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Description of key information

A sub-chronic oral toxicity study was performed in rats, according to OECD guideline 408 with the structurally similar DIPA. The NOAELs of 100 mg/kw bw/day (equivalent to 56 mg/kg bw day) for males and 500 mg/kg bw/day (equivalent to 282 mg/kg bw/day) for females were established.

In a combined repeated dose oral toxicity study with a reproduction/developmental screening study in rats, performed according to OECD guideline 422, a NOAEL for general systemic toxicity of 300 mg/kw bw/day was established for males, based on some indications for a mild anemic process. For females, the NOAEL was 1000 mg/kg bw/day, the highest dose tested.

The endpoint repeated dose toxicity is assessed in a weight of evidence approach, based on the adaptation justification attached in section 13 in IUCLID.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
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
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
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.
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
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
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
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.
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
Critical effects observed:
no

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

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.

Endpoint:
short-term repeated dose 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
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
22 March 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF and BA 1449
- Purity test date: 04 Jan 2006
- concetration of given stock solution:65.4 g isopropanolamine hydrochloride per 100 g aqueous solution

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (freezer for reanalysis)
- Stability under test conditions: confirmed by reanalysis
- Solubility and stability of the test substance in the solvent/vehicle: stability of aq. solution confirmed by reanalysis

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test material was weighed up and dissolved in tap water

FORM AS APPLIED IN THE TEST: solution
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, Research Models and Services, Germany Gmbh
- Age at study initiation: 11-13 weeks
- Weight at study initiation: 267.4-309.0 g (males) and 186.7-217.7 g (females)
- Number of animals: 96 (12 per sex per dose group)
- Housing: Individually in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm2), with the following exceptions: for the overnight mating the females were put into the cages of the males; from day 18 p.c. until day 4 p.p. the pregnant animals and their litters were housed in Makrolon type M III cages (floor area about 800 cm2). The M III cages were also supplied by Becker & Co.. Pregnant females were provided with nesting material (cellulose wadding) toward the end of pregnancy.
- Diet (e.g. ad libitum): Ground Kliba maintenance diet mouse/rat “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
tap water
Details on oral exposure:
TEST ITEM
- Dosing solution: pH 6.0 - 7.5

VEHICLE
- Amount of vehicle: 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- The solutions were analyzed twice and determined to be 0, 1.61, 5.11, 15.73 g/100mL using a content 68.9g/100g, which was determined by potentiometric titration and 0, 1.62, 4.67, 15.60 g/100 mL using 69.0 g/100g, also determined by potentiometric titration.
Duration of treatment / exposure:
38 days (males), 45 days (females)
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
equivalent to 67 mg/kg bw/day Isopropanolamine
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
equivalent to 202 mg/kg bw/day Isopropanolamine
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
equivalent to 673 mg/kg bw/day Isopropanolamine
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on range finding study
- Fasting period before blood sampling for clinical biochemistry: 16 to 20 hours before necropsy
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (littering and lactation behaviour evaluated in the mornings
- Cage side observations included any signs of morbidity, pertinent behavioral changes and signs of overt toxicity, documented for each animal

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Parameters: abnormal behavior during “handling”, fur, skin, salivation, nose discharge, lacrimation, pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, feces (appearance/consistency), urine, other findings

BODY WEIGHT: Yes
- Time schedule for examinations:once a week at the same time of the day (in the morning);
- Time schedule exceptions: during the mating period the parental females were weighed on the day of positive evidence day 0 p.c. and on days 7, 14 and 20 p.c.; females with litter: day day 0 p.p. and on day 4 p.p.; females without a litter: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- once a week (in a period of 7 days) for male and female parental animals
- exceptions: not determined during the mating period; F0 females with evidence of sperm were determined on days 0, 7, 14 and 20 p.c.; F0 females, which gave birth to a litter were determined on days 0 and 4 p.c.
- not determined in females without positive evidence of sperm and females without litter

OPHTHALMOSCOPIC EXAMINATION: Yes
- within the detailed clinical observations

HAEMATOLOGY and CLINICAL CHEMISTRY:
- hematology (5 animals/sex/group) with EDTA-K3 as anticoagulant: Leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, reticulocytes
- Prothrombin time (Hepato Quick's test)
- Clinical chemistry (5 animals/sex/group): alanine aminotransferase, aspartate aminotrasferase, alkaline phosphatase, gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

URINANALYSIS: YES
- on day 38 (males) and 45 (females) volume, color, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment
- Parameters: volume, color, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity (urine refractometer), sediment (microscopically)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on study day 36 in male and on study day 43 in female, first 5 male/group and first 5 female/group
- Dose groups that were examined: all dose groups
- Battery of functions tested: Functional observational battery (FOB):
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations (at least for 2 minutes): behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE
- Male animals: after blood from 5 F0 males per group was sampled, on study day 38 necropsy of all male animals was performed
- Female animals: after blood from 5 F0 females per group was sampled, on study day 45 necropsy of all female animals was performed

GROSS NECROPSY
- Gross necropsy was not further specified.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated in Table 1 were prepared for microscopic examination.
- The animals/organs were weighed: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, seminal vesicle, prostate, ovaries, uterus, thymus, spleen, brain, heart
Statistics:
Due to limitations of characters, details for statistics are given as tables (2, 3, 4) under part 'other information'.

CLINICAL EXAMINATIONS
- DUNNETT-test (two-sided) for the hypothesis of equal means
- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions
- Pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians
- Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (twosided) for the equal medians

CLINICAL PATHOLOGY
- Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians
- Pair-wise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions

HISTOPATHOLOGY
- Means and standard deviations AND KRUSKAL-WALLIS and WILCOXON test
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- no test substance-related or spontaneous clinical findings observed in the male and female animals of 100 mg/kg body weight/day test group
- salivation after treatment was seen in all high dose male animals (1000 mg/kg body weight/day) during study weeks 1-5, finding persisted in the respective males only for a few minutes after daily gavage dosing
- urine discoloration was observed in all male animals of the high dose group during the whole study period (week 0-5) and in all mid dose male animals (300 mg/kg body weight/day) during study weeks 1-5 and females, for all high dose females during the whole study period (week 0 - 6) and in all mid dose females during study weeks 1-6
- 7 out of 12 female animals of test group 3 showed salivation after treatment during study weeks 1-6, finding persisted in the respective females only for a few minutes after daily gavage dosing
- detailed clinical observations on study days 0, 7, 14, 21, 28, 35 and additionally day 42 for female animals did not reveal any additional, substance-induced abnormalities in the male and female animals of the test groups 0-3 (0, 100, 300 and 1000 mg/kg body weight/day)

The clinical findings, i.e. transient salivation and discolored urine are considered to be substance-induced, but are without any toxicological relevance and are not assessed as being adverse.
It is assumed that the temporary salivation was induced by the bad taste of the test substance (small droplets of the test substance solution might be adjacent on the tip of the gavage) or minor local affection of the upper digestive tract (without showing a morphological correlate at pathology). The urine discoloration is possibly related to a chemical reaction of the test substance or its metabolites with the bedding or with components of the air.
Mortality:
no mortality observed
Description (incidence):
There were no substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gains of all male and female (+ during the gestation, lactation period and after weaning) animals of all substance-treated groups (100, 300 and 1000 mg/kg body weight/day) were comparable to that of the concurrent control group during the whole study taking normal biological variability into account.
The statistically significantly increased body weight gains of the low and mid dose females (100 and 300 mg/kg body weight/day) during premating weeks 1-2 are considered to be spontaneous in nature and not to be adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no effects observed for food consumption compared to the control group.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- slightly, but statistically significantly decreased hemoglobin and hematocrit values in the peripheral blood of high dose males
- no treatment related effects were seen in the other hematology parameters of males and in the hematology investigations of females
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
- differences in serum enzyme activities were not evident at any dose level in either males or females
- blood chemistry results showed statistically significantly increased urea concentrations in the serum of high dose males and significantly higher albumin levels in the serum of high dose females
- marginally increased urea concentrations were also seen in the high dose females
- the increase in females was not sufficient to be statistically identified
- no treatment related changes were found in the other blood chemistry parameters
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
- decreased amounts of urine with increased specific gravity in all animals, more pronounced in females than in males
- no treatment related effects were seen in the other urine parameters examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related or spontaneous findings in the male and female animals of test groups 1-3 during FOB observations.
There were statistically significantly increased motor activity observed in males which were without a clear relation to dosing. This effect has no toxicological relevance because the values from the substance-treated males fit to the historical control range (mean value: 245.1; range: 207.6–299.5) of male rats of this strain at a comparable age.
There were no statistically significant or clearly dose-dependent deviations concerning the overall motor activity (summation of all intervals) in the females.
A substance-induced origin for this finding is excluded with certainty because of its scattered occurrence without any relation to dosing.
Thus, the motor activity of the substance-treated males and females did not show any toxicological relevant deviations.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Detailed information on changes (absolute and relative) of organ weights are given in the table 5 and 6 under any other information.

ABSOLUTE WEIGHT EFFECTS
- statistically increased brain weight of the mid dose group of female animals
This effect is thought to be incidental in nature, as no relevant microscopic findings were noted and no dose response relationship was observed.
- statistically increased thymus weights of females of the mid and top dose group
This effect is also thought to be incidental as no microscopic findings were noted.

RELATIVE WEIGHT EFFECTS
- statistically significant increased brain and kidney weights of females of the mid dose group
This effect was considered incidental in nature, as there was no dose-response relationship and no relevant microscopic findings were noted.
- statistically increased thymus weights of females of the mid and top dose group
This effect is also thought to be incidental as no microscopic findings were noted.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- additional statistically significant intergroup differences in the results of clinical pathology testing
- these deviations were marginal, incidental or inconsistent, when compared with the other sex, and/or lack dose-response relationship

These findings were considered to be of no toxicological significance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Further details to histopathological findings are given in the table 7.
- liver: males of the top dose group revealed a diffuse hepatocellular hypertrophy
- reproductive organs: no histopathological findings observed
Details on results:
Hematology determinations in high dose males revealed slightly, but statistically significantly reduced hemoglobin and hematocrit values, which are indicative of a mild anemic process. This is considered as an adverse substance-induced effect.
No treatment-related effects were noted in hematology investigations of females and serum enzyme examinations of both sexes.

The most prominent findings in clinical pathology were the reduced amounts of urine with subsequently increased specific gravity excreted by treated animals of either sex. The decreased urinary volume and the increased urinary specific gravity, however, are not considered as markers of kidney toxicity or an impairment of renal function. These findings are regarded non-renal in nature and are possibly due to decreased water intake.
A reduction in water intake could also well account for the slightly increased urea and albumin levels of the high dose males and/or females.
It is concluded that the changes in urinalysis and in blood chemistry examination are not caused by a direct toxic effect of the test compound and are not adverse in nature. This assessment is supported by the fact, that absolute and relative kidney weights as well as gross and histopathological evaluations of this organ did not give any indications for substance-induced alterations.

The livers of female animals of the top dose did not reveal any histological findings. Nevertheless, the increase in liver weight in the top dose females is thought to be substance-related.
These liver findings are regarded to be non-adverse in nature, but are considered to mirror adaptive responses to the test substance administration. Moreover, liver enzymes in these rats remained unaffected (see clinical biochemistry), but showed the usual range of biological variation.
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
equivalent to 202 mg/kg bw/day Isopropanolamine
Sex:
male
Basis for effect level:
haematology
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
equivalent to 673 mg/kg bw/day Isopropanolamine
Sex:
female
Remarks on result:
other: No effects observed for female animals.
Critical effects observed:
no

Table 5: Mean absolute weights of statistically significant changes in organs.

Absolute Weights

Male

Female

Group

1

2

3

1

2

3

Liver

+3%

-3%

+22%*

-4%

-4%

+17%*

Brain

-

-

-

0%

+4%*

-1%

Adrenal Glands

+4%

+4%

+19%*

-

-

-

Thymus

-

-

-

0%

+28%*

+26%*

*values were statistically significant different, P = 0.05

Table 6: Relative absolute weights of statistically significant changes in organs.

Relative Weights

Male

Female

Group

1

2

3

1

2

3

Liver

+3%

+1%

+23%*

-2%

0%

+16%*

Brain

-

-

-

+2%

+8%

-2%

Adrenal Glands

0%

+5%

+15%*

-

-

-

Thymus

-

-

-

+3%

+33%*

+25%**

Kidneys

-

-

-

+3%

+9%*

+4%

*values were statistically significant different, P <= 0.001, (**p= 0.05)

Table 7: Histopathological findings in the liver

Liver

Male animals

Female animals

Group

0

1

2

3

0

1

2

3

Organs examined

12

12

12

12

12

 

 

12

Hypertrophy, diffuse

0

0

0

9

0

 

 

0

Conclusions:
- Only the findings of decreased hemoglobin and hematocrit at 1000 mg/kg bw/day (equivalent to 202 mg/kg bw/day Isopropanolamine) in males are considered compound-related
- The other clinical and pathological findings appear to be incidental and not dose-related
- NOAEL for general, systemic toxicity of the test substance is 300 mg/kg body weight/day (equivalent to 673 mg/kg bw/day Isopropanolamine) for the F0 parental males based on some indications for a mild anemic process
Executive summary:

The hydrochloride salt of Isopropanolamine was administered orally via gavage to groups of 12 male and 12 female Wistar rats (F0 animals) at doses of 100 (67 mg/kg bw/day Isopropanolamine), 300 (202 mg/kg bw/day Isopropanolamine) and 1000 mg/kg of body weight/day (673 mg/kg bw/day Isopropanolamine) in order to detect possible effects of the test substance on the integrity and performance of the reproductive system of both sexes. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and no observed adverse effect level (NOAEL) after repeated oral administration, according to the OECD 422 guideline. Control animals were dosed daily with the vehicle (tap water).

The duration of treatment covered a 2 week premating period and mating period in both sexes, approximately 2 weeks post-mating in males, and the entire gestation period and 4 days of lactation in females.

F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). Mating pairs were from the same dose group.

A detailed clinical observation was performed in all animals before test substance administration and thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly during premating and after the mating period and during the gestation and lactation periods in dams. In general, body weights of F0 animals were determined once a week; however, during gestation and lactation, F0 females were weighed on days 0, 7, 14 and 20 post coitum, on the parturition day and day 4 post partum. Near the end of the administration period a functional observational battery was performed and motor activity was measured in 5 randomly selected parental males and females per group, respectively. Blood was sampled from 5 randomly selected parental males and 5 parental females per group for hematological and clinical chemistry examination. All surviving F0 parental animals were sacrificed by decapitation, under CO2 anesthesia, and were assessed by gross pathology. Organ weights were recorded and a histopathological examination was performed.

The following test substance-related, adverse effects/findings were noted:

- 1000 mg/kg body weight/day:

F0 parental animals: statistically significantly reduced hemoglobin and hematocrit values (i.e. indications of a mild anemic process) in the F0 males

F0 parental animals and F1 pups: no adverse effects/findings

Thus, under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for general, systemic toxicity of the test substance is 300 mg/kg body weight/day (202 mg/kg bw/day Isopropanolamine) for the F0 parental males based on some indications for a mild anemic process.

The NOAEL for parental females (F0) was found to be 1000 mg/kg body weight/day (673 mg/kg bw/day Isopropanolamine).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
56 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

No information available.

Additional information

Sub-chronic 90d study in rats

No sub-chronic oral toxicity studies are available for MIPA. Based on the weight of evidence approach, the 90-day oral toxicity study for DIPA (according to OECD guideline 408 and under GLP) is assumed to cover this endpoint. In this 90-day toxicity study, Fischer 344 rats (10/sex/dose) were administered DIPA at 0, 100, 500 or 1000 mg/kg bw/day (equivalent to 0, 56, 282 and 564 mg/kg bw/day MIPA) via their drinking water. Additional groups (10/sex) were maintained on untreated water for an additional 28 days to assess recovery from DIPA-induced effects after initially receiving the control or high-dose water (0 or 1000 mg/kg bw/day) for at least 90 days. Rats given 1000 mg/kg bw/day drank less water than lower dose groups, with a corresponding decrement in food consumption and body weight. Urine specific gravity increased, and urine volume decreased for this group as well; both were considered to be related to reduced water intake. Serum cholesterol was slightly increased, and serum phosphorous was slightly decreased in the main study group at 1000 mg/kg-bw/day; neither effect was noted in the recovery group. Changes in albumin in treated groups were also observed. Absolute and relative kidney weights were increased, males were affected to a larger degree. After the 28-day recovery, the changes in kidney weights were approximately one half that present at the end of the dosing period and slight renal tubule degeneration with regeneration was seen at the end of recovery in the 1000 mg/kg bw/day group. The only effect found in the 500 mg/kg bw/day group was increased absolute and relative kidney weights for males and females, without any histopathologic correlate. Absolute kidney weights were increased in the 100 mg/kg bw/day male group, but these rats weighed more than the controls, and the relative kidney weights were not statistically different than controls. Thus, NOAELs of 100 and 500 mg/kg bw/day DIPA were established for males and females, respectively; equivalent to 56 and 282 mg/kg bw/day MIPA.

Reproduction/Developmental screening study in rats

In a combined repeated dose oral toxicity study with a reproduction/developmental screening study (according to OECD guideline 422 and under GLP), Wistar rats (12/sex/dose) were administerd the hydrochloride salt of MIPA (CAS No. 7780-04-3) at 0, 100, 300 or 1000 mg/kg bw/day (equivalent to 0, 67, 202, 673 mg/kg bw/day MIPA) by oral gavage. The duration of treatment covered a 2-week premating period and mating period in both sexes, approximately 2 weeks post-mating in males, and the entire gestation period and 4 days of lactation in females, resulting in a total of 38 and 45 exposure days for males and females, respectively. Clinical pathology examinations revealed slightly, but statistically significant reduced hemoglobin and hematocrit values, which are indicative of a mild anemic process, in high dose males. Other (minor) clinical and pathological findings appear to be incidental and not dose-related. Thus, the NOAELs for general, systemic toxicity of 300 and 1000 mg/kg bw/day MIPA HCl were established for males and females, respectively; equivalent to 202 and 673 mg/kg bw/day MIPA.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.