery examination was conducted (study week 3). All ocular examinations were conducted using an indirect ophthalmoscope (or other equivalent suitable equipment), preceded by pupillary dilation with an appropriate mydriatic agent.
Statistics:
All analyses were conducted using two-tailed tests for significance levels of
5% and 1 % comparing the vehicle control and test article-treated groups to the control group by sex. All means are presented with standard deviations and the number of sampling units (N) used to calculate the means. Body weight, body weight change, food consumption and efficiency,
clinical laboratory and absolute and relative organ weight data were subjected to a one-way analysis of variance (ANOVA), followed by Dunnett's test if the ANOVA revealed statistical significance (p<0.05).
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
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:
not examined
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):
no effects observed
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
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Critical effects observed:
not specified

Clinical observations and survival

Except for the deaths two animals (one male, one female, thought to be not treatment related) there were no test article-related deaths. There were no test article-related clinical observations. Clinical findings observed in the test article-treated groups were noted similarly in the control group, were not present in a dose-related manner, were noted at a low incidence, typically in single animals and/or were those types of clinical signs commonly observed in rats of this strain and age.

Dermal observations

Low incidences of test article-related erythema and/or edema, generally graded as very slight, were observed for DMA, DMS, DMG and DBE treatment groups in at least one sex. Erythema was observed for one to four males in each of the 300 and 1000 mg/kg/day DMA and DBE groups, the 1000 mg/kg/day DMS group and all three DMG groups. For females, erythema was noted for one to five animals in all DMA and DMG groups and for the 100 and 1000 mg/kg/day DBE groups. Erythema was not observed for females treated with DMS at any dose level. Edema was limited to single males in the 1000 mg/kg/day DMA, DMS and DMG groups and one female in the 1000 mg/kg/day DMG group. Erythema and edema were not observed until the second week of dosing for any dose group and were not observed in the control group. The most prevalent sign of dermal irritation observed during the dosing period was eschar (scab) formation, mostly focal. Focal eschar formation (scabbing) was typically first observed near the end of the first week or beginning of the second week of dosing. This finding was observed at higher incidences than in the control group for all groups treated with DMA, DMG and DBE and the 300 and 1000 mg/kg/day DMS male groups. The number of animals with focal eschar/eschar was greater in the DMG and DBE groups than in the DMA and DMS groups. However, the most severe scores recorded were for male no. 41569 (1000 mg/kg/day DMS group), which was observed with eschar for eight consecutive days beginning on study day 6, reduced to focal eschar on the day of primary necropsy. In addition, fissuring was noted for one 1000 mg/kg/day DMA group female on the final day of the dosing period. The only other dermal finding was desquamation, generally first observed during the second week of dosing. Desquamation was noted for all groups, but with the highest incidence in the test article-treated groups. This increased incidence was considered to be possibly test article-related for most dosage levels in both sexes for DMA, DMG and DBE. The following table summarizes the dermal findings during the dosing period:

Males

Control

DMA

DMS

DMG

DBE

Dermal Finding

1

2

3

4

5

6

7

8

9

10

11

12

13

Erythema

0

0

1

2

0

0

2

1

4

4

0

1

2

Edema

0

0

0

1

0

0

1

0

0

1

0

0

0

Focal EscharlEschar

1

2

7

8

1

4

4

9

9

9

7

7

6

Desquamation

4

5

9

9

6

6

5

8

9

8

9

10

9

Females

Cntrol

DMA

DMS

DMG

DBE

Dermal Finding

1

2

3

4

5

6

7

8

9

10

11

12

13

Erythema

0

1

2

2

0

0

0

1

3

5

1

0

3

Edema

0

0

0

0

0

0

0

0

0

1

0

0

0

Focal Eschar/Eschar

2

4

4

4

0

2

0

6

6

7

5

2

10

Fissuring

0

0

0

1

0

0

0

0

0

0

0

0

0

Desquamation

4

6

7

6

1

3

4

8

9

8

6

7

9

DMA, dimethyl adipate; DMS, dimethyl succinate; DMG, dimethyl glutarate; DBE, dibasic ester.

Body weights

 

Mean body weights were unaffected by dermal exposure to DBE. Occasional statistically significant variations from the control group in mean body weight gain and/or mean cumulative gain were noted in the test article-treated groups. However, no consistent effects were observed and mean body weights were comparable to the control throughout the study.

 

Food consumption

 

Food consumption was unaffected by exposure to any of the test articles. Statistically significant changes in food efficiency were only observed during the recovery period and were considered random occurrences.

Clinical pathology

No test article-related effects on hematologic parameters were observed. A few statistically significant (p 0.05) differences from the control group were noted; however, the changes were not observed in a dose-related manner or were not of sufficient magnitude to be toxicologically relevant.

Serum chemistry

 

There were no test article-related changes in serum chemistry parameters. Slight increases (p 0.05) in mean sorbitol dehydrogenase levels were noted in the 300 mg/kg/day DMG and 1000 mg/kg/day DBE group females when compared to the control group. However, the changes were slight, present in only one sex, changes in other liver enzymes measured (i.e., ALP, ALT, AST or GGT) were not observed and there were no corresponding macroscopic or microscopic findings in the liver. Therefore, these changes were mostlikely random occurrences unrelated to test article treatment. Other statistically significant (p 0.05) changes from the control group were not observed in a dose-related manner and/or were not observed with

sufficient magnitude to be toxicologically relevant.

Urinalysis

No test article-related effects on urinalysis parameters were noted. Specific gravity was decreased (p 0.05) 100 mg/kg/day DBE group females. These changes were slight and not observed in a dose-related manner. Therefore, a relationship to test article exposure was not apparent.

Ophthalmalogical examinations

No oculopathic lesions indicative of a toxic effect were observed. All findings were typical for this species and strain.

Anatomic pathology

A pale liver was noted for the 300 mg/kg/day DBE group male that was found dead on study day 10. There were no internal macroscopic findings for the 100 mg/kg/day DBE group female that was found dead. At the study week 2 primary necropsy, scabbing of the treated skin was observed for one 300 mg/kg/day DMA group male, one 1000 mg/kg/day DMS group male, two 1000 mg/kg/day DMG group males and one male in each of the DBE groups. Findings for the treated skin of females consisted of scabbing for one 1000 mg/kg/day DMA group female and dark red  areas for one 1000 mg/kg/day DBE group female. No microscopic findings correlated with the dark red areas noted for the 1000 mg/kg/day DBE group female. Scabbing of the treated skin was not observed for any animals at the study week 4 recovery necropsy.  No other test article-related macroscopic findings were noted at the scheduled necropsies.

Organ weight

No changes attributed to test article exposure were observed in organ weights. Mean and relative (to final body weight and brain weight) lung weights were increased (p 0.05) in the 100 mg/kg/day DBE group females.

Microscopic Examination

The cause of death for the 300 mg/kg/day DBE group male and 100 mg/kg/day DBE group female could not be determined microscopically.  It was considered not treatment related.

Conclusions:
Based on the results of this study, the no-observed effect level (NOEL) for systemic toxicity of DBE when administered dermally to male and female rats for 14 consecutive days was 1000 mg/kg/day.
Executive summary:

A 14-day dermal toxicity study was performed in the rat in accordance with the OECD Guideline 410 and under GLP Regulations. There were no test article or treatment related deaths or clinical observations following administration of DBE for 14 consecutive days. Minimal to mild dermal irritation was observed for all treatment levels near the end of the first week or during the second week of dosing. Scabbing of the treated skin was observed at necropsy for one male in each of the DBE-treatment groups and dark red areas on the treated skin was observed for one female in the 1000 mg/kg/day DBE group; however,  microscopic findings did not indicate a generalized reaction of the treated skin to DBE. There were no other test article-related microscopic findings. Body weights and food consumption were unaffected by DBE treatment. There were no test article-related changes in hematology, serum chemistry, urinalysis, ophthalmoscopy, neurobehaviour or organ weight parameters. Based on these study results, the no-observed-effect level (NOEL) for systemic toxicity for dermal application of DBE for 14 consecutive days was considered to be 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
February 24 to December 20, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to to OECD Guideline 410; Study under EPA GLP Regulation 40 CFR &92;
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
14 days exposure period, followed by 14 days recovery period; no separate limit test performed;
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
other: Crl:CD (SD)IGS BR
Sex:
male/female
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test articles were applied seven days per week for two weeks to the shaved intact dorsal skin of each rat. The application sites were covered with gauze, occluded with plastic wrap and secured with non-irritating tape for a period of six hours per exposure. Following the exposure period, the application sites were gently washed with a solution of non-
irritating soap and disposable paper towels to remove residual test article. Each test article group consisted of 10 males and 10 females. Dosage levels were 100, 300 and 1000 mg/kg/day. The test article was administered undiluted based on specific gravity at varying dosage volumes. A concurrent control group received deionized water on a comparable regimen. The animals were examined twice daily for mortality and moribundity.

Clinical and dermal observations were performed daily prior to and following exposure and once each day of the recovery period. Detailed physical examinations were performed weekly. Ophthalmological examinations were performed prior to dosing and during study weeks 1 and 3. Clinical pathology evaluations (hematology, serum chemistry and urinalysis) were performed prior to the scheduled primary necropsy. Complete necropsies were performed for all animals and selected organs weighed from animals at the primary and recovery necropsies. Selected tissues were examined microscopically.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Six (6) hours per day, seven (7) days per week for two (2) weeks;
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw;
Basis:
nominal per unit area
No. of animals per sex per dose:
10 male animals per dose;
10 female animals per dose;
excluding satelitte animals;
Control animals:
yes
Details on study design:
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
no data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, before and after exposure, once daily in recovery period;
DERMAL IRRITATION (if dermal study): Yes, mild;
- Time schedule for examinations: daily, and at necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, selected organs;
Other examinations:

Body weights

Individual body weights were recorded twice weekly, beginning
approximately 10 days prior to test article administration. Mean body weight
changes were calculated for each corresponding interval.

A final (fasted) body weight was collected for each animal on the day of
scheduled necropsy.

Food consumption

Individual food consumption was measured weekly, beginning
approximately 10 days prior to test article administration. Food intake was
calculated as g/animal/day for the corresponding body weight intervals.


Clinical pathology

Blood and urine samples for clinical pathology evaluation were collected
from all surviving animals at the time of the primary necropsy (study week 2). Blood was collected from a lateral tail vein or the retro-orbital sinus. Urine was collected overnight using metabolism cages. The animals were fasted overnight prior to collection of blood samples. Clinical pathology methods, procedures and references are presented in Appendix F of the attached complete study report, as is the list of the clinical parameters in hematology, serum chemistry, and urinanalysis, examined in this study (Study report DBE WIL 385001, (2000), pages 28 & 29).

Ophthalmological examinations

Ocular examinations were conducted on all animals prior to initiation of test
article administration (study week -1) and during study week 1. Even though no ocular changes attributed to test article administration were noted at study week 2, a recovery examination was conducted (study week 3). All ocular examinations were conducted using an indirect ophthalmoscope (or other equivalent suitable equipment), preceded by pupillary dilation with an appropriate mydriatic agent.
Statistics:
All analyses were conducted using two-tailed tests for significance levels of
5% and 1 % comparing the vehicle control and test article-treated groups to the control group by sex. All means are presented with standard deviations and the number of sampling units (N) used to calculate the means. Body weight, body weight change, food consumption and efficiency,
clinical laboratory and absolute and relative organ weight data were subjected to a one-way analysis of variance (ANOVA), followed by Dunnett's test if the ANOVA revealed statistical significance (p<0.05).
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
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:
not examined
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):
no effects observed
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
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Critical effects observed:
not specified

Clinical observations and survival

Except for the deaths two animals (one male, one female, thought to be not treatment related) there were no test article-related deaths. There were no test article-related clinical observations. Clinical findings observed in the test article-treated groups were noted similarly in the control group, were not present in a dose-related manner, were noted at a low incidence, typically in single animals and/or were those types of clinical signs commonly observed in rats of this strain and age.

Dermal observations

Low incidences of test article-related erythema and/or edema, generally graded as very slight, were observed for DMA, DMS, DMG and DBE treatment groups in at least one sex. Erythema was observed for one to four males in each of the 300 and 1000 mg/kg/day DMA and DBE groups, the 1000 mg/kg/day DMS group and all three DMG groups. For females, erythema was noted for one to five animals in all DMA and DMG groups and for the 100 and 1000 mg/kg/day DBE groups. Erythema was not observed for females treated with DMS at any dose level. Edema was limited to single males in the 1000 mg/kg/day DMA, DMS and DMG groups and one female in the 1000 mg/kg/day DMG group. Erythema and edema were not observed until the second week of dosing for any dose group and were not observed in the control group. The most prevalent sign of dermal irritation observed during the dosing period was eschar (scab) formation, mostly focal. Focal eschar formation (scabbing) was typically first observed near the end of the first week or beginning of the second week of dosing. This finding was observed at higher incidences than in the control group for all groups treated with DMA, DMG and DBE and the 300 and 1000 mg/kg/day DMS male groups. The number of animals with focal eschar/eschar was greater in the DMG and DBE groups than in the DMA and DMS groups. However, the most severe scores recorded were for male no. 41569 (1000 mg/kg/day DMS group), which was observed with eschar for eight consecutive days beginning on study day 6, reduced to focal eschar on the day of primary necropsy. In addition, fissuring was noted for one 1000 mg/kg/day DMA group female on the final day of the dosing period. The only other dermal finding was desquamation, generally first observed during the second week of dosing. Desquamation was noted for all groups, but with the highest incidence in the test article-treated groups. This increased incidence was considered to be possibly test article-related for most dosage levels in both sexes for DMA, DMG and DBE. The following table summarizes the dermal findings during the dosing period:

Males

Control

DMA

DMS

DMG

DBE

Dermal Finding

1

2

3

4

5

6

7

8

9

10

11

12

13

Erythema

0

0

1

2

0

0

2

1

4

4

0

1

2

Edema

0

0

0

1

0

0

1

0

0

1

0

0

0

Focal EscharlEschar

1

2

7

8

1

4

4

9

9

9

7

7

6

Desquamation

4

5

9

9

6

6

5

8

9

8

9

10

9

Females

Cntrol

DMA

DMS

DMG

DBE

Dermal Finding

1

2

3

4

5

6

7

8

9

10

11

12

13

Erythema

0

1

2

2

0

0

0

1

3

5

1

0

3

Edema

0

0

0

0

0

0

0

0

0

1

0

0

0

Focal Eschar/Eschar

2

4

4

4

0

2

0

6

6

7

5

2

10

Fissuring

0

0

0

1

0

0

0

0

0

0

0

0

0

Desquamation

4

6

7

6

1

3

4

8

9

8

6

7

9

DMA, dimethyl adipate; DMS, dimethyl succinate; DMG, dimethyl glutarate; DBE, dibasic ester.

Body weights

 

Mean body weights were unaffected by dermal exposure to DBE. Occasional statistically significant variations from the control group in mean body weight gain and/or mean cumulative gain were noted in the test article-treated groups. However, no consistent effects were observed and mean body weights were comparable to the control throughout the study.

 

Food consumption

 

Food consumption was unaffected by exposure to any of the test articles. Statistically significant changes in food efficiency were only observed during the recovery period and were considered random occurrences.

Clinical pathology

No test article-related effects on hematologic parameters were observed. A few statistically significant (p 0.05) differences from the control group were noted; however, the changes were not observed in a dose-related manner or were not of sufficient magnitude to be toxicologically relevant.

Serum chemistry

 

There were no test article-related changes in serum chemistry parameters. Slight increases (p 0.05) in mean sorbitol dehydrogenase levels were noted in the 300 mg/kg/day DMG and 1000 mg/kg/day DBE group females when compared to the control group. However, the changes were slight, present in only one sex, changes in other liver enzymes measured (i.e., ALP, ALT, AST or GGT) were not observed and there were no corresponding macroscopic or microscopic findings in the liver. Therefore, these changes were mostlikely random occurrences unrelated to test article treatment. Other statistically significant (p 0.05) changes from the control group were not observed in a dose-related manner and/or were not observed with

sufficient magnitude to be toxicologically relevant.

Urinalysis

No test article-related effects on urinalysis parameters were noted. Specific gravity was decreased (p 0.05) 100 mg/kg/day DBE group females. These changes were slight and not observed in a dose-related manner. Therefore, a relationship to test article exposure was not apparent.

Ophthalmalogical examinations

No oculopathic lesions indicative of a toxic effect were observed. All findings were typical for this species and strain.

Anatomic pathology

A pale liver was noted for the 300 mg/kg/day DBE group male that was found dead on study day 10. There were no internal macroscopic findings for the 100 mg/kg/day DBE group female that was found dead. At the study week 2 primary necropsy, scabbing of the treated skin was observed for one 300 mg/kg/day DMA group male, one 1000 mg/kg/day DMS group male, two 1000 mg/kg/day DMG group males and one male in each of the DBE groups. Findings for the treated skin of females consisted of scabbing for one 1000 mg/kg/day DMA group female and dark red  areas for one 1000 mg/kg/day DBE group female. No microscopic findings correlated with the dark red areas noted for the 1000 mg/kg/day DBE group female. Scabbing of the treated skin was not observed for any animals at the study week 4 recovery necropsy.  No other test article-related macroscopic findings were noted at the scheduled necropsies.

Organ weight

No changes attributed to test article exposure were observed in organ weights. Mean and relative (to final body weight and brain weight) lung weights were increased (p 0.05) in the 100 mg/kg/day DBE group females.

Microscopic Examination

The cause of death for the 300 mg/kg/day DBE group male and 100 mg/kg/day DBE group female could not be determined microscopically.  It was considered not treatment related.

Conclusions:
Based on the results of this study, the no-observed effect level (NOEL) for systemic toxicity of DBE when administered dermally to male and female rats for 14 consecutive days was 1000 mg/kg/day.
Executive summary:

A 14-day dermal toxicity study was performed in the rat in accordance with the OECD Guideline 410 and under GLP Regulations. There were no test article or treatment related deaths or clinical observations following administration of DBE for 14 consecutive days. Minimal to mild dermal irritation was observed for all treatment levels near the end of the first week or during the second week of dosing. Scabbing of the treated skin was observed at necropsy for one male in each of the DBE-treatment groups and dark red areas on the treated skin was observed for one female in the 1000 mg/kg/day DBE group; however,  microscopic findings did not indicate a generalized reaction of the treated skin to DBE. There were no other test article-related microscopic findings. Body weights and food consumption were unaffected by DBE treatment. There were no test article-related changes in hematology, serum chemistry, urinalysis, ophthalmoscopy, neurobehaviour or organ weight parameters. Based on these study results, the no-observed-effect level (NOEL) for systemic toxicity for dermal application of DBE for 14 consecutive days was considered to be 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

There is only one repeated dose toxicity study on DMA. This is the inhalation study designated as the key study. In addition to this study, other studies using the mix of dibasic esters or the individual esters are provided to give an overall weight of evidence for the repeated dose toxicity of Dimethyl adipate. Justification for this reliance on data from the other dibasic esters is given in the category justification attached to the section 13 summary.

 

Oral route:

One study of reliability 4 according to Klimisch criteria (due to limited documentation) is available and was selected as a supporting study. In this sub-acute study, dibasic ester blend was administered in feed at 10000, 20000 or 50000 ppm (equivalent to 980, 1841 or 3958 mg/kg bw/day, respectively, based on average food consumption and body weight changes over the dosing period). Initial and sporadic decreases in body weight and body weight gain were observed at 20000 and 50000 ppm. At the end of a 2-week recovery period, body weight had returned to normal, except at 50000 ppm where it was still 7% lower than in controls. The lower bodyweight in the mid and high dose animals appears to be related more to the palatability of the feed rather than a general toxic effect. This is demonstrated by the decreased food consumption in the mid and high dose groups. As such it is likely that the mid dose tested (20000 ppm equivalent to 1841 mg/kg) did not produce systemic toxicity and can be considered as a NOAEL (since bodyweight recovered after cessation of test material administration). In the highest dose tested there was not a full recovery of body weight after 14 days so it is not possible to rule out some systemic toxicity not otherwise apparent from the other parameters assessed in the study. Due to the drop in body weight the overall No Observed Effect Level is the lowest dose of 10000 ppm (equivalent to 980 mg/kg bw).

 

Dermal route:

One study of reliability 1 according to Klimisch cotation criteria is available. In this sub-acute study, dibasic ester blend was administered dally at 0, 100, 300 or 1000 mg/kg bw. There was a low incidence of local findings illustrative of minimal to mild dermal irritation as erythema and/or edema (graded as very slight) and an increased incidence of focal eschar formation (scabbing) or desquamation in all groups applied the dibasic ester blend, starting around the end of the first week of dosing. No test article-related findings were seen in clinical observations, food consumption, clinical pathology, urinalysis, ophthalmology or anatomic pathology. The NOEL for systemic toxicity was therefore 1000 mg/kg over 14 days of dermal application to rats.

 

Inhalation route:

Three studies are available. A 90-day study on the individual components of dibasic ester blend (dimethyl glutarate at 10,50 or 400 mg/m3, dimethyl succinate at 400 mg/m3 and dimethyl adipate at 400 mg/m3) of reliability 1 according to Klimisch citation criteria was selected as the key study. In this study, only a single concentration of DMA was used, therefore the data from each of the other structurally related materials are used to support the identification of a NOEL.

There was a depression in bodyweight gain in the male animals dosed with DMA. There were also fluctuations in female body weight and bodyweight gain throughout the study. These observations were considered to be treatment related and consistent with those observed with the other two dibasic esters. The depression in male bodyweight gain is also consistent with an apparent decrease in feed efficiency. There were no clinically significant or toxicologically relevant clinical pathology findings in the animals at any dose group for any material. There were no adverse neurobehavioural findings in any group. There were no test compound related adverse changes in organ weights or gross observations. The key finding of the study that was considered to be treatment related was histopathological findings in the olfactory mucosa and upper respiratory tract. These findings were observed in the high dose group of DMA, DMG and DMS. The NOEC for these findings was therefore the mid dose group for DMG. Making the assumption that each of these substances would be similarly cytotoxic to the respiratory mucosa, the NOEC for DMA is also assumed to be 50 mg/m3.

 

 

In a similar 90-day study, of reliability 1 according to Klimisch citation criteria and selected as a supporting study, the concentrations tested were 20, 76 and 390 mg/m3 of dibasic ester blend. Olfactory epithelial lesions similar to those seen in the key study were observed at 76 and 390 mg/m3 in both genders, but also in females exposed to 20 mg/m3 and control females. A peer-review of nasal tissues by certified pathologists was performed (Ref. Pathology peer review of nasal tissue slides from two 90-day studies on behalf of Invista SARL, Experimental Pathology Laboratories, Inc., EPL Report No.: 851-001,18 September 2009). The peer review showed that olfactory epithelial lesions in the control females were seen with a roughly similar incidence and severity than in the females exposed to 20 mg/m3. This similarity between 20 mg/m3 and control females casts doubt on whether these lesions at this level of exposure are clearly related to the test substance. Furthermore, several male and female rats showed squamous metaplasia of the respiratory epithelium. This change which was clearly unrelated to the test substance may be a confounding factor in evaluating low-dose effects. Therefore, the study is regarded as inconclusive as to the respiratory local changes and 20 mg/m3 is likely a NOEC in this study, and 76 mg/m3 likely a LOAEC, which is consistent with the NOEC of 50 mg/m3 determined in the key study.

 

In the third study of unassignable reliability (4 according to Klimisch cotation criteria) selected as a supporting study, male rats were exposed (whole-body exposure) to 0, 100, 300 or 900 mg/m3 for 14 days (6 hours/day, 5 days/week). Changes in relative weights of lungs and liver at the end of the exposure period were noted at 900 mg/m3 with no histopathological correlates, and therefore with no clear toxicological significance. A tentative NOEL of 300 mg/m3 over 14 days can be set.

 

Irritating effects on the respiratory epithelium, and atrophy/degeneration of the nasal and olfactory epithelium are critical effects consistently observed with various ester derivatives (butyl acrylate, methyl acrylate, methylmethacrylate, methyl acetate, ethyl acrylate, lactate esters) These effects are thought to be related to a mechanism common between esters (including dibasic esters) which involves hydrolysis by unspecific carboxylesterases located in the nasal/olfactory epithelium to release corresponding acids and alcohols. However, there are well-known differences in the anatomy and physiology of the nasal and olfactory epithelia between rats and humans. Although some variability in the experimental results of carboxylesterase activity exists, depending on the methodology and the substrate used, there is a general trend supporting lower nasal esterase activity towards esters from human tissues compared to those from rat tissues. This has also been supported by results in primate nasal tissues using ethyl acrylate (Ref. Frederick CBet al. Use of a hybrid computational fluid dynamics and physiologically based inhalation model for interspecies dosimetry comparisons of ester vapours. Toxicol Appl Pharmacol. 183(1): 23-40, 2002). The respiratory local effects should therefore be of a lower concern in the human situation. These effects are thought to be overpredictive in the rat model as compared to humans, because of the morphological and physiological differences.

Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: other

Justification for classification or non-classification

Based on the classification criteria of Annex VI Directive 67/548/EEC, no R37 classification is warranted because no signs of immediate or massive upper respiratory tract irritation are observed following inhalation of dibasic ester blend in rats or humans.

 

Based on the classification criteria of EU Regulation 1272/2008 (CLP), 'STOT – single exposure Cat. 3' classification is NOT warranted because nasal lesions appear to be subchronic effects in rats. 'STOT – repeated exposure' classification is NOT warranted because the toxicological relevance of rat findings to humans is limited based on interspecies differences.

 

No classification is warranted based on repeat-dose experimental data on dibasic esters.