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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
ADAPTATION ACCORDING TO REACH ANNEX XI, section 1 - see justification attached to IUCLID section 13.2.
- Justification for WoE: RDT and Toxicity to reproduction

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2007
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- purity ranged from 98% - 99.6% (GC/FID)
- Source of test material: The Dow Chemical Company (Midland, MI)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: drinking water formulations were found to be stable for at least 25 days
- Solubility of the test substance in the solvent: solubility in drinking water tested

FORM AS APPLIED IN THE TEST: solution with municipal drinking water

OTHER SPECIFICS:
- pH: adjusted with HCl to match pH of control water

Test animals

Species:
rat
Strain:
Fischer 344
Remarks:
DuCrl
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc. (Kingston, New York and Raleigh, North Carolina)
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: not specified
- Housing: singly housed
- Diet: ad libitum, LabDiet #5002 Certified Rodent Diet (PMI Nutrition International, St. Louis, MO)
- Water: ad libitum (formulated with DIPA for the substance administration), drinking water
- Acclimation period: acclimated to the laboratory

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 45-60
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12


Fischer 344 rats were selected because of their general acceptance, and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.

FURTHER DETAILS
Physical and Acclimation
Each animal was evaluated by a laboratory veterinarian to determine their general health status and acceptability for study purposes upon arrival at the laboratory. The animals were housed 2-3 per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for approximately one week prior to the start of the study.

Housing
Animals were housed one per cage in stainless steel cages after assignment to the study. The room relative humidity and temperature were maintained within a range of 46.8 - 56.5% and 21 - 24.4 °C, respectively. A 12-hour light/dark photocycle was maintained for the animal room with lights on at 6:00 a.m. and off at 6:00 p.m., and room air was exchanged approximately 12 - 15 times/hour. Cages had wire-mesh floors that were suspended above cageboards and contained a feed crock and a water bottle.

Randomization and Identification
Animals were stratified by pre-exposure body weight and then randomly assigned to treatment groups using a computer program. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) which were correlated to unique alphanumeric identification numbers.

Feed and Water
Animals were provided LabDietâ Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility.

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Remarks:
substance solution with drinking water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

WATER PREPARATION
- Rate of preparation of drinkiung water (frequency): prepared weekly
- Rate of adjustment of concentration in water: weekly
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- concentration of test material in the drinking water was adjusted weekly
- test material intake, calculated from DIPA concentrations, body weights and water consumption were between 98% and 106% of targeted levels
Duration of treatment / exposure:
90 days (13 weeks)
Frequency of treatment:
continuous via water intake
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
equivalent to 56 mg/kg bw/day Isopropanolamine
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
equivalent to 282 mg/kg bw/day Isopropanolamine
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
equivalent to 564 mg/kg bw/day Isopropanolamine
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: dose levels were based upon a prior 2-week drinking water study
- Rationale for selecting satellite groups: assess recovery from any treatment-related effects
- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
no positive control

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pre-exposure and weekly throughout the study
- all animals, examination included cage-side, hand-held and open-field observations

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION
- Time schedule for examinations: weekly

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prestudy and during the last week of treatment
- indirect ophthalmoscopy

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes, with methoxyflurane or CO2
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked according to guideline OECD 408

CLINICAL CHEMISTRY: Yes
- for details refer to haematology

URINALYSIS: Yes
- Time schedule for collection of urine: collected overnight from all non-fasted rats 1 week prior to necropsy
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- all rats fasted overnight were anesthetized by the inhalation of methoxyflurane or CO2
- adrenal glands, brain, heart, kidneys, liver, epididymides, uterus, thymus, spleen and ovaries or testes were weighed
- ratios of organ weight to terminal body weight were calculated

HISTOPATHOLOGY: Yes
- extensive set of organs consistent with regulatory guidelines (OECD 408)
- including all organ systems and gonads and secondary sex organs of both sexes were examined histopathologically from the control and high dose animals with the kidneys, liver, lungs, urinary bladder and all gross lesions examined from the remaining rats
Statistics:
- all continuous data: means and standard deviations (first examined for equality of variance using Bartlett’s test (a = 0.01))
- statistical interactions (i.e., time by dose or sex by dose) were identified in several of the tests: alpha levels for interaction terms were set a priori at 0.01–0.10, with Bonferroni’s correction
- alpha level for comparison of individual dose groups to controls was set a priori at 0.05

- weekly body weights, feed and water consumption, clinical pathology parameters, terminal body weight, organ weights: parametric or non-parametric ANOVA, followed by Dunnett’s or Wilcoxon’s test for comparison to controls
- detailed clinical observations: Z-test of proportions

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs observed in any test group compared to the control group.
Mortality:
no mortality observed
Description (incidence):
No animals died up to the highes dose tested.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- 1000 mg/kg/day: rats weighed slightly less than controls (refer to table 1) and gained 4-5 % less body weight after 13 weeks of dosing
- considered secondary adaptive effects
There were no statistically-significant changes observed in any test group compared to the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- 1000 mg/kg/day: rats consumed slightly less feed than controls ( ca. 2–3%)
There were no statistically-significant changes observed in any test group compared to the control group.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
- 1000 mg/kg/day, males and females: total water consumption decreased 7.5% and 18% relative to controls
Ophthalmological findings:
no effects observed
Description (incidence and severity):
- no treatment-related changes in ophthalmic parameters observed
Haematological findings:
no effects observed
Description (incidence and severity):
- hematologic parameters and prothrombin times were unaffected in all dose groups
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 1000 mg/kg/day, males: minimal, but statistically identified, differences noted for cholesterol (increased), albumin (decreased) and phosphorus (decreased)
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- 1000 mg/kg/day: increased urine specific gravity and the females had decreased urine volume
- considered secondary adaptive effects
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 500, 1000 mg/kg/day: absolute and relative kidney weights were increased, absolute kidney weight was increased for males given 100 mg/kg/day
- 1000 mg/kg bw/day: relative kidney weight of males ca. 21% greater than controls while the females were ca. 14% greater than controls
Gross pathological findings:
no effects observed
Description (incidence and severity):
- no gross pathologic effects related to treatment observed in any dose group
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
- no histopathologic effects related to treatment observed in any dose group
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
- no histopathologic effects related to treatment observed in any dose group
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Satellite groups (4-week recovery period):
- water consumption of the treated rats remained below controls by approximately the same degree as at the end of the dosing period
- all other affected endpoints demonstrated reversibility
- absolute and relative kidney weights were still above controls, but the differences were about one-half of those immediately following 13 weeks dosing
- mean relative kidney weights were increased 11% (males) and 6.6% (females) relative to controls
- no treatment-related renal histopathological changes were observed
Details on results:
Despite the kidney weight increase, there were no gross or histopathologic effects related to treatment.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
equivalent to 56 mg/kg bw/day Isopropanolamine
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
equivalent to 282 mg/kg bw/day Isopropanolamine
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1: Effects of DIPA given in drinking water to F344 rats for 13 weeks.

   Dose level (mg/kg/day                     
   Males           Females         
   0  100  500  1000  0  100  500  1000
 Body weight - day 90 (g)  348.5  354.2  357.6  340.9  196.7  194.6  195.1  192.1
 Body weight gain (g)  176.5  182.1  185.8  169.3  85.4  83.3 83.2  80.8 
Water consumption (g)  2048.8  2061.2  2119.0  1985.1  1890.6 1999.3  2045.8  1549.9 
 Feed consumption (g)  1554.1 1598.8   1593.6  1508.3  1127.0 1138.3  1138.1  1107.9 
 Cholesterol (mg/dL)  60  60  64  70*  19 18  18  21 
 Albumin (g/dL)  3.5 3.5  3.5  3.4*  95 98 97  93 
 Phosphorus (mg/dL)  10.5 10.6  10.4  9.7*   3.4 3.5  3.4  3.3 
 Serum urea nitrogen (mg/dL)  17  17  17  17  10.2  9.5  9.5  9.6
 Urine volume (mL)  4.5  4.4  4.9  4.6  2.9  3.7  3.0  1.4
 Urine specific gravity  1.067  1.068 1.072  1.079*   1.070 1.065   1.070 1.114* 
 Terminal body weight (g)  319.7  329.7  332.9  319.5  179.9 179.4  181.2  181.1 
 Kidney body weight (g)  2.146  2.357* 2.494*  2.594*   1.276 1.301  1.374*  1.466* 
 Relative kidney weight (g/100g)  0.671  0.716  0.749*  0.813*  0.709 0.726  0.759*   0.810*
                 

Statisticially different from control mean by Dunnett´s test: α =0.05

Applicant's summary and conclusion

Conclusions:
The only effect found in the 500 mg/kg bw/day DIPA (equivalent to 282 mg/kg bw/day MIPA) group was increased relative and absolute kidney weights without any histopathologic correlate.
The NOAEL was 500 mg/kg bw/day for females and 100 mg/kg bw/day (equivalent to 56 mg/kg bw/day) for males.
Executive summary:

Ten male and ten female Fischer 344 rats per group were given drinking water formulated to supply 0, 100, 500, or 1000 milligrams Diisopropanolamine (DIPA) per kilogram body weight per day (mg/kg/day) for at least 90 days to evaluate the potential for systemic toxicity of DIPA. The doses are calculated to equivalent doses of Monoisopropanolamin (MIPA) of 0, 56, 282 and 564 mg/kg bw/day.

Standard toxicologic parameters were evaluated during the in-life phase of the study with clinical and anatomic pathology investigations at termination. Additional groups of ten/sex were maintained on untreated drinking water for at least an additional 28 days after initially receiving the control or high-dose water (0 or 1000 mg/kg bw/day) for at least 90 days to assess recovery from effects induced by DIPA. Rats of either sex given 1000 mg DIPA/kg bw/day for at least 90 days had few effects, all of which were of minimal degree, attributed to DIPA.

While there were no treatment-related clinical signs, rats given this dose level drank slightly less water (females had a greater decrement than males) with corresponding decrements of feed consumption and body weights considered secondary to the water aversion. The body weight decrements were only ca. 2% at termination and were never statistically identified. Urine specific gravity was increased in both sexes while urine volume was decreased for females; both of which were adaptive effects to the decreased water consumption. Serum cholesterol was slightly increased while serum phosphorus was slightly decreased for male rats given 1000 mg/kg bw/day. These effects were not present after the 28-day recovery period. Kidney weights, both absolute and relative to body weight, were increased with the males affected to a greater degree (male relative kidney weight increased ca. 21% and female increased ca. 14%). However, histopathologic effects were not found. After four weeks drinking untreated water, the increase in kidney weight was about one-half of that present at the end of the dosing period for both males and females.

This limit dose level, 1000 mg/kg bw/day (equivalent to 564 mg/kg bw/day), was considered to be a LOAEL. The only effect found in rats given 500 mg/kg bw/day (equivalent to 282 mg/kg bw/day) was increased absolute and relative kidney weights with males again having a greater increase than females (male relative kidney weight increased ca. 12% and female increased ca. 7%), also without any histopathologic correlate. The absolute kidney weight of males given 100 mg/kg bw/day (equivalent to 56 mg/kg bw/day) was also increased and was statistically identified; however, these rats weighed more than controls and the relative kidney weight was similar to controls.

Thus, the 500 mg/kg bw/day (equivalent to 282 mg/kg bw/day) dose level was considered the NOAEL for females, while 100 mg/kg bw/day (equivalent to 56 mg/kg bw/day) was the NOAEL for males.