2,2'-oxydi(ethylamine)

Endpoint summary

Description of key information

Repeated dose toxicity: oral:
No repeated dose toxicity via the oral route was available. The study is waived based on following justification:
A key study is available for the dermal and inhalation route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the oral route of exposure.
Repeated dose toxicity: dermal:
A 90 day dermal study was conducted with the read-across substance BDMAEE in rabbits according to OECD guideline 411 with a reliability score of 2. The NOAEL for systemic effects was considered to be greater than the highest tested dose of 8.0 mg/kg/day.  All dose levels tested resulted in varying levels of irritation at the exposure site.
Repeated dose toxicity: inhalation:
A 90 day whole body vapour inhalation study was conducted with the read-across substance BDMAEE in rats similar to OECD guidelines with a reliability score of 2.  Doses tested  were 0, 0.23, 1.25 and 5.8 ppm. There were signs of ocular and respiratory irritation at all doses. Evidence of minor, non-specific systemic toxicity (weight loss, changes in urinalysis and increase in relative weight of the testes and adrenals in males) was only observed at the highest dose of 5.8 ppm. No target organ toxicity was observed at any dose level. The NOAEC for systemic effects is therefore considered to be 1.25 ppm. LOAEC value of 1.51 mg/m³ (0.23 ppm) was obtained. Various signs of the eyes and respiratory tract irritation at all concentrations were observed.

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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-04-11 - 1993-08-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

GLP study conducted according to internal laboratory standards and methods similar to OECD guidelines.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

This project was conducted according to the protocol prepared by the Bushy Run Research Center (BRRC) and amendments 1, 2, 3, and 4.
Four groups, each consisting of 15 male and 15 female Sprague Dawley rats, were exposed 6 hours per day, 5 days per week, for 14 weeks to the test substance vapor at target concentrations of 0 (control), 0.22, 1.25, or 5.8 ppm. Ten rats per sex from each group were sacrificed after the exposure regimen; the remaining 5 rats per sex were sacrificed after a 6-week recovery period. A further 4 male and 4 female rats were added to the control and 5.8 ppm groups and assigned for perfusion-fixation sacrifice. Animals were sacrificed following the 6-hour exposure regimen on exposure day 1, 3, 5 or 10 (1/sex/group/sacrifice) and used for electron microscopic examination of nasal mucosa. Monitors for toxic effects included clinical observations, body weight, food and water consumption, hematology and serum clinical chemistry evaluations, urinalysis, urine chemistries, organ weights, and ophthalmic, gross pathologic and microscopic evaluations.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)
- Molecular formula (if other than submission substance): (CH3)2NCH2CH2OCH2CH2N(CH3)2
- Molecular weight (if other than submission substance): 160.26
- Substance type: Pure active substance
- Physical state: Light yellow liquid, slight amine odor
- Analytical purity: 97%
- Other: Complete solubility in water
- Lot/batch No.: S-0044117, reference no. 24VCB-65 A and B, BRCC (Sample numbers 51-486 and 51-487)
- Impurities (identity and concentrations): Unknown component (prestudy) 1.263% in sample 24VCB65A and 1.199% in sample 24VCB65B; unnknown component (post study) 0.901%.
- Composition of test material, percentage of components: Composition of test material, percentage of components: (prestudy analysis of sample 24VCB65A): dimethylamine 0.015%, N-methylmorpholine (NMM) 0.014%, dimethylethanolamine (DMEA) 0.492%, bis(2-dimethylaminoethyl)ether (A-99) 97.026%, N-methylaminoethoxy-N,N-dimethylethylamine 0.082%, 2-(2-dimethylaminoethoxy)ethanol (DMEE) 1.108%, unknowns 1.263%. (poststudy analysis of sample 24VCB65B): dimethylamine 0.017%, N-methylmorpholine (NMM) 0.014%, dimethylethanolamine (DMEA) 0.483%, bis(2-dimethylaminoethyl)ether (A-99) 97.114%, N-methylaminoethoxy-N,N-dimethylethylamine 0.084%, 2-(2-dimethylaminoethoxy)ethanol (DMEE) 1.089%, unknowns 1.199%.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc. (Frederick, MD)
- Age at study initiation: 35 days
- Weight at study initiation: Males in exposure groups 0, 0.22, 1.25, and 5.8 ppm had mean weights of 255.1, 254.1, 255.1, and 254.3 g, respectively. Females in exposure groups 0, 0.22, 1.25, and 5.8 ppm had mean weights of 169.6, 169.5, 167.2, and 168.5 g, respectively.
- Fasting period before study: Food and water withheld during exposure
- Housing: Housed two per cage in stainless steel, wire-mesh cages, 23.5 cm x 20 cm x 18 cm for approximately two weeks (acclimation period) and individually. The recovery animals were individually housed in the same size cages in a Relocatable Containment Systeme Unit housed during the 14-week exposure regimen. During the exposures, the animals were individually housed, separated by sex and exposure group, in stainless steel, wire-mesh cages (35 cm x 17 cm x 18 cm).
- Diet (e.g. ad libitum): Powdered certified Agway Prolab Animal Diet Rat, Mouse, Hamster 3200 (Agway Inc.); ad libitum
- Water (e.g. ad libitum): Water (Municipal Authority of Westmoreland County, Greensburg, PA) provided by an automatic watering system was available ad libitum, except during the water consumption measurement period.
- Acclimation period: Two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Mean chamber temperature: 22-25 deg C. General conditions in Evaporator: 0.22 ppm exposure group (top): 46, 50, 55 deg C (bottom): 44, 33, 30 deg C; 1.25 ppm exposure group (top): 41, 45, 50 deg C (bottom): 33, 36, 40; 5.80 ppm exposure group (top): 68, 51, 49 deg C (bottom): 48, 39, 39 deg C. Housing conditions: 18-22 deg C.
- Humidity (%): Mean chamber relative humidity: 36-57%, Housing conditions: 41-62%.
- Air changes (per hr): 21 air changes per hr
- Photoperiod (hrs dark / hrs light): 12-hr photoperiod

IN-LIFE DATES: From: 12/5/1988 To: 3/9-10/1989 (necropsy at 14 wks) or 4/21/1989 (group with additional 6 wks of recovery)

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: N/A

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel with glass windows, approximately 4320 liters
- Method of holding animals in test chamber: Whole body exposure
- Source and rate of air: N/A
- Method of conditioning air: N/A
- System of generating particulates/aerosols: Liquid test substance was metered with a Sage Model 355 syringe pump into a heated glass evaporator. The temperature in the evaporator was maintained at the lowest level sufficient to vaporize the liquid. The resultant vapor was carried into the chamber by a countercurrent air stream that entered the bottom of the evaporator.
- Temperature, humidity, pressure in air chamber: Evaporator temperatures ranged from 42 to 66 deg C.
- Air flow rate: approximately 1500 liters/minute
- Air change rate: 21 air changes per hour
- Method of particle size determination: N/A
- Treatment of exhaust air: N/A

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations of the test substance were analyzed approximately once every 30 to 120 minutes (depending on the target concentration) by gas chromatography of impinger solutions containing samples of the chamber atmosphere.
- Samples taken from breathing zone: No

VEHICLE (if applicable)
- Justification for use and choice of vehicle: N/A
- Composition of vehicle: N/A
- Type and concentration of dispersant aid (if powder): N/A
- Concentration of test material in vehicle: N/A
- Lot/batch no. of vehicle (if required): N/A
- Purity of vehicle: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of the test substance were analyzed approximately once every 30 to 120 minutes (depending on the target concentration) by gas chromatography of impinger solutions containing samples of the chamber atmosphere.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

1.51 mg/m³ air (nominal)

Remarks:

0.22 ppm

Dose / conc.:

8.2 mg/m³ air (nominal)

Remarks:

1.25 ppm

Dose / conc.:

38 mg/m³ air (nominal)

Remarks:

5.8 ppm

No. of animals per sex per dose:

15/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale: A series of acute inhalation exposures with the test substance were conducted at the BRRC Laboratory. When the test substance was generated dynamically (heated evaporator or bubbler method), the 6-hour LC50 value (with 95% confidence limits) for male and female Sprague Dawley rats was 166 (155 to 178) ppm. Clinical signs of ocular and respiratory irritation were observed in all exposure groups (68, 149, 200, and 248 ppm). The principal gross lesions observed at necropsy were dark red discoloration of the lungs, and encrustation around the eyes and nose. Rats exposed for 6 hours to test substance vapor generated statically survived the 14-day postexposure period. However, under static generation conditions, the test substance chamber concentration was only 24 ppm because the high chamber relative humidity (82%) lowered the vapor concentration.
- Rationale for animal assignment (if not random): Computer-based randomization program. At the time of group assignment, only animals with body weights within two standard deviations of the mean for each sex were used in the study. Any animal observed to be in poor health was rejected from group assignment.
- Rationale for selecting satellite groups: Ten rats per sex from each group were sacrificed after the exposure regimen; the remaining 5 rats per sex were sacrificed after a 6-week recovery period. A further 4 male and 4 female rats were added to the control and 5.8 ppm groups and assigned for perfusion-fixation sacrifice.
- Post-exposure recovery period in satellite groups: 6 weeks
- Section schedule rationale (if not random): Computer-based randomization program

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before and after exposure on an individual basis, during exposure on a group basis.
- Cage side observations checked: Overt clinical signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight collection and just preceding sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to initiation of the first exposure, also weighed on the morning preceding the second day (day 2), and days 5, 8, 9 and 12 (prior to sacrifice). Control animals were weighed on the morning preceding the third (Day 3) exposure due to unexpected weight losses in all groups on the morning preceding the second exposure. Animals designated for the 21-day recovery were weighed once each week during the
recovery period (Days 19, 26, and 33).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; Food consumption was measured for approximately 15 hours following 8 exposures for the male rats and following 9 exposures for the female rats, while in metabolism cages.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was measured for approximately 15 hours following 8 exposures for the male rats and following 9 exposures for the female rats, while in metabolism cages.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the first exposure, the eyes were examined by a veterinarian using an external light source and a magnifying lens. During the second week of the exposure regimen, eyes were examined immediately after the ninth (and final) exposure (all groups) and were examined again the following morning (high and intermediate test and control groups). Recovery group animals were also examined 2 weeks following the last exposure.
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes, food was removed at the start of blood collection.
- How many animals: All animals
- Parameters checked: erythrocyte count, platelet count, hemoglobin, leukocyte count, hematocrit, differential leukocyte count, mean corpuscular volume, reticulocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the time of scheduled sacrifice.
- Animals fasted: Yes, food was removed at the start of blood collection.
- How many animals: all animals
- Parameters checked: glucose, calcium, urea nitrogen, phosphorus, creatinine, sodium, total protein, potassium, albumin, chloride, globulin, total bilirubin, direct bilirubin, indirect bilirubin, aspartate aminotransferase, alanine aminotransferase, creatine kinase, lactic dehydrogenase, gamma-glutamyl transferase, sorbitol dehydrogenase, alkaline phosphatase.

URINALYSIS: Yes
- Time schedule for collection of urine: Approximately 15 hours following each exposure.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, food was removed at the start of blood collection.
- Parameters checked: Volume, color, turbidity, osmolality, microscopic constituents, pH, protein (quantitative), protein, creatinine, glucose, sodium,
ketone, potassium, bilirubin, chloride, blood, creatinine, clearance urobilinogen (estimate).


NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A
- Battery of functions tested: sensory activity / grip strength / motor activity / other: N/A

OTHER: Animals were sacrificed following the 6-hour exposure regimen on exposure day 1, 3, 5 or 10 (l/sex/group/sacrifice) and used for electron microscopic examination of nasal mucosa.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; all animals except those assigned to the recovery group were sacrificed the morning of the day following the final exposure. The remaining rats were sacrificed 3 weeks later. A complete necropsy was performed on each animal and all tissues were fixed in 10% neutral buffered formalin or Bouin's fixative for histologic evaluations. Five rats/sex from the control and high concentration groups were perfused with formaldehyde:glutaraldehyde solution for fixation of the lungs, liver, coronary artery, skin (dorsum of head and ear), spleen, and cornea for possible examination by electron microscopy.

HISTOPATHOLOGY: Yes; tissues fixed for microscopic examination included: brain, nasal turbinates, 1arynx, trachea, lungs, eyes, liver, kidneys, adrenals, spleen, testes, ovaries, lymph nodes (submandibular), heart, thymus, skin (dorsum of head, ears, and eyelids), gross lesions.

Other examinations:

The brain, liver, kidneys, lungs, spleen, and heart from all animals and the testes from all males were weighed at necropsy. Organ weights were recorded as absolute weights and as a percentage of both body and brain weights.

Statistics:

Results of quantitative continuous variables (e.g., body weight change) were intercompared for the three exposure groups and one control group by use of Bartlett's test for homogeneity of variances, analysis of variance (ANOVA), and Duncan's multiple range test. The latter was used, if F for the ANOVA was significant, to delineate which groups differed from the control. If Bartlett's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, if necessary, by t-tests. A two-sample t-test was used to compare the control group to the high concentration exposure group for the recovery animals. The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs of ocular and respiratory irritation were observed during the 14-week exposure and 6-week recovery periods and included swollen periocular tissue in rats from all exposure groups and periocular and perinasal encrustation in rats from the 5.8 ppm group. Periocular encrustation was also observed in males from the 1.25 ppm group. An additional sign observed primarily in animals from the 5.8 ppm group was cloudiness in the eyes. Detailed ophthalmic examination revealed keratitis in males from the 5.8 ppm group and females from the 1.25 and 5.8 ppm groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortalities occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight losses were observed for both sexes from the 5.8 ppm group at the end of the first week of exposure. Body weight gain in males from the 5.8 ppm group was statistically significantly lower throughout the 14-week exposure period; the percent decreased in body weight gain at week 14 was 18%. Body weight gain in females from the 5.8 ppm group was slightly reduced during the 14-weeek study period, although not statistically significant; the percent decrease in body weight gain at week 14 was 9%. Absolute body weight in the males from the 5.8 ppm group was slightly lower throughout the study; however, a statistical difference was achieved only at week 14. The percent decrease in body weight at week 14 was 6.5%. No statistically significant differences in absolute body weight were observed in females from the 5.8 ppm group. Also, no effects on body weight were observed in males and females from the 0.23 and 1.25 ppm groups during the study.
During the 6-week recovery period, body weight gain for both sexes from the 5.8 ppm group indicated some recovery (body weight gain was 14.5 and 5.2% lower than the controls at the end of the recovery period for males and female animals, respectively. No statistical differences were observed in body weight for the 5.8 ppm group during the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant decrease in food consumption was observed in females from the 5.8 ppm group at the end of the 14-week exposure regimen; the percent decrease in fool consumption was 18%. No statistically significant differences in food consumption were observed in males of any exposure group.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in water consumption was observed in males from the 5.8 group at the end of the exposure regimen; the percent increase in water consumption was 21%. No other differences in water consumption were observed in males and females from any exposure group.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

During week 13, keratitis (mostly in mild degree) was observed in all rats from the 5.8 ppm group. In addition, minimal keratitis was observed in 6 females from the 1.25 ppm group.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum chemistry revealed no consistent or concentration-related effects.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis results showed slight decreases in creatinine, sodium, potassium, and chloride in both sexes from the 5.8 ppm group at 14 weeks.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Exposure-related increases in relative organ weights were observed for the adrenals and testes of males from the 5.8 ppm group at the 14-week time point, but not after the 6-week recovery period. These organs showed no histologic abnormalities.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, color changes of the eyes (5.8 ppm group at the 14-week sacrifice only) and swollen periocular tissue (1.25 and/or 5.8 ppm groups at both 14- and 20-week sacrifices) were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Exposure-related microscopic lesions were seen only in tissues receiving direct contact with the chemical (exposed skin, eyes, ears, and the upper respiratory tract) and consisted of cytoplasmic vacuolation of the exposed epithelium, accompanied by varying degrees of necrosis, inflammation, and degeneration of superficial and deeper tissues. Lesions involving the nasal cavities were present in rats of both sexes from all exposure groups at both 14- and 20-week sacrifices, while lesions involving the other tissues were generally observed only in the 5.8 ppm group at the 14-week sacrifice. Electron microscopic evaluations of the anterior nasal turbinates of the 5.8 ppm group rats showed lesions consisting of intracytoplasmic membrane-bound vacuoles in the mucosal epithelium after one exposure, and in the mucosa and submucosa after 3 and 5 exposures (this evaluation was not performed after 10 exposures). The vacuoles appear to be derived from the coalescence of many small secretory vesicles produced by the Golgi apparatus.

Other effects:

not specified

Key result

Dose descriptor:

LOAEC

Remarks:

Local Toxicity

Effect level:

<= 1.51 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Various signs of irritation of the eye and respiratory tract at all concentrations were observed.

Key result

Dose descriptor:

NOAEC

Remarks:

Systemic Toxicity

Effect level:

8.2 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Minor, nonspecific systemic toxicity at 38 mg/m3 and no target organ toxicity.

Critical effects observed:

no

Conclusions:

Repeated exposure of rats to the test substance vapor at 0.23, 1.25, and 5.8 ppm for 14 weeks produced no mortality, indications of possible minor, nonspecific systemic toxicity at 5.8 ppm and no target organ toxicity, but various signs of irritation of the eye and respiratory tract at all concentrations was observed. Thus, a NOEL could not be determined for irritancy under the conditions of this study. However, a NOAEC for systemic effects is considered to be 1.25 ppm (8.2 mg/m3).

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Following the read across strategy, this endpoint is covered by a key study performed with the structural analogue BDMAEE (N, N, N', N'-tetramethyl-2,2'-oxybis(ethylamine)). The justification for read across is attached in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

LOAEC

Remarks:

Local Toxicity

Effect level:

<= 1.51 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Various signs of irritation of the eye and respiratory tract at all concentrations were observed.

Key result

Dose descriptor:

NOAEC

Remarks:

Systemic Toxicity

Effect level:

8.2 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Minor, nonspecific toxicity at 38 mg/m3 and no target toxicity

Key result

Critical effects observed:

no

Conclusions:

No reliable data for this endpoint is available with the test substance BAEE. Data generated with the analogue substance BDMAEE is used for endpoint coverage.
Repeated exposure of rats to the test substance vapor at 0.23, 1.25, and 5.8 ppm for 14 weeks produced no mortality, indications of possible minor, nonspecific systemic toxicity at 5.8 ppm and no target organ toxicity, but various signs of irritation of the eye and respiratory tract at all concentrations was observed. Thus, a NOEL could not be determined for irritancy under the conditions of this study. However, a NOAEC for systemic effects is considered to be 1.25 ppm (8.2 mg/m3). The same is assumed for the test substance BAEE.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

8.2 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-04-11 - 1993-08-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

GLP study conducted according to internal laboratory standards and methods similar to OECD guidelines.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

This project was conducted according to the protocol prepared by the Bushy Run Research Center (BRRC) and amendments 1, 2, 3, and 4.
Four groups, each consisting of 15 male and 15 female Sprague Dawley rats, were exposed 6 hours per day, 5 days per week, for 14 weeks to the test substance vapor at target concentrations of 0 (control), 0.22, 1.25, or 5.8 ppm. Ten rats per sex from each group were sacrificed after the exposure regimen; the remaining 5 rats per sex were sacrificed after a 6-week recovery period. A further 4 male and 4 female rats were added to the control and 5.8 ppm groups and assigned for perfusion-fixation sacrifice. Animals were sacrificed following the 6-hour exposure regimen on exposure day 1, 3, 5 or 10 (1/sex/group/sacrifice) and used for electron microscopic examination of nasal mucosa. Monitors for toxic effects included clinical observations, body weight, food and water consumption, hematology and serum clinical chemistry evaluations, urinalysis, urine chemistries, organ weights, and ophthalmic, gross pathologic and microscopic evaluations.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)
- Molecular formula (if other than submission substance): (CH3)2NCH2CH2OCH2CH2N(CH3)2
- Molecular weight (if other than submission substance): 160.26
- Substance type: Pure active substance
- Physical state: Light yellow liquid, slight amine odor
- Analytical purity: 97%
- Other: Complete solubility in water
- Lot/batch No.: S-0044117, reference no. 24VCB-65 A and B, BRCC (Sample numbers 51-486 and 51-487)
- Impurities (identity and concentrations): Unknown component (prestudy) 1.263% in sample 24VCB65A and 1.199% in sample 24VCB65B; unnknown component (post study) 0.901%.
- Composition of test material, percentage of components: Composition of test material, percentage of components: (prestudy analysis of sample 24VCB65A): dimethylamine 0.015%, N-methylmorpholine (NMM) 0.014%, dimethylethanolamine (DMEA) 0.492%, bis(2-dimethylaminoethyl)ether (A-99) 97.026%, N-methylaminoethoxy-N,N-dimethylethylamine 0.082%, 2-(2-dimethylaminoethoxy)ethanol (DMEE) 1.108%, unknowns 1.263%. (poststudy analysis of sample 24VCB65B): dimethylamine 0.017%, N-methylmorpholine (NMM) 0.014%, dimethylethanolamine (DMEA) 0.483%, bis(2-dimethylaminoethyl)ether (A-99) 97.114%, N-methylaminoethoxy-N,N-dimethylethylamine 0.084%, 2-(2-dimethylaminoethoxy)ethanol (DMEE) 1.089%, unknowns 1.199%.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc. (Frederick, MD)
- Age at study initiation: 35 days
- Weight at study initiation: Males in exposure groups 0, 0.22, 1.25, and 5.8 ppm had mean weights of 255.1, 254.1, 255.1, and 254.3 g, respectively. Females in exposure groups 0, 0.22, 1.25, and 5.8 ppm had mean weights of 169.6, 169.5, 167.2, and 168.5 g, respectively.
- Fasting period before study: Food and water withheld during exposure
- Housing: Housed two per cage in stainless steel, wire-mesh cages, 23.5 cm x 20 cm x 18 cm for approximately two weeks (acclimation period) and individually. The recovery animals were individually housed in the same size cages in a Relocatable Containment Systeme Unit housed during the 14-week exposure regimen. During the exposures, the animals were individually housed, separated by sex and exposure group, in stainless steel, wire-mesh cages (35 cm x 17 cm x 18 cm).
- Diet (e.g. ad libitum): Powdered certified Agway Prolab Animal Diet Rat, Mouse, Hamster 3200 (Agway Inc.); ad libitum
- Water (e.g. ad libitum): Water (Municipal Authority of Westmoreland County, Greensburg, PA) provided by an automatic watering system was available ad libitum, except during the water consumption measurement period.
- Acclimation period: Two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Mean chamber temperature: 22-25 deg C. General conditions in Evaporator: 0.22 ppm exposure group (top): 46, 50, 55 deg C (bottom): 44, 33, 30 deg C; 1.25 ppm exposure group (top): 41, 45, 50 deg C (bottom): 33, 36, 40; 5.80 ppm exposure group (top): 68, 51, 49 deg C (bottom): 48, 39, 39 deg C. Housing conditions: 18-22 deg C.
- Humidity (%): Mean chamber relative humidity: 36-57%, Housing conditions: 41-62%.
- Air changes (per hr): 21 air changes per hr
- Photoperiod (hrs dark / hrs light): 12-hr photoperiod

IN-LIFE DATES: From: 12/5/1988 To: 3/9-10/1989 (necropsy at 14 wks) or 4/21/1989 (group with additional 6 wks of recovery)

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: N/A

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel with glass windows, approximately 4320 liters
- Method of holding animals in test chamber: Whole body exposure
- Source and rate of air: N/A
- Method of conditioning air: N/A
- System of generating particulates/aerosols: Liquid test substance was metered with a Sage Model 355 syringe pump into a heated glass evaporator. The temperature in the evaporator was maintained at the lowest level sufficient to vaporize the liquid. The resultant vapor was carried into the chamber by a countercurrent air stream that entered the bottom of the evaporator.
- Temperature, humidity, pressure in air chamber: Evaporator temperatures ranged from 42 to 66 deg C.
- Air flow rate: approximately 1500 liters/minute
- Air change rate: 21 air changes per hour
- Method of particle size determination: N/A
- Treatment of exhaust air: N/A

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations of the test substance were analyzed approximately once every 30 to 120 minutes (depending on the target concentration) by gas chromatography of impinger solutions containing samples of the chamber atmosphere.
- Samples taken from breathing zone: No

VEHICLE (if applicable)
- Justification for use and choice of vehicle: N/A
- Composition of vehicle: N/A
- Type and concentration of dispersant aid (if powder): N/A
- Concentration of test material in vehicle: N/A
- Lot/batch no. of vehicle (if required): N/A
- Purity of vehicle: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of the test substance were analyzed approximately once every 30 to 120 minutes (depending on the target concentration) by gas chromatography of impinger solutions containing samples of the chamber atmosphere.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

1.51 mg/m³ air (nominal)

Remarks:

0.22 ppm

Dose / conc.:

8.2 mg/m³ air (nominal)

Remarks:

1.25 ppm

Dose / conc.:

38 mg/m³ air (nominal)

Remarks:

5.8 ppm

No. of animals per sex per dose:

15/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale: A series of acute inhalation exposures with the test substance were conducted at the BRRC Laboratory. When the test substance was generated dynamically (heated evaporator or bubbler method), the 6-hour LC50 value (with 95% confidence limits) for male and female Sprague Dawley rats was 166 (155 to 178) ppm. Clinical signs of ocular and respiratory irritation were observed in all exposure groups (68, 149, 200, and 248 ppm). The principal gross lesions observed at necropsy were dark red discoloration of the lungs, and encrustation around the eyes and nose. Rats exposed for 6 hours to test substance vapor generated statically survived the 14-day postexposure period. However, under static generation conditions, the test substance chamber concentration was only 24 ppm because the high chamber relative humidity (82%) lowered the vapor concentration.
- Rationale for animal assignment (if not random): Computer-based randomization program. At the time of group assignment, only animals with body weights within two standard deviations of the mean for each sex were used in the study. Any animal observed to be in poor health was rejected from group assignment.
- Rationale for selecting satellite groups: Ten rats per sex from each group were sacrificed after the exposure regimen; the remaining 5 rats per sex were sacrificed after a 6-week recovery period. A further 4 male and 4 female rats were added to the control and 5.8 ppm groups and assigned for perfusion-fixation sacrifice.
- Post-exposure recovery period in satellite groups: 6 weeks
- Section schedule rationale (if not random): Computer-based randomization program

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before and after exposure on an individual basis, during exposure on a group basis.
- Cage side observations checked: Overt clinical signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight collection and just preceding sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to initiation of the first exposure, also weighed on the morning preceding the second day (day 2), and days 5, 8, 9 and 12 (prior to sacrifice). Control animals were weighed on the morning preceding the third (Day 3) exposure due to unexpected weight losses in all groups on the morning preceding the second exposure. Animals designated for the 21-day recovery were weighed once each week during the
recovery period (Days 19, 26, and 33).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; Food consumption was measured for approximately 15 hours following 8 exposures for the male rats and following 9 exposures for the female rats, while in metabolism cages.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was measured for approximately 15 hours following 8 exposures for the male rats and following 9 exposures for the female rats, while in metabolism cages.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the first exposure, the eyes were examined by a veterinarian using an external light source and a magnifying lens. During the second week of the exposure regimen, eyes were examined immediately after the ninth (and final) exposure (all groups) and were examined again the following morning (high and intermediate test and control groups). Recovery group animals were also examined 2 weeks following the last exposure.
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes, food was removed at the start of blood collection.
- How many animals: All animals
- Parameters checked: erythrocyte count, platelet count, hemoglobin, leukocyte count, hematocrit, differential leukocyte count, mean corpuscular volume, reticulocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the time of scheduled sacrifice.
- Animals fasted: Yes, food was removed at the start of blood collection.
- How many animals: all animals
- Parameters checked: glucose, calcium, urea nitrogen, phosphorus, creatinine, sodium, total protein, potassium, albumin, chloride, globulin, total bilirubin, direct bilirubin, indirect bilirubin, aspartate aminotransferase, alanine aminotransferase, creatine kinase, lactic dehydrogenase, gamma-glutamyl transferase, sorbitol dehydrogenase, alkaline phosphatase.

URINALYSIS: Yes
- Time schedule for collection of urine: Approximately 15 hours following each exposure.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, food was removed at the start of blood collection.
- Parameters checked: Volume, color, turbidity, osmolality, microscopic constituents, pH, protein (quantitative), protein, creatinine, glucose, sodium,
ketone, potassium, bilirubin, chloride, blood, creatinine, clearance urobilinogen (estimate).


NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A
- Battery of functions tested: sensory activity / grip strength / motor activity / other: N/A

OTHER: Animals were sacrificed following the 6-hour exposure regimen on exposure day 1, 3, 5 or 10 (l/sex/group/sacrifice) and used for electron microscopic examination of nasal mucosa.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; all animals except those assigned to the recovery group were sacrificed the morning of the day following the final exposure. The remaining rats were sacrificed 3 weeks later. A complete necropsy was performed on each animal and all tissues were fixed in 10% neutral buffered formalin or Bouin's fixative for histologic evaluations. Five rats/sex from the control and high concentration groups were perfused with formaldehyde:glutaraldehyde solution for fixation of the lungs, liver, coronary artery, skin (dorsum of head and ear), spleen, and cornea for possible examination by electron microscopy.

HISTOPATHOLOGY: Yes; tissues fixed for microscopic examination included: brain, nasal turbinates, 1arynx, trachea, lungs, eyes, liver, kidneys, adrenals, spleen, testes, ovaries, lymph nodes (submandibular), heart, thymus, skin (dorsum of head, ears, and eyelids), gross lesions.

Other examinations:

The brain, liver, kidneys, lungs, spleen, and heart from all animals and the testes from all males were weighed at necropsy. Organ weights were recorded as absolute weights and as a percentage of both body and brain weights.

Statistics:

Results of quantitative continuous variables (e.g., body weight change) were intercompared for the three exposure groups and one control group by use of Bartlett's test for homogeneity of variances, analysis of variance (ANOVA), and Duncan's multiple range test. The latter was used, if F for the ANOVA was significant, to delineate which groups differed from the control. If Bartlett's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, if necessary, by t-tests. A two-sample t-test was used to compare the control group to the high concentration exposure group for the recovery animals. The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs of ocular and respiratory irritation were observed during the 14-week exposure and 6-week recovery periods and included swollen periocular tissue in rats from all exposure groups and periocular and perinasal encrustation in rats from the 5.8 ppm group. Periocular encrustation was also observed in males from the 1.25 ppm group. An additional sign observed primarily in animals from the 5.8 ppm group was cloudiness in the eyes. Detailed ophthalmic examination revealed keratitis in males from the 5.8 ppm group and females from the 1.25 and 5.8 ppm groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortalities occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight losses were observed for both sexes from the 5.8 ppm group at the end of the first week of exposure. Body weight gain in males from the 5.8 ppm group was statistically significantly lower throughout the 14-week exposure period; the percent decreased in body weight gain at week 14 was 18%. Body weight gain in females from the 5.8 ppm group was slightly reduced during the 14-weeek study period, although not statistically significant; the percent decrease in body weight gain at week 14 was 9%. Absolute body weight in the males from the 5.8 ppm group was slightly lower throughout the study; however, a statistical difference was achieved only at week 14. The percent decrease in body weight at week 14 was 6.5%. No statistically significant differences in absolute body weight were observed in females from the 5.8 ppm group. Also, no effects on body weight were observed in males and females from the 0.23 and 1.25 ppm groups during the study.
During the 6-week recovery period, body weight gain for both sexes from the 5.8 ppm group indicated some recovery (body weight gain was 14.5 and 5.2% lower than the controls at the end of the recovery period for males and female animals, respectively. No statistical differences were observed in body weight for the 5.8 ppm group during the recovery period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significant decrease in food consumption was observed in females from the 5.8 ppm group at the end of the 14-week exposure regimen; the percent decrease in fool consumption was 18%. No statistically significant differences in food consumption were observed in males of any exposure group.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in water consumption was observed in males from the 5.8 group at the end of the exposure regimen; the percent increase in water consumption was 21%. No other differences in water consumption were observed in males and females from any exposure group.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

During week 13, keratitis (mostly in mild degree) was observed in all rats from the 5.8 ppm group. In addition, minimal keratitis was observed in 6 females from the 1.25 ppm group.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum chemistry revealed no consistent or concentration-related effects.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis results showed slight decreases in creatinine, sodium, potassium, and chloride in both sexes from the 5.8 ppm group at 14 weeks.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Exposure-related increases in relative organ weights were observed for the adrenals and testes of males from the 5.8 ppm group at the 14-week time point, but not after the 6-week recovery period. These organs showed no histologic abnormalities.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, color changes of the eyes (5.8 ppm group at the 14-week sacrifice only) and swollen periocular tissue (1.25 and/or 5.8 ppm groups at both 14- and 20-week sacrifices) were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

Exposure-related microscopic lesions were seen only in tissues receiving direct contact with the chemical (exposed skin, eyes, ears, and the upper respiratory tract) and consisted of cytoplasmic vacuolation of the exposed epithelium, accompanied by varying degrees of necrosis, inflammation, and degeneration of superficial and deeper tissues. Lesions involving the nasal cavities were present in rats of both sexes from all exposure groups at both 14- and 20-week sacrifices, while lesions involving the other tissues were generally observed only in the 5.8 ppm group at the 14-week sacrifice. Electron microscopic evaluations of the anterior nasal turbinates of the 5.8 ppm group rats showed lesions consisting of intracytoplasmic membrane-bound vacuoles in the mucosal epithelium after one exposure, and in the mucosa and submucosa after 3 and 5 exposures (this evaluation was not performed after 10 exposures). The vacuoles appear to be derived from the coalescence of many small secretory vesicles produced by the Golgi apparatus.

Other effects:

not specified

Key result

Dose descriptor:

LOAEC

Remarks:

Local Toxicity

Effect level:

<= 1.51 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Various signs of irritation of the eye and respiratory tract at all concentrations were observed.

Key result

Dose descriptor:

NOAEC

Remarks:

Systemic Toxicity

Effect level:

8.2 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Minor, nonspecific systemic toxicity at 38 mg/m3 and no target organ toxicity.

Critical effects observed:

no

Conclusions:

Repeated exposure of rats to the test substance vapor at 0.23, 1.25, and 5.8 ppm for 14 weeks produced no mortality, indications of possible minor, nonspecific systemic toxicity at 5.8 ppm and no target organ toxicity, but various signs of irritation of the eye and respiratory tract at all concentrations was observed. Thus, a NOEL could not be determined for irritancy under the conditions of this study. However, a NOAEC for systemic effects is considered to be 1.25 ppm (8.2 mg/m3).

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Following the read across strategy, this endpoint is covered by a key study performed with the structural analogue BDMAEE (N, N, N', N'-tetramethyl-2,2'-oxybis(ethylamine)). The justification for read across is attached in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

LOAEC

Remarks:

Local Toxicity

Effect level:

<= 1.51 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Various signs of irritation of the eye and respiratory tract at all concentrations were observed.

Key result

Dose descriptor:

NOAEC

Remarks:

Systemic Toxicity

Effect level:

8.2 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Minor, nonspecific toxicity at 38 mg/m3 and no target toxicity

Key result

Critical effects observed:

no

Conclusions:

No reliable data for this endpoint is available with the test substance BAEE. Data generated with the analogue substance BDMAEE is used for endpoint coverage.
Repeated exposure of rats to the test substance vapor at 0.23, 1.25, and 5.8 ppm for 14 weeks produced no mortality, indications of possible minor, nonspecific systemic toxicity at 5.8 ppm and no target organ toxicity, but various signs of irritation of the eye and respiratory tract at all concentrations was observed. Thus, a NOEL could not be determined for irritancy under the conditions of this study. However, a NOAEC for systemic effects is considered to be 1.25 ppm (8.2 mg/m3). The same is assumed for the test substance BAEE.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

1.51 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982-08-25 - 1984-11-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP study with methods similar to OECD 411 conducted according to internal laboratory methods.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)
- Substance type: Pure active substance
- Physical state: liquid, yellow/transparent
- Analytical purity: 99.6%
- Lot/batch No.: Reference no. 51DKE115 / Sample no. 45-190

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rabbits (70 of each sex) received from Langshaw Farms, Augusta, MI.
- Age at study initiation: 12-13 wks
- Weight at study initiation: 2-3 kg
- Fasting period before study:
- Housing: Individually housed in stainless steel cages with wire floors
- Diet (e.g. ad libitum): Oregon State University diet provided by Langshaw Farms. For the first 5 days, each rabbit received 6 oz of diet; the next 5 days 7 oz received; the next 5 days 8 oz received. After study initiation food was available ad libitum.
- Water (e.g. ad libitum): Water (Municipal Authority of Westmoreland County, Greensburg, PA), ad libitum to all animals.
- Acclimation period: Approximately two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.8-21.1 deg C
- Humidity (%): 40-60%
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): N/A

IN-LIFE DATES: From: 1982-08-25 To: 1982-09-24

Type of coverage:

occlusive

Vehicle:

other: Deionized water

Details on exposure:

TEST SITE
- Area of exposure: Dorsal area of trunk
- % coverage: 25-30% of body area
- Type of wrap if used: 4-inch square, 8-ply gauze patch held together on the back by using strips of two sided tape.
- Time intervals for shavings or clipplings: Fur was clipped shortly before study initiation and trimmed as necessary during the study.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dose was wipped away witha gauze patch dampened with warm water and then blotted with a disposable wiper.
- Time after start of exposure: Six hours after dosing

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1.0 mL
- Concentration (if solution): 2.0%, 0.7%, 0.2%
- Constant volume or concentration used: yes, constant volume for several dose concentration groups.
- For solids, paste formed: no

VEHICLE
- Justification for use and choice of vehicle (if other than water): dieoniced water
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, certain rabbits because of their tendency to remove the sheeting and gauze patches, had extra tape placed over the polyethylene sheeting or wore Elizabethan collar during exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of all concentrations and deionized water were analyzed on a monthly basis.

Duration of treatment / exposure:

13 weeks (90 days)

Frequency of treatment:

6 hrs/day, 5 days/wk

Dose / conc.:

2 other: % (nominal per unit area)

Remarks:

1.98 % analytical; male/female

Dose / conc.:

0.7 other: % (nominal per unit area)

Remarks:

0.75% analytical; male/female

Dose / conc.:

0.2 other: % (nominal per unit area)

Remarks:

0.25% analytical; male/female

Dose / conc.:

0 other: %

No. of animals per sex per dose:

10/sex/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The exposure concentration levels for this 90-day dermal study were based on the results of a 9-day study where the test substance was given as aqueous dilutions in deionized water using constant volumes, and a water control group was used. Each rabbit received 1 mL/day of the dilutions. The concentrations of the test substance were 2.0% (8 mg/kg/day nominal), 0.7% (2.8 mg/kg/day nominal), and 0.2% (0.74 mg/kg/day nominal). All percentages were calculated on a weight/ weight basis. Water was chosen as the diluent and negative control substance because the test substance is soluble in water at the concentrations used. Since the lowest concentration in the 9-day study, 2.5% produced local and systemic toxcity, the upper concentration chosen for the subchronic study was below that concentration.
- Rationale for animal assignment (if not random): By a formal randomization system
- Rationale for selecting satellite groups: High dose and control groups for male and female rabbits were used to assess the reversibility of any induced toxic effects.
- Post-exposure recovery period in satellite groups: one month
- Section schedule rationale (if not random): At random

Positive control:

N/A

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (on weekdays)
- Cage side observations checked: Toxicologic and pharmaccologic signs and dermal irritation; mortality and morbidity only (weekends)

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule: N/A

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Immediately prior to dosing

BODY WEIGHT: Yes
- Time schedule for examinations: First day of exposure, weekly thereafter and preceding sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/week: Yes; measured weekly for males and twice weekly for females, after week 1. food consumption was not measured during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
- Time schedule for examinations: N/A

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to study initiation and again before the 90-day sacrifice. Blood was also taken from the male rabbits prior the the recovery sacrificed, but not remale rabbits.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All animals
- Parameters checked: Hematocrit, hemoglobin, erythrocyte count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total leukocyte count, differential leukocyte count, platelet count and reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to study initiation and again before the 90-day sacrifice. Blood was also taken from the male rabbits prior the the recovery sacrificed, but not remale rabbits.
- Animals fasted: No data
- How many animals: All animals
- Parameters checked: Glucose, creatinine, bilirubin (total), serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, albumin, total protein, globulin, gamma-glutamyl transpeptidase, creatine phosphokinase, calcium, sodium, potassium, chloride, total CO2 and phosphorus.

URINALYSIS: Yes
- Time schedule for collection of urine: For non-recovery male rabbits urine was collected during weeks 6, 10, and 13. Urine was collected from recovery male and female rabbits during week 12. Urine was collected from non-recovery female rabbits during weeks 5, 9, and 13. Urine was not collected from males or females during the recovery period or prior to the recovery sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: Volume, specific gravity, total protein, glucose, ketones, bilirubin, blood, urobilinogen, and amount and nature of sediment.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A
- Battery of functions tested: sensory activity / grip strength / motor activity / other: N/A

OTHER: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, upon necropsy the following tissues were examined grossly and fixed in commercially prepared 10% neutral buffered formalin from all dosage groups and control groups: All lesions, Brain - 3 coronal sections (cerebrum, cerebellum, brain stem), Spinal cord* - 3 levels, Sciatic nerve, Eyes*, Pituitary, Salivary glands (submaxillary), Heart and aorta, Thymus, Thyroid (with parathyroids), Trachea, Lungs with mainstem bronchi, Esophagus
Stomach, Small and large intestine, Adrenals, Pancreas, Liver and gall bladder, Kidneys, Testes and epididymis (males), Prostate, Accessory sex glands, Uterus and ovaries (females), Mammary gland*, Urinary bladder, Spleen, Mediastinal or mesenteric lymph node, Sternum* with marrow, Gastrocnemius muscle, Any other target tissue, and Treated and untreated skin.
* Tissues with asterisk were examined microscopically only if indicated by other findings.

HISTOPATHOLOGY: Yes, Histopathological examination was limited to all tissues from the high dose and control groups and target tissues from the other groups (treated skin only).

Other examinations:

ORGAN WEIGHTS: At the 90-day sacrifice, weights were recorded for the liver, kidneys, brain, testes, and adrenals of all animals. Based on the evaluation of these weights, no organ weights were recorded at the recovery sacrifice.

Statistics:

For body weight changes, food consumption values, and organ weights, both absolute and relative to final body weight, group means were compared by use of Bartlett’s test for homogeneity of variances, and when Bartlett’s test showed homogeneity, a standard analysis of variance. If F for analysis of variance was significant, Duncan’s multiple range test was used to delineate which groups differed from the controls. If Bartlett’s test showed heterogeneity, Levene’s test for equal variances was used, and if variances were equal, a standard analysis of variance was performed. Then the analysis of variance test showed a signification F value, polled variance t-tests were used. When levene’s test showed unequal variances, means were compared by Welch and Brown-Forsythe analysis of variance test for groups with unequal variances. When either of these tests showed significance, separate variance t-tests were used to differentiate between groups.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

-The incidence of erythema, edema, desquamation and fissuring were directly related to the concentration of the test substance, which is reflected in the similar pattern of Draize scores. Open sores at the application site occurred primarily at the highest concentration.
- The signs of skin irritation subsided gradually after termination of treatment and in most cases had disappeared by the time of sacrifice of the recovery animals.

Dermal irritation:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Description (incidence):

- There were no deaths or signs of systemic toxicity among the male rabbits of any dose group or controls. Skin irritation in males was observed including: erythema, desquamation, edema and fissuring or cracking of the skin occurring in a dose-related pattern.
- One female rabbit was sacrificed moribund after 12 days on study and a second female was found dead after 20 days. All other female rabbits survived until schedule sacrifice. Clinical signs included erythema, edema, desquamation, and some fissuring.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- There were no treatment-related effects on body weight.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- There were no treatment-related effects on food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- There was a statistically significant increase in absolute but not relative testicular weight at the 0.7% concentration. Since there was no significant effect at the highest concentration, the observed difference was not considered biologically significant. No other significant differences were observed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- There were no biologically significant effects with respect to clinical pathology. Occasional statistically significant differences were considered not biologically important because they were not dose-related or because the differences occurred with respect to only one of the two control groups.
- No biologically significant gross lesions were observed. The only treatment-related lesions were found on the treated skin. Epidermal vacuolization occurred in 10 males and 10 females at the highest concentration (2%) of the test substance, and in 2 males and 7 females at 0.7% of the test substance. No lesions were observed in the control groups. Acanthosis occurred at approximately the same incidence in all the male dosage groups, but was more pronounced at the highest concentration of the test substance. Among the females the condition was twice as prevalent at the two highest concentrations of the test substance as in the controls. In addition, the lesion was generally more severe in the high dose animals than in the controls or lower dosage groups. The incidence of dermatitis was increased in females at all concentrations of the test substance, but the incidence among the treated males was not increased.
- No other treatment-related lesions were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

>= 8 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: There was an absence of any indication of systemic toxicity at all dose levels. All dose levels tested resulted in varying levels of irritation at the exposure site.

Critical effects observed:

no

Conclusions:

Daily recurrent application of the test substance to intact skin of rabbits, over a period of 90 days at concentrations of 2.0, 0.7, and 0.2% produced local dermal inflammation (including: erythema, edema, desquamation, fissuring, and by microscopic examination epidermal vacuolization and acanthosis were observed) but no signs of toxicity.

Reference 2

Endpoint:

sub-chronic toxicity: dermal

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP study with methods similar to OECD 411 conducted according to internal laboratory methods.

Justification for type of information:

Following the read across strategy, this endpoint is covered by a key study performed with the structural analogue BDMAEE (N, N, N', N'-tetramethyl-2,2'-oxybis(ethylamine)). The justification for read across is attached in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

>= 8 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: There was an absence of any indication of systemic toxicity at all dose levels. All dose levels tested resulted in varying levels of irritation at the exposure site.

Key result

Critical effects observed:

no

Conclusions:

No reliable data for this endpoint is available for the test substance. Data generated with the analogue substance BDMAEE is used for endpoint coverage.
Daily recurrent application of the test substance to intact skin of rabbits, over a period of 90 days at concentrations at 2.0, 0.7, and 0.2% produced local dermal inflammation (including: erythema, edema, desquamation, fissuring, and by microscopic examination epidermal vacuolization and acanthosis were observed) but no signs of toxicity. The same is assumed for the test substance BAEE.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982-08-25 - 1984-11-05

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP study with methods similar to OECD 411 conducted according to internal laboratory methods.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N,N',N'-tetramethyl-2,2'-oxybis(ethylamine)
- Substance type: Pure active substance
- Physical state: liquid, yellow/transparent
- Analytical purity: 99.6%
- Lot/batch No.: Reference no. 51DKE115 / Sample no. 45-190

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rabbits (70 of each sex) received from Langshaw Farms, Augusta, MI.
- Age at study initiation: 12-13 wks
- Weight at study initiation: 2-3 kg
- Fasting period before study:
- Housing: Individually housed in stainless steel cages with wire floors
- Diet (e.g. ad libitum): Oregon State University diet provided by Langshaw Farms. For the first 5 days, each rabbit received 6 oz of diet; the next 5 days 7 oz received; the next 5 days 8 oz received. After study initiation food was available ad libitum.
- Water (e.g. ad libitum): Water (Municipal Authority of Westmoreland County, Greensburg, PA), ad libitum to all animals.
- Acclimation period: Approximately two weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.8-21.1 deg C
- Humidity (%): 40-60%
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): N/A

IN-LIFE DATES: From: 1982-08-25 To: 1982-09-24

Type of coverage:

occlusive

Vehicle:

other: Deionized water

Details on exposure:

TEST SITE
- Area of exposure: Dorsal area of trunk
- % coverage: 25-30% of body area
- Type of wrap if used: 4-inch square, 8-ply gauze patch held together on the back by using strips of two sided tape.
- Time intervals for shavings or clipplings: Fur was clipped shortly before study initiation and trimmed as necessary during the study.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dose was wipped away witha gauze patch dampened with warm water and then blotted with a disposable wiper.
- Time after start of exposure: Six hours after dosing

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1.0 mL
- Concentration (if solution): 2.0%, 0.7%, 0.2%
- Constant volume or concentration used: yes, constant volume for several dose concentration groups.
- For solids, paste formed: no

VEHICLE
- Justification for use and choice of vehicle (if other than water): dieoniced water
- Amount(s) applied (volume or weight with unit): N/A
- Concentration (if solution): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A

USE OF RESTRAINERS FOR PREVENTING INGESTION: Yes, certain rabbits because of their tendency to remove the sheeting and gauze patches, had extra tape placed over the polyethylene sheeting or wore Elizabethan collar during exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of all concentrations and deionized water were analyzed on a monthly basis.

Duration of treatment / exposure:

13 weeks (90 days)

Frequency of treatment:

6 hrs/day, 5 days/wk

Dose / conc.:

2 other: % (nominal per unit area)

Remarks:

1.98 % analytical; male/female

Dose / conc.:

0.7 other: % (nominal per unit area)

Remarks:

0.75% analytical; male/female

Dose / conc.:

0.2 other: % (nominal per unit area)

Remarks:

0.25% analytical; male/female

Dose / conc.:

0 other: %

No. of animals per sex per dose:

10/sex/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The exposure concentration levels for this 90-day dermal study were based on the results of a 9-day study where the test substance was given as aqueous dilutions in deionized water using constant volumes, and a water control group was used. Each rabbit received 1 mL/day of the dilutions. The concentrations of the test substance were 2.0% (8 mg/kg/day nominal), 0.7% (2.8 mg/kg/day nominal), and 0.2% (0.74 mg/kg/day nominal). All percentages were calculated on a weight/ weight basis. Water was chosen as the diluent and negative control substance because the test substance is soluble in water at the concentrations used. Since the lowest concentration in the 9-day study, 2.5% produced local and systemic toxcity, the upper concentration chosen for the subchronic study was below that concentration.
- Rationale for animal assignment (if not random): By a formal randomization system
- Rationale for selecting satellite groups: High dose and control groups for male and female rabbits were used to assess the reversibility of any induced toxic effects.
- Post-exposure recovery period in satellite groups: one month
- Section schedule rationale (if not random): At random

Positive control:

N/A

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily (on weekdays)
- Cage side observations checked: Toxicologic and pharmaccologic signs and dermal irritation; mortality and morbidity only (weekends)

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule: N/A

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Immediately prior to dosing

BODY WEIGHT: Yes
- Time schedule for examinations: First day of exposure, weekly thereafter and preceding sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/week: Yes; measured weekly for males and twice weekly for females, after week 1. food consumption was not measured during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No
- Time schedule for examinations: N/A

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to study initiation and again before the 90-day sacrifice. Blood was also taken from the male rabbits prior the the recovery sacrificed, but not remale rabbits.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All animals
- Parameters checked: Hematocrit, hemoglobin, erythrocyte count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, total leukocyte count, differential leukocyte count, platelet count and reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to study initiation and again before the 90-day sacrifice. Blood was also taken from the male rabbits prior the the recovery sacrificed, but not remale rabbits.
- Animals fasted: No data
- How many animals: All animals
- Parameters checked: Glucose, creatinine, bilirubin (total), serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, alkaline phosphatase, albumin, total protein, globulin, gamma-glutamyl transpeptidase, creatine phosphokinase, calcium, sodium, potassium, chloride, total CO2 and phosphorus.

URINALYSIS: Yes
- Time schedule for collection of urine: For non-recovery male rabbits urine was collected during weeks 6, 10, and 13. Urine was collected from recovery male and female rabbits during week 12. Urine was collected from non-recovery female rabbits during weeks 5, 9, and 13. Urine was not collected from males or females during the recovery period or prior to the recovery sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: Volume, specific gravity, total protein, glucose, ketones, bilirubin, blood, urobilinogen, and amount and nature of sediment.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: N/A
- Dose groups that were examined: N/A
- Battery of functions tested: sensory activity / grip strength / motor activity / other: N/A

OTHER: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, upon necropsy the following tissues were examined grossly and fixed in commercially prepared 10% neutral buffered formalin from all dosage groups and control groups: All lesions, Brain - 3 coronal sections (cerebrum, cerebellum, brain stem), Spinal cord* - 3 levels, Sciatic nerve, Eyes*, Pituitary, Salivary glands (submaxillary), Heart and aorta, Thymus, Thyroid (with parathyroids), Trachea, Lungs with mainstem bronchi, Esophagus
Stomach, Small and large intestine, Adrenals, Pancreas, Liver and gall bladder, Kidneys, Testes and epididymis (males), Prostate, Accessory sex glands, Uterus and ovaries (females), Mammary gland*, Urinary bladder, Spleen, Mediastinal or mesenteric lymph node, Sternum* with marrow, Gastrocnemius muscle, Any other target tissue, and Treated and untreated skin.
* Tissues with asterisk were examined microscopically only if indicated by other findings.

HISTOPATHOLOGY: Yes, Histopathological examination was limited to all tissues from the high dose and control groups and target tissues from the other groups (treated skin only).

Other examinations:

ORGAN WEIGHTS: At the 90-day sacrifice, weights were recorded for the liver, kidneys, brain, testes, and adrenals of all animals. Based on the evaluation of these weights, no organ weights were recorded at the recovery sacrifice.

Statistics:

For body weight changes, food consumption values, and organ weights, both absolute and relative to final body weight, group means were compared by use of Bartlett’s test for homogeneity of variances, and when Bartlett’s test showed homogeneity, a standard analysis of variance. If F for analysis of variance was significant, Duncan’s multiple range test was used to delineate which groups differed from the controls. If Bartlett’s test showed heterogeneity, Levene’s test for equal variances was used, and if variances were equal, a standard analysis of variance was performed. Then the analysis of variance test showed a signification F value, polled variance t-tests were used. When levene’s test showed unequal variances, means were compared by Welch and Brown-Forsythe analysis of variance test for groups with unequal variances. When either of these tests showed significance, separate variance t-tests were used to differentiate between groups.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

-The incidence of erythema, edema, desquamation and fissuring were directly related to the concentration of the test substance, which is reflected in the similar pattern of Draize scores. Open sores at the application site occurred primarily at the highest concentration.
- The signs of skin irritation subsided gradually after termination of treatment and in most cases had disappeared by the time of sacrifice of the recovery animals.

Dermal irritation:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Description (incidence):

- There were no deaths or signs of systemic toxicity among the male rabbits of any dose group or controls. Skin irritation in males was observed including: erythema, desquamation, edema and fissuring or cracking of the skin occurring in a dose-related pattern.
- One female rabbit was sacrificed moribund after 12 days on study and a second female was found dead after 20 days. All other female rabbits survived until schedule sacrifice. Clinical signs included erythema, edema, desquamation, and some fissuring.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- There were no treatment-related effects on body weight.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- There were no treatment-related effects on food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- There was a statistically significant increase in absolute but not relative testicular weight at the 0.7% concentration. Since there was no significant effect at the highest concentration, the observed difference was not considered biologically significant. No other significant differences were observed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- There were no biologically significant effects with respect to clinical pathology. Occasional statistically significant differences were considered not biologically important because they were not dose-related or because the differences occurred with respect to only one of the two control groups.
- No biologically significant gross lesions were observed. The only treatment-related lesions were found on the treated skin. Epidermal vacuolization occurred in 10 males and 10 females at the highest concentration (2%) of the test substance, and in 2 males and 7 females at 0.7% of the test substance. No lesions were observed in the control groups. Acanthosis occurred at approximately the same incidence in all the male dosage groups, but was more pronounced at the highest concentration of the test substance. Among the females the condition was twice as prevalent at the two highest concentrations of the test substance as in the controls. In addition, the lesion was generally more severe in the high dose animals than in the controls or lower dosage groups. The incidence of dermatitis was increased in females at all concentrations of the test substance, but the incidence among the treated males was not increased.
- No other treatment-related lesions were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

>= 8 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: There was an absence of any indication of systemic toxicity at all dose levels. All dose levels tested resulted in varying levels of irritation at the exposure site.

Critical effects observed:

no

Conclusions:

Daily recurrent application of the test substance to intact skin of rabbits, over a period of 90 days at concentrations of 2.0, 0.7, and 0.2% produced local dermal inflammation (including: erythema, edema, desquamation, fissuring, and by microscopic examination epidermal vacuolization and acanthosis were observed) but no signs of toxicity.