Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
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).

Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
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 justifications attached to IUCLID section 13.2.
- Justification WoE_RDT_Reprotox_Feb2019
- Justification RA_CAS 122-20-2_Dev.tox.2ndspecies_Nov2018
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Test 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)
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
- Diet: Ground Kliba maintenance diet mouse/rat “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: ad libitum from drinking bottles
- 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 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/100 mL using a content 68.9 g/100 g, which was determined by potentiometric titration and 0, 1.62, 4.67, 15.60 g/100 mL using 69.0 g/100 g, also determined by potentiometric titration
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy and the following day "day 1" post coitum
- After successful mating each pregnant female was caged: in Makrolon type M III cages (floor area about 800 cm2)from day 18 p.c. until day 4 p.p., supplied with nesting material (cellulose wadding) toward the end of pregnancy
- Females were allowed to litter and rear their pups until day 4 after parturition
Duration of treatment / exposure:
38 days (males), 45 days (females)
Frequency of treatment:
daily
Duration of test:
39-47 days
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
Maternal examinations:
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

POST-MORTEM EXAMINATIONS: Yes
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

OTHER:
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
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
For all surviving pups (after sacrifice on day 4 post partum), all stillborn pups and those pups that died before schedule:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No
- Head examinations: No
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
Indices:
FEMALE REPRODUCTION AND DELIVERY DATA
Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x100
* defined as the number of females with implants in utero

Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

Post implantation loss (%) = ([number of implantations - number of pups delivered] / number of implantations) x 100


OFFSPRING VIABILITY INDICES
Viability index (%) = (number of live pups on day 4 after birth / number of liveborn pups on the day of birth) x 100

Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100
Historical control data:
There are historical control data from various studies with the same species and strain available in-house.
The included paramters are among others: mean maternal body weights during gestation and lactation, reproduction and litter indices as indicated before, pup weights and pup necropsy data and motor activity observations.
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
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations during gestation / lactation:
- discolored urine was recorded for all high and mid dose females (1000 and 300 mg/kg body weight/day) during the whole gestation and lactation period
- in 9 out of 12 high dose females salivation after treatment was noted during gestation
- in 10 out of 12 high dose females salivation after treatment was noted during lactation

No test substance-related clinical findings occurred in the female animals of test group 1 (100 mg/kg body weight/day) during the lactation period.

Both findings are not assessed as adverse or toxic effects for reasons described under overall clinical findings above.
Number of abortions:
not specified
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
- postimplantation loss in the high dose group slightly exceeded the historical control range, however, the mean number of F1 pups delivered per dam remained unaffected (12.9 / 11.6 / 10.7 and 11.5 pups/dam at 0, 100, 300 and 1000 mg/kg body weight/day)
- no statistically significant differences between the groups for the postimplantation loss
Dead fetuses:
no effects observed
Description (incidence and severity):
- rate of liveborn pups: live birth indices ranging between 99% (high and low dose) and 100% (control and mid dose)
- rate of stillborn pups was comparable between the groups
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
- gestation index reached 100% in all test groups
Other effects:
no effects observed
Description (incidence and severity):
- mean number of implantation sites was comparable between all test groups including the controls and did not show any relation to dosing (13.6 / 12.4 / 11.8 and 13.2 implants/dam in test groups 0 - 3 (0, 100, 300 and 1000 mg/kg body weight/day))
Details on maternal toxic effects:
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.

Considering the unaffected implantation and litter size and the absence of any statistically significant differences between the groups for the postimplantation loss, no test substance-induced intrauterine embryo-/fetolethality is assumed. The rate of liveborn pups was also not affected by the test substance administration as indicated by live birth indices ranging between 99% (high and low dose) and 100% (control and mid dose). Moreover, the rate of stillborn pups was comparable between the groups.
Thus, the administration of the Hydrochloride salt of Isopropanolamine did not adversely affect reproduction and delivery of the F0 females.
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
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
Remarks on result:
other: No effects observed for female animals.
Dose descriptor:
NOAEL
Remarks:
for developmental toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
equivalent to 673 mg/kg bw/day Isopropanolamine
Remarks on result:
other: No effects observed for maternal developmental toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
- mean pup body weights in test group 3 (1000 mg/kg body weight/day) were statistically significantly increased on post partum day 1 (males, females and males + females) and day 4 (males and males + females)
- mean pup body weights in test groups 1 and 2; 100 and 300 mg/kg body weight/day were comparable to the concurrent control values
- mean pup body weight changes in test groups 1, 2 and 3; 100, 300 and 1000 mg/kg body weight/day did not show any statistically significant differences

- body weight and body weight changes: spontaneous in nature, are not of toxicological relevance and are not considered as adverse effects
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
- mean number of delivered pups per dam and the rate of liveborn and stillborn pups were evenly distributed among the groups
Changes in sex ratio:
no effects observed
Description (incidence and severity):
- sex distribution and sex ratios of live F1 pups on the day of birth and 4 days post partum did not show biologically relevant differences between controls and test groups
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
- unaffected litter size between the groups
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
- viability index as indicator for pup mortality between days 0 - 4 p.p. varied between 98% (control) and 100% (test group 2)
- no compound related changes
External malformations:
no effects observed
Description (incidence and severity):
- numbers of runts were 0 /0 /0 /2 in test groups 0-3 (0, 100, 300 and 1000 mg/kg body weight/day), fully in the range of the biological variation inherent in the strain of rats
- scattered occurrence without a clear relation to dosing of post mortem autolysis
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- very few findings as situs inversus, misshapen spleen, infarct of liver, empty stomach, and dilated renal pelvis were of scattered occurrence without a clear relation to dosing
- substance-induced origin for these findings is excluded due to existence in the historical control data at comparable or even higher incidences
Details on embryotoxic / teratogenic effects:
No test substance-induced signs of developmental toxicity were noted in the progeny of the F0 parents up to and including 1000 mg/kg body weight/day. The number of delivered F1 pups/litter, their postnatal survival and their body weights remained unaffected by the test substance. Clinical and/or gross necropsy examinations of the F1 pups revealed only findings, which were considered to be spontaneous in nature.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
equivalent to 673 mg/kg bw/day Isopropanolamine
Sex:
male/female
Remarks on result:
other: No effects observed for F1 animals.
Abnormalities:
no effects observed
Developmental effects observed:
no

- 1000 mg/kg body weight/day, F1 pups: no adverse effects/findings

- 100 and 300 mg/kg body weight/day, F1 pups: no adverse effects/findings

Conclusions:
- Only the findings of decreased hemoglobin and hematocrit at 1000 mg/kg bw/day (equivalent to 673 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.
- The NOAEL for developmental toxicity in the F1 progeny is 1000 mg/kg bw/day.
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 (673 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. Mating was discontinued as soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females.

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. Pups were sexed on day 0 post partum and weighed one day after birth and on day 4 post partum. Their viabilitys recorded twice daily on each workday or only in the morning on Saturday and Sunday. All pups were sacrificed with CO2 on day 4 post partum and examined macroscopically for external and visceral findings.

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

F1 pups: no adverse effects/findings

- 100 and 300 mg/kg body weight/day:

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 reproductive performance and fertility is 1000 mg/kg body weight/day

(673 mg/kg bw/day Isopropanolamine) for the F0 parental rats. The NOAEL 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).

The NOAEL for developmental toxicity was 1000 mg/kg body weight/day (673 mg/kg bw/day Isopropanolamine).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
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

Test animals

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
- Diet: Ground Kliba maintenance diet mouse/rat “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: ad libitum from drinking bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
tap water
Details on exposure:
TEST ITEM
- Dosing solution: pH 6.0 - 7.5

VEHICLE
- Amount of vehicle: 10 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: in Makrolon type M III cages (floor area about 800 cm2)from day 18 p.c. until day 4 p.p., supplied with nesting material (cellulose wadding) toward the end of pregnancy
- Females were allowed to litter and rear their pups until day 4 after parturition
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/100 mL using a content 68.9 g/100 g, which was determined by potentiometric titration and 0, 1.62, 4.67, 15.60 g/100 mL using 69.0 g/100 g, also determined by potentiometric titration
Duration of treatment / exposure:
38 days (males), 45 days (females)
Frequency of treatment:
daily
Details on study schedule:
Premating exposure period:
- Male: 13 days
- Female: 13 days
Doses / concentrationsopen allclose all
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
Positive control:
None

Examinations

Parental animals: Observations and examinations:
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

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:
- 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)

Functional observation battery (FOB) and motor activity measurement:
- on study day 36 in first 5 male/group and on study day 43 in first 5 female/group
Sperm parameters (parental animals):
Parameters examined in F0 male parental generations: testes weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, weight gain (PND 1-4), presence of gross anomalies, anogenital distance (AGD)

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
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
Postmortem examinations (offspring):
- Organs examined at necropsy (F1 pups): all surviving pups, all stillborn pups and those pups that died before schedule

SACRIFICE
- The F1 offsprings were sacrificed on day 4 post partum.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external and internal (eviscerated) examinations including and their organs were assessed macroscopically.
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
Reproductive indices:
MALE MATING AND FERTILITY INDICES

Male mating index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility* / number of males placed with females) x 100
* defined by a female with implants in utero


FEMALE REPRODUCTION AND DELIVERY DATA

Female mating index (%) = (number of females mated* / number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x100
* defined as the number of females with implants in utero

Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

Post implantation loss (%) = ([number of implantations - number of pups delivered] / number of implantations) x 100
Offspring viability indices:
Viability index (%) = (number of live pups on day 4 after birth / number of liveborn pups on the day of birth) x 100

Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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
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
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations during gestation / lactation:
- discolored urine was recorded for all high and mid dose females (1000 and 300 mg/kg body weight/day) during the whole gestation and lactation period
- in 9 out of 12 high dose females salivation after treatment was noted during gestation
- in 10 out of 12 high dose females salivation after treatment was noted during lactation

No test substance-related clinical findings occurred in the female animals of test group 1
(100 mg/kg body weight/day) during the lactation period.

Both findings are not assessed as adverse or toxic effects for reasons described under overall clinical findings above.

One sperm positive control female (No. 102), one sperm positive low dose female (No. 123) and one sperm positive mid dose female (No. 136) did not deliver any F1 pups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- mean duration until sperm was detected (day 0 p.c.) amounted to 3.2 / 3.2 / 2.7 and 2.5 days (0, 100, 300 and 1000 mg/kg body weight/day)

These values reflect the normal range of biological variation inherent in the strain used in this study.
Reproductive performance:
no effects observed
Description (incidence and severity):
Male and female fertility indices are given in table 8 and 9. Mating was confirmed for all F0 males placed with females.

- not generating F1 pups: one control male (No. 2 mated with female No. 102), one low dose male (No. 23 mated with female No. 123 – 100 mg/kg body weight/day) and one mid dose male (No. 36 mated with female No. 136 – 300 mg/kg body weight/day)
- female rats not becoming pregnant (but sperm positive): control female No. 102 (mated with male No. 2), low dose female No. 123 (mated with male No. 23) and mid dose female No. 136 (mated with male No. 36)
- not pregnant females failed to show a morphological correlate for the apparent infertility for females Nos. 102 (control) and 123 (low dose)
- mid dose female No. 136 had a minimal diffuse inflammation of the uterus, which may well account for the observed infertility (see also Pathology)

The fertility indices for male and female varied between 92% (0, 100 and 300 mg/kg body weight/day) and 100% (1000 mg/kg body weight/day). These values reflect the normal range of biological variation inherent in the strain of rats used for this study. All respective values were within the range of the historical control data and do not show any relation to dosing.

Details on results (P0)

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.

The fertility indices observed in the study reflect the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. Furthermore, gross and histopathological examinations of these males failed to show a relevant morphological correlate for the apparently impaired fertility (see also Pathology).

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
for systemic toxicity
Effect level:
300 mg/kg bw/day
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
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.
Dose descriptor:
NOAEL
Remarks:
for reproductive performance and fertility
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks:
equivalent to 673 mg/kg bw/day Isopropanolamine
Sex:
male/female
Remarks on result:
other: no substance-related effects on fertility indices observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
- numbers of runts: 0 /0 /0 /2 in test groups 0-3 (0, 100, 300 and 1000 mg/kg body weight/day)
This finding is fully in the range of the biological variation inherent in the strain of rats used for this study.
- no adverse clinical signs up to scheduled sacrifice on day 4 post partum
Mortality / viability:
no mortality observed
Description (incidence and severity):
- viability index between days 0 - 4 p.p. varied between 98% (control) and 100% (test group 2)
- no compound related changes
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- 1000 mg/kg body weight/day: mean b.w. data were statistically significantly increased on post partum day 1 (males, females and males + females) and day 4 (males and males + females)
- 100 and 300 mg/kg body weight/day: mean b.w. data were comparable to the concurrent control values
- mean pup body weight changes in all test groups: did not show any statistically significant differences
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio:
- sex distribution and sex ratios of live F1 pups on the day of birth and 4 days post partum did not show biologically relevant differences between controls and test groups

Details on results (F1)

The statistically significantly increased mean body weights at the top dose pups as well as all other differences between controls and substance-treated group in body weight data do not have any toxicological relevance and are not considered as adverse effects. Instead, they are spontaneous in nature.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks:
equivalent to 673 mg/kg bw/day Isopropanolamine
Sex:
male/female
Remarks on result:
other: No test substance-induced signs of developmental toxicity were noted in the progeny of the F0 parents up to and including 1000 mg/kg body weight/day.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

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

Table 8: Male fertility indices (F0 males) in %:

 

Test group 0

(0 mg/kg bw/day)

Test group 1

(100 mg/kg bw/day)

Test group 2

(300 mg/kg bw/day)

Test group 3

(1000 mg/kg bw/day)

concerning F1 litters

92

92

92

100

Table 9: Female fertility indices (F0 females) in %:

 

Test group 0

(0 mg/kg bw/day)

Test group 1

(100 mg/kg bw/day)

Test group 2

(300 mg/kg bw/day)

Test group 3

(1000 mg/kg bw/day)

concerning F1 litters

92

92

92

100

Applicant's summary and conclusion

Conclusions:
- The NOAEL for reproductive performance and fertility is 1000 mg/kg bw/day (equivalent to 673 mg/kg bw/day Isopropanolamine) for the F0 parental rats
- The NOAEL for general, systemic toxicity is 300 mg/kg body weight/day (equivalent to 202 mg/kg bw/day Isopropanolamine) for the F0 parental males based on some indications of a mild anemic process and 1000 mg/kg body weight/day for the F0 parental females
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 (673 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. Mating was discontinued as soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females.

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. Pups were sexed on day 0 post partum and weighed one day after birth and on day 4 post partum. Their viability was recorded twice daily on each workday or only in the morning on Saturday and Sunday. All pups were sacrificed with CO2 on day 4 post partum and examined macroscopically for external and visceral findings.

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

F1 pups: no adverse effects/findings

- 100 and 300 mg/kg body weight/day:

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 reproductive performance and fertility is 1000 mg/kg body weight/day

(673 mg/kg bw/day Isopropanolamine) for the F0 parental rats. The NOAEL 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.