Reaction mass of 2,2'-(ethylenedioxy)diethanol and 3,6,9,12,15-pentaoxaheptadecane-1,17-diol and 3,6,9,12-tetraoxatetradecane-1,14-d

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

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

Adequacy of study:

supporting study

Study period:

prior to or in 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP/Guideline study

Justification for type of information:

Read across is based on the category approach. Please refer to attached category document.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tetraethylene Glycol

IUPAC Name:

Tetraethylene Glycol

Constituent 2

Reference substance name:

3,6,9-trioxaundecane-1,11-diol

EC Number:

203-989-9

EC Name:

3,6,9-trioxaundecane-1,11-diol

Cas Number:

112-60-7

Molecular formula:

C8H18O5

IUPAC Name:

2,2'-[oxybis(ethane-2,1-diyloxy)]diethanol

Test material form:

not specified

Details on test material:

A 20% solution of DBNPA (Lot#: MM890624) in tetraethylene glycol (TEG from V-310 & V-311) was supplied with analyses by the Specialty Chemicals Department, The Dow Chemical Company. The composition of the solution by nuclear magnetic resonance analysis was as follows:
2,2-Dibromo-3-nitrilopropionamide 20.2 +/- 1.0%
Tetraethylene glycol (TEG) 64 +/- 2%
Water 16.0 +/- 0.7%
The composition of the DBNPA solution by reversed-phase high pressure liquid chromatography analysis was 18.9 +/- 0.5 wt% DBNPA

Tetraethylene glycol was used as the vehicle control at a dose of 3 ml/kg

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Fischer 344 rats (approximately six weeks of age) were purchased from Charles River Breeding Laboratories, Inc., Kingston, New York. Extra animals were ordered to ensure that a sufficient number of animals of acceptable health and weight were available to conduct the study as designed. This strain of rat was selected because of its general acceptance and suitability for toxicity testing, the availability of historical data and the reliability of the commercial supplier.

Upon arrival at the laboratory, the rats were examined for health status by the laboratory veterinarian and acclimated to the laboratory environment according to the Standard Operating Procedures of the Subchronic/Chronic Toxicity Section. During acclimation the rats were weighed and randomly assigned one/cage into exposure groups using a computer-generated randomization procedure based on body weights. Identification was accomplished by inserting a unique alphanumeric metal tag in one ear of each rat.

The animal rooms of the testing facility were designed to maintain adequate environmental conditions concerning temperature, humidity, and photocycle and were regulated for the species tested. A basal diet of Purina Certified Rodent Chow #5002 (Ralston Purina Co., St. Louis, MO) and tap water was available ad libitum during the prestudy and study periods. The feed was analyzed by the Ralston Purina Co. to confirm that it was nutritionally adequate and to quantitate the levels of selected contaminants associated with the formulation process. Water analysis was performed according to the Standard Operating Procedures of The Toxicology Research Laboratory.

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

other: tetraethylene glycol

Details on exposure:

The test solution was applied as received (undiluted) at a volume of approximately 0.4,1.2 or 4.0 ml/kg body weight/day. The vehicle control was applied at a rate of 3.0 ml/kg/day. Dose volumes were determined weekly and were based on the most recent individual animal body weight.

All rats were acclimated to an elastic wrap (used to hold the test material in dermal contact) at least four times prior to the start of the study. An area
approximately 5 X 5 cm on the back of each rat was clipped free of hair prior to study initiation and as necessary thereafter. An absorbent gauze patch was used to hold the test material/vehicle or vehicle control in dermal contact. The wraps were removed approximately six hours after application and the test site was wiped, if necessary, with a water-dampened disposable towel to remove any residual test material.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test formulation was shown to be stable over 116 days. After 116 days, the concentration of DBNPA in TTEG was 96% of day 1 values.

Duration of treatment / exposure:

6 hrs/day

Frequency of treatment:

5 days/week excluding holidays

Doses / concentrations

Remarks:

Doses / Concentrations:
0.4, 1.2 or 4.0 ml/kg/day of the DBNPA test solution
Basis:
nominal per unit body weight

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

Groups of 20 adult rats (10/sex/dose) received topical applications five days/week for 13 consecutive weeks, excluding holidays. One group was designated a sham control and was wrapped only. The vehicle control received only the TEG (needed to solubilize the DBNPA) at the rate of 3.0 ml/kg/day, which corresponds to a dose level of approximately 3360 mg TEG/kg/day. The remaining dose groups received 0.4,1.2 or 4.0 ml/kg/day of the DBNPA test solution, which corresponds to dose levels of approximately 103,309 or 1031 mg DBNPA/kg/day, respectively, and 327,980 or 3266 mg TEG/kg/day, respectively. The dose volume of 4.0 ml/kg represents the maximum amount of material that could be practically maintained at the application site. The dose levels selected for the 13-week study were chosen based on the rationale delineated in the subsequent section.

Dose Level Selection and Results of Dermal Probe Studies
DBNPA was not sufficiently soluble in water to allow the use of water as the vehicle control. Therefore, TEG, the solvent used in the manufacture and use of formulations containing DBNPA, was used to solubilize the DBNPA (therefore enabled the conduct of this dermal toxicity study).

A probe study was initially conducted with DBNPA in TEG to generate information for the basis of appropriate dose level selection for this 13-week
study. In the probe study, groups of five male and five female Fischer 344 rats initially received ten daily dermal applications of the following preparations at the rate of 2.0 &kg: 2.5%, 5%, 10% and 20% DBNPA. This corresponded to dose levels of 64,128,256 and 512 mg DBNPA/kg/day, respectively.

During the course of these ten daily applications, there were no discernible indications of treatment-related adverse effects at the application site or on clinical in-life parameters being monitored for systemic toxicity (Dow, 1990). Thus, the decision was made to extend the probe study for an additional two weeks, with the same groups of males and females given the same dosing regimen, but at the higher application rate of 4.0 ml/kg. This corresponded to dose levels of approximately 128,256,512 and 1031 mg DBNPA/kg/day, respectively.

The day following the last application, the rats were submitted for termination and gross necropsy. The in-life examinations indicated several male and female rats in various treatment groups had a minimal inflammatory reaction at the application site consisting of erythema and edema. At necropsy, localized scab formation, interpreted as evidence of a minor inflammatory reaction, was noted at the dermal test site of 3/5 males given the 10% and 3/5 males given the 20% DBNPA concentration (supplying 1031 mg DBNPA/kg/day). An increased degree of discoloration of the haircoat at the dermal test site was attributed to the physical effects of repeated dermal application of the DBNPA and/or TEG to all groups of males and the
two highest dose groups of females.

These data were interpreted to indicate the maximum dose for the 13-week study to be 4 ml/kg/day, supplying 1031 mg DBNPA/kg/day and 3266 mg TEG/kg/day.

The maximum dose selected for this 13-week study was based on the following considerations:
(1) relative insolubility of DBNPA in water, precluding the use of water as the vehicle,
(2) the required use of a suitable solvent (TEG) to solubilize the technical grade active ingredient, DBNPA,
(3) the use of a 20% concentration of DBNPA in TEG which represents the approximate concentration of the technical grade material that is manufactured and used,
(4) the maximum dose volume of 4 ml/kg which could be practically maintained at the application site, and
(5) the minor inflammatory reaction that was noted at the application site of some rats in the highest dose level of the probe study which was considered tolerable over the course of a 13week study.

Based on these factors, a maximal dose of 1031 mg DBNPA/kg/day was selected for the 13-week study.

Positive control:

Not applicable.

Examinations

Observations and examinations performed and frequency:

Body Weights and Feed Consumption.
All animals were weighed prior to the first application and approximately weekly thereafter. Feed consumption was determined weekly for all animals.

Observations and Records,
Ophthalmological Examination. The eyes of all animals were examined by the laboratory veterinarian prior to the start of the study utilizing penlight illumination. Eyes were also examined upon termination of the study at terminal necropsy (see Pathology section).

In-Life Observations. A careful clinical examination was conducted on all animals prior to the start of the study and at least weekly thereafter for the duration of the study. This examination included thorough evaluations of the skin, fur, mucous membranes, respiration, nervous system function (e.g., tremors, convulsions) and behavior pattern. An additional observation for morbidity, mortality and the availability of feed and water was made each day of the work-week as well as twice daily on weekends and holidays.

Functional Observational Battery. A functional observational battery (FOB) was conducted by a trained observer on all surviving rats during the week prior to necropsy. The observer (blind to the identity of the animal) assessed the following parameters upon examination of each individual rat: eyes (pupil size, palpebral fissure, lacrimation, etc.), respiration (increased, wheezing, etc.), movement/behavior (bizarre behavior, vocalization, tremors, convulsions, muscle tone, etc.), skin and fur (rough haircoat, perineal soiling, piloerection, bluish, etc.), mucous membranes, general appearance (salivation, nasal discharge, obese, etc.), and feces and urine (decreased, dark, mucoid, reddish, etc.). The rat was then placed into an observation box (a clear plastic box with approximately 50 x 50 cm floor and 25 cm high walls) for approximately 20 seconds. Parameters evaluated during this observation period included: movement/behavior (gait, pattern, activity level, coordination, etc.) and sensory responsiveness (touch, auditory, nociceptive).

Evaluation of Dermal Application Site.
The condition of the skin at the application site was subjectively evaluated weekly using this laboratory's modification of the acute dermal irritation scoring system recommended by the Organization for Economic Co-operation and Development (OECD, 1981b):

Erythema and Eschar:
Within normal limits ...............................................................0
Very slight erythema (barely perceptible) ...............................1
Well-defined erythema ...........................................................2
Moderate-to-severe erythema ..................................................3
Severe erythema to slight eschar formation ..............................4
Edema:
Within normal limits ...................................................................0
Very slight (barely perceptible) ................................................1
Well-defined (edges raised) ................................................ .....2
Moderate (raised -1 mm) ...................................................... ...3
Severe (raised >1 mm) ..............................................................4
Scaling and Fissuring:
Within normal limits ................................................................. ..0
Slight scaling ................................................................................1
Moderate-to-severe scaling ......................................................2
Slight fissuring.. ...........................................................................3
Moderate-to-severe fissuring ...................................................4
Necrosis, scabs/crusts and scars:
- = condition not present
+ = condition present

Necrosis, scabs and/or scars were noted if present; however, they were not graded.
Necrosis, scabs and/or scars were noted if present; however, they were not graded.

Sacrifice and pathology:

Hematoloy Blood samples were collected from the orbital sinus of fasted rats lightly anesthetized with methoxyflurane approximately one week prior to necropsy. The following hematologic parameters were evaluated for each animal: hematocrit (HCT), hemoglobin concentration (HGB), erythrocyte count (RBC), total leukocyte count (WBC), and platelet counts (PLAT). Blood smears were prepared and stained with Wright's stain for all animals from which blood samples were collected. Complete blood smear examinations were conducted manually on high dose and both controls (sham and vehicle control) which included differential leukocyte counts (the number of leukocytes counted was specified if other than 100 cells were counted) and an assessment of erythrocyte, leukocyte and platelet morphology. Hematologic determinations were made on an ELT-8, Ortho Instruments, Westwood, MA.

Clinical Chemistry. Blood samples were collected from the orbital sinus of fasted rats lightly anesthetized with methoxyflurane approximately one week prior to necropsy. The following parameters were evaluated for each animal: alkaline phosphatase activity (AP), alanine arninotransferase activity (ALT), aspartate aminotransferase activity (AST), total protein (TI?), albumin (ALB), globulin (GLOB - calculated), total bilirubin (TBILI), glucose (GLU), urea itrogen (UN), cholesterol (CHOL), triglycerides (TRIG), creatinine (CREAT), phosphorus (I?), calcium (CA), sodium (NA), potassium (K) and chloride (CL). All analyses were conducted with a Monarch 2000 automated chemistry analyzer (Instrumentation Laboratory Inc., Lexington, MA.). Globulin values were calculated as the difference between total protein and albumin levels and a Beckman E4A flame photometer (Beckman Instruments Inc., Brea, CA) was used to determine Na, K and Cl levels.

Urinalysis. Urine samples were obtained from all nonfasted rats approximately one week prior to necr0psy.b~m anual compression of the urinary bladder. The following parameters were evaluated for each urine sample: specific gravity (T. S. Meter, American Optical, Keene, NH) and a semiquantitative estimate of bilirubin, glucose, ketones, blood, pH, protein and urobilinogen (Urotron test strip analyzer, BioDynamics, Indianapolis, IN). In addition, the color and appearance, as well as the presence of microsediment in a pooled sample from each dose group was determined.

Pathology. All animals were fasted overnight prior to necropsy. Each animal was weighed, anesthetized with methoxyflurane and euthanatized. To
prevent terminal aspiration of blood, the trachea was exposed and clamped prior to decapitation. Eyes were examined by visual inspection of the cornea, lens and other internal components via placement of a moistened glass slide on the corneal surface using a fluorescent light. All observations made on the eyes were recorded with the gross necropsy observations. A complete gross necropsy of all rats was performed by a veterinary pathologist.

Weights of the liver, kidneys, testes, and brain were recorded and the organ weight to final body weight ratios were calculated for each animal. A complete set of tissues plus any additional gross lesions were collected from each animal and preserved in neutral, phosphate-buffered 10% formalin. Lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin solution by tracheal instillation using a hand-operated syringe. The nasal cavity was flushed by a retrograde infusion of the formalin solution via the pharyngeal duct. Sections of the integument were collected from the dermal test site as well as immediately adjacent to the dermal test site. These sections of the integument were supplemental to the section of skin and subcutis routinely collected from the abdominal / inguinal region.

A histologic examination via light microscopy by a veterinary pathologist included a complete set of tissues and organs, including lesions from all ten male and female rats from the sham control group, the vehicle control group and the high dose group. Histopathologic examination was extended to the intermediate and low dose animals and included 1) integument from the dermal test site, 2) integument immediately adjacent to the test site, and 3) all gross lesions noted in any organ or tissue at necropsy. Tissues were prepared for light microscopic evaluation by standard processing procedures, sectioned at approximately 6 p an d stained with hematoxylin and eosin

Other examinations:

No additional information available.

Statistics:

Descriptive statistics (means and standard deviations) were reported for differential leukocyte counts and feed consumption. Body weights, absolute and relative organ weights, urine specific gravity, clinical chemistry data, and appropriate hematology data were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett's test, exploratory data analyses were performed by a parametric or non-parametric analysis of variance (ANOVA), followed respectively by Dunnett's test or the Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons. Statistical outliers were identified by a sequential test, but were only excluded from analyses for documented, scientifically sound reasons unrelated to treatment with the test material. A separate comparison of sham control to vehicle control was conducted using the same statistical methods. If statistical significance was noted between the two control groups during the comparison a succeeding comparison of all treatment groups to the sham control and all treatment groups to the vehicle control was conducted.

The nominal alpha levels used and test references are as follows:
Bartlett's test (Winer, 1971) ...............................................................a = 0.01
Parametric ANOVA (Steel and Torrie, 1960) ...................................a = 0.10
Non-parametric ANOVA (Hollander and Wolfe, 1973). ................ a = 0.10
Dunnett's test (Winer, 1971).............................................................. a = 0.05, two-sided
Wilcoxon Rank-Sum test (Hollander and Wolfe, 1973) ..................a = 0.05, two-sided
Bonferroni correction (Miller, 1966)
Outlier test (Grubbs, 1969)................................................................ a = 0.02, two-sided

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

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:

no effects observed

Details on results:

NOTE (It is difficult to separate the DBNPA data from the vehicle only data without rewriting the report. Therefore some but not all of the DBNPA is included here with emphasis placed on the vehicle only to control (animals wrapped only)
In-Life Observations
There were no in-life signs of systemic toxicity associated with repetitive dermal application of DBNPA or the vehicle control, TEG, even under conditions maximizing the amount of each material that could be reasonably applied. There were sporadic observations of lacerations on the chest which were self-inflicted by the animals chewing on the wraps used to hold the test material in place. These self-inflicted lacerations occurred in one male sham control, two highdose males, one female sham control, one female vehicle control and three high-dose females. These lesions appeared to be self-limiting and were treated topically when the severity of the wound deemed it necessary in the judgement of study personnel. The lesions were resolved by study termination. On test day 85 one female control was found at the end of the day as having been wrapped too tightly which resulted in rapid respiration in this rat; the wrap was removed, the animal regained a normal respiration pattern, and was dosed and wrapped the following day. There was no further in-life involvem ent with this animal. However, all hematology values obtained from the blood sample taken at necropsy from this animal were subsequently eliminated from the control group mean; elimination of these values was attributed to the wrapping incident on test day 85.

The test site of the sham control and vehicle control groups was within normal limits throughout the observation period.

A prestudy ophthalmic exam conducted on all male and female rats showed that the eyes of all rats had either no visible lesions or small corneal opacities which are common to rats of this age and strain.

No treatment-related observations were noted in the functional observational battery during this examination.

Bodv Weights and Feed Consumption
Mean body weights and feed consumption from all male and female rats of all dose groups were comparable to concurrent controls. Some statistical
differences were identified for the female rat vehicle control group and the low-dose females versus the female sham controls, and for high-dose female rats versus the female vehicle controls. These differences were not dose related nor were they observed in the males. They were considered representative of normal biological variation and considered not toxicologically significant.

Clinical Pathology
There were no treatment-related effects observed in hematologic parameters, urinalysis parameters or clinical chemistry determinations of male and female rats of any dose level. Statistically significant differences were identified in various clinical chemistry parameters of the vehicle control group compared to the sham control group and the DBNPA treated groups compared to sham control and vehicle control groups. These statistically identified differences were not considered toxicologically significant since there was no doseresponse relationship, the differences were not demonstrated in the other sex of a comparable dose level, the differences between either control group and DBNPA treated groups were very slight (i.e., ALB for males given 309 mg DBNPA/kg/day was 3.8 g/dl identified as statistically significant versus ALB for male sham controls of 3.7 g/dl) or the differences were not consistently identified versus sham controls and vehicle controls.

Organ and Organ/Bodv Weight Ratio.
The mean terminal body weights and absolute and relative liver, kidney, brain and testes weights of rats given DBNPA were comparable to those of the controls and demonstrated no treatment-related effect. There were statistically identified differences in relative kidney and liver weights of the vehicle control males versus the male sham controls, relative brain weights of low-dose male rats versus the male sham controls, absolute kidney and liver weights of female vehicle controls versus female sham controls, absolute kidney weights of high-dose females versus female vehicle controls, absolute liver weights of low- and intermediate-dose females versus female sham controls, relative brain weights of female vehicle controls versus female sham controls, relative brain weights of high-dose females versus the female vehicle controls, and relative liver weights of intermediate- and high-dose females versus female sham controls. These differences were considered of no toxicological significance since there was no dose-response relationship, the differences were not identified in the other sex of a comparable dose level or differences identified in absolute organ weights versus relative organ weights did not correspond and they occurred in the absence of any histopathologic alterations.

Pathology
The dermal test site was unremarkable for all male and female rats treated with the sham (water) control, the vehicle (TEG-solvent) control or the intermediate and lower dose groups (103,309 mg DBNPA/kg/day) of the test material. The other observations made at the time of gross necropsy consisted of a low incidence of diverse observations with no relationship to treatment or dosage. All these other observations (excluding dermal test site) were considered spontaneous in origin and unrelated to treatment with the test material or the vehicle solvent.

Histopathologically, the dermal test site was unremarkable for all males and females treated with the sham control (water), vehicle control (solvent) or the intermediate and low dose groups (309 and 103 mg DBNPA/kg/day). Histopathologic examination of integument away from the dermal test site as well as all internal organs and tissues revealed no observations that were considered related to repetitive treatment with the vehicle control (solvent) or any of the three dose levels of the test material (1031,309 or 103 mg DBNPA/kg/day). The various observations that were made on certain sections of liver, lungs, cornea of eyes, heart, kidneys, nasal tissues, ovaries, stomach, testes, ovaries and thyroid were isolated inflammatory and/or degenerative processes typically encountered as spontaneous occurrences in rats of this age and derivation. These observations were noted in control and treated groups, with no relation to treatment with the vehicle solvent or test material.

The occurrence of interstitial inflammation of the lacrimal glands in some rats of control and treatment groups were considered secondary artifacts due to the previous collection of blood from the orbital venous sinus for determination of hematologic and serum chemistry parameters.

Effect levels

Dose descriptor:

NOEL

Effect level:

3 360 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no treatment related effects noted between the sham exposed and vehicle control group (TEG group) which received ~3360 mg/kg/day dermally.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

No additional information available.

Applicant's summary and conclusion

Conclusions:

The NOEL in a 13 week rat dermal study is 3360 mg/kg/day of TEG when compared to the sham exposed control group.

Executive summary:

2,2-Dibromo-3-nitrilopropionamide (DBNPA) was evaluated for systemic toxicity following repeated dermal application for six hours/day, five days/week for 13 weeks, excluding holidays. Groups of ten Fischer 344 rats/sex/dose received a 20% solution of DBNPA in tetraethylene glycol (TEG) vehicle at targeted dose levels of 0 (sham control), 103,309 or 1031 mg DBNPA/kg/day. A comparable vehicle control group of male and female rats received only the TEG (needed to solubilize the DBNPA) via dermal application at a dose level of approximately 3360 mg TEG/kg/day. Parameters evaluated included general appearance and demeanor, topical response of skin at the application site, body weights, feed consumption, hematology, clinical chemistry, urinalysis, selected organ weights, gross pathologic and histopathologic observations. In addition, a functional observational battery (FOB) was conducted after twelve weeks of exposure.

There was a topical response of the skin that was dose-dependent and limited to the application site. The dermal irritation was characterized by erythema, edema and scabs/crusts at the test site of most high-dose male and female rats. Transient erythema and/or edema was observed in four male and two female rats given 309 mg DBNPA/kg/day. The test site of the sham control and vehicle control groups was within normal limits throughout the observation period. Gross and histopathologic examination of rats given 1031 mg DBNPA/kg/day showed a localized response at the dermal test site. This localized response did not occur in the rats given 103 or 309 mg DBNPA/kg/day or the controls.

In regard to systemic toxicity, there were no in-life signs of systemic toxicity observed during the study and all groups of rats had comparable rates of weight gain. The FOB revealed no differences among the control and treated groups of rats. There were no treatment-related changes in feed consumption, hematology, clinical chemistry, urinalysis parameters or organ weights. In addition, there were no systemic gross or histopathologic lesions attributed to treatment with DBNPA or the vehicle control.