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Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 March to 8 September 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted according to GLP
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: U.S. FDA Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food - 21 CFR 314.50(d)(2)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
1,1',1''-nitrilotripropan-2-ol
EC Number:
204-528-4
EC Name:
1,1',1''-nitrilotripropan-2-ol
Cas Number:
122-20-3
Molecular formula:
C9H21NO3
IUPAC Name:
1,1',1''-nitrilotripropan-2-ol
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): Triisopropanolamine (TIPA)
- Molecular formula (if other than submission substance): C9H21NO3
- Molecular weight (if other than submission substance): 191.27
- Smiles notation (if other than submission substance): CC(O)CN(CC(C)O)CC(C)O
- InChl (if other than submission substance): InChI=1/C9H21NO3/c1-7(11)4-10(5-8(2)12)6-9(3)13/h7-9,11-13H,4-6H2,1-3H3
- Structural formula attached as image file (if other than submission substance): see attached illustration.
- Substance type: No data
- Physical state: No data
- Analytical purity: 98.76%
- Impurities (identity and concentrations): Diisopropanolamine, monopropanolamine, water and ammonia. (Conc not specified.)
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Lot/batch No.: No data
- Expiration date of the lot/batch: No data
- Stability under test conditions: No data
- Storage condition of test material: No data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
124 male and 124 female Crl:CD BR rats were acquired from Charles River Breeding Laboratories, Kingston, New York on 2/17/87. These rats were designated as the parental generation (P) rats to be mated in order to expose the first filial (F1) generation in utero prior to beginning the 90-day feeding phase. The rats were weighed the day after arrival and were 42.0-68.4 and 28.2-50.4 grams for males and females respectively. The rats were born 1/26/87 and were 22 days old upon arrival and 42 days old at study start. This strain was selected on the bases of extensive experience with that strain and its suitability with respect to longevity, hardiness, sensitivity, and low incidence of spontaneous diseases. Upon arrival at Haskell Laboratory, all rats were removed from shipping cartons and housed three per cage, sexes separate, in stainless steel, wire-mesh cages suspended above Upjohn Deotized Animal Cage Boards (DACB®) or R2 Reemay®-backed cage boards. During a quarantine period of 14 days, the rats were fed IPCRC and provided tap water ad libitum. The rats were temporarily identified with colored tail marks and cage identification, weighed at approximately three-day intervals, and observed with respect to weight gain and any gross signs of disease or injury. During the pretest period and throughout the study, the animal room was maintained on a 12-hour light/12-hour dark cycle. The target room temperature and relative humidity were 23±2°C and 50±10%, respectively. After release from quarantine by the laboratory veterinarian, 100 rats of each sex, selected on the bases of body weight gain and freedom from any clinical signs of disease or injury, were divided by computerized-stratified randomization into 4 groups of 25 male and 4 groups of 25 female rats so that there were no statistically significant differences among group body weight means within a sex. The order of rats within each group was randomized. Each group of rats within a sex was designated as either a control, low-, intermediate-, or high-concentration group. After assignment to treatment groups, each rat within a sex was identified by a number tattooed on the tail. During tattooing, but prior to administration of TIPA, one male rat in the high-concentration group was accidentally killed. This rat was replaced with a healthy rat of approximately the same weight that had not been selected during grouping. Because a rat of similar weight was used, mean group weight and differences between groups did not change. After grouping, all rats were individually housed in stainless steel, wire-mesh cages suspended above Upjohn DACB® or R2 Reemay®-backed cage boards. During the premating phase and throughout the study except during mating, male and female parental generation rats were housed on separate cage racks. Rats not selected for the study were sacrificed and discarded without pathological evaluation.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
water
Details on exposure:
During the test period, parent and offspring rats in each group were fed an Irradiated Purina Certified Rodent Chow #5002 (IPCRC) diet that contained 0, 500, 2000 or 7500 ppm TIPA. Throughout the study, all rats were fed the diet of their respective treatment group and provided tap water ad libitum. Before diets were prepared, TIPA was dried with Drierite drying agent under vacuum for several days. All compound was stored in containers which were purged with nitrogen after opening.
TIPA was added to 200 ml of distilled water and mixed on a magnetic stirrer until dissolved. Dissolved TIPA was added to IPCRC and mixed in a Stephan high-speed mixer (model# VCM-80E) for 3 minutes. When this mixer was unavailable, due to a mechanical problem (6/29/87 through 8/4/87), diets were mixed in a Hobart low-speed mixer (model# M-802) for 15 minutes. Control diets, with 200 ml distilled water added, were subjected to the same mixing conditions. All diets were prepared weekly and refrigerated until used.
At the beginning of the study, samples of diet (approximately 50 g each) were collected from each concentration of diet prepared with the test material. These samples were analyzed to verify concentration, homogeneity, and stability of TIPA in the test diets. Concentration/stability samples were collected from the middle of the mixing vessel. They were either frozen the day of preparation, frozen after 24 hours at room temperature, frozen after 17 days at room temperature, or frozen after 17 days of refrigeration. Homogeneity samples were collected from the top, middle, and bottom of the mixing vessel and frozen the day of preparation. A sample of freshly prepared control diet was also collected the day of diet preparation. Diet samples were analyzed by the Molecular and Genetic Toxicology Section of Haskell Laboratory. Samples for the determination of homogeneity and stability of TIPA in the diets were also taken at the end of the P gestation phase. Stability samples were inadvertently stored for 18 days prior to freezing rather than 17 days. This is not considered to have adversely affected the outcome of the study. Homogeneity samples were taken when the diet mixer was changed to low-speed instead of high-speed (6/29/87). When the high-speed mixer was again used to prepare diets (8/17/87) another set of homogeneity samples was collected. Since this was nine days prior to the start of sacrifice, these samples were considered end-of-study samples. At the request of the sponsor to provide data on samples that were never frozen, these samples were extracted on the day of collection and analyzed the following day. Stability samples were not collected at the end of the study since the two sets of stability samples already collected from this study and the two sets of stability samples from a previous study indicated TIPA remained stable in the diet.
Details on mating procedure:
Five weeks after initiation of the P premating phase, P rats were mated to produce F1 litters. For mating, each P female rat was housed with a randomly assigned male until evidence of copulation was obtained (copulation plug) or for a maximum of seven days. If copulation was not detected within seven days, the female was housed for a second period of up to seven days with a different male which had copulated during the previous week. Upon detection of a copulation plug (designated day 0 of pregnancy), each female was returned to individual housing in its assigned cage. On day 14 of gestation, females were housed individually in polypropylene cages with bed-o'cobs cage bedding. Female rats that did not show copulation plugs by the end of the second seven-day mating period were assumed pregnant and were housed individually in the polypropylene cages at the end of this period. Female rats were observed at least twice daily for signs of delivery starting several days prior to expected parturition.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Methanol was added to each diet sample and TIPA was extracted by sonication, filtered, evaporated under nitrogen, and derivatized with Pyridine and N-Trimethylsilylimidazole (TMSI). The reaction was completed by warming the solutions to 80°C for 10 (0, 500 ppm) or 20 minutes (2000, 7500 ppm). Recovery efficiencies were determined at the 500 ppm level by adding 5 mL of a 1000 µg/mL TIPA stock solution to control diet and at the 2000 and 7500 ppm levels by adding 20.3 and 75.9 mg H-16,648, respectively, to 10.0 g control diet. Extraction and preparation of the recovery samples were performed as described above. Calibration curves were created using standarrd solutions of TIPA or TEA. Peak area ratios were calculated (TIPA to TEA) and used for quantitation by linear regression analysis. The dietary concentration of TIPA was determined by multiple injection with a Hewlett-Packard 5880A gas chromatograph.
Duration of treatment / exposure:
Twenty-five parental generation rats/sex/group were fed TIPA in diet for five weeks. After five weeks of feeding, they were mated to produce offspring which were fed diets containing TIPA for 90 days after weaning.
Frequency of treatment:
Daily.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0; 500; 2000 or 7500 ppm TIPA
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0; 39.7; 160; 609 mg/kg bw/day for males of the F1 generation; 0, 43.7; 182; 700 mg/kg/day for females of the F1 generation.
Basis:
other: mean daily intake of TIPA over the 90-day feeding period
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
Dose levels for this study were selected after consultation with the FDA. Dose levels were based upon acute and subchronic studies in rats conducted with TIPA in drinking water. On an acute basis, the single dose oral LD50 of TIPA in rats was 5994 mg/kg bw. When rats were given 140 to 1350 mg TIPA/kg bw/day in drinking water for 30 days, 1350 mg/kg bw/day resulted in growth reduction and decreased food consumption. A dose level of 260 mg/kg bw/day produced unspecified histopathologic changes in some rats. No effects were seen at 140 mg/kg bw/day. In a 90-day study in rats, dose levels of 770 mg TIPA/kg bw/day in drinking water produced kidney effects consisting of dilation of Bowman's capsule and convoluted tubules, as well as marked cloudy swelling of liver parenchyma. A level of 220 mg/kg bw/day produced unspecified pathological changes in some animals. A concentration of 110 mg/kg bw/day for 90 days revealed no microscopic changes. In the present study, the intermediate- and high-dose levels (2000 and 7500 ppm in diet) were expected to produce mean daily intakes in rats of about 200 and 700 mg/kg bw/day, respectively. The intermediate-concentration level was selected to be similar to one at which pathological changes were seen in some rats on a previous study (220 mg/kg bw/day); the high-concentration level was selected to be comparable to a level at which pathological changes were seen in all rats (770 mg/kg bw/day).
Positive control:
None

Examinations

Parental animals: Observations and examinations:
Clinical Observations and Mortality:
Cage-site examinations to detect moribund or dead rats and abnormal behavior and appearance among rats were conducted at least once daily throughout the study. During the premating phase of the study, each rat was individually handled at each weighing and carefully examined for abnormal behavior and appearance.

Body Weights
All rats were weighed once a week during the five-week premating phase of the study.

Food Consumption, Food Efficiency, and Intake of TIPA
The amount of food consumed by each test group during each weighing interval was determined during the premating phase. From these determinations and body weight data, mean individual daily food consumption, food efficiency, and intake of TIPA were calculated.
Oestrous cyclicity (parental animals):
Not assessed.
Sperm parameters (parental animals):
Not assessed.
Litter observations:
Litter counts and weights were determined collectively by sex on day 4 postpartum, prior to and after culling and on day 14 postpartum. 21 Days after birth (weaning) individual pup weights were recorded.
Clinical signs, counts, mortality, and group body weights of F1 offspring were recorded prior to weaning. At weaning, 20 randomly selected F1 rats/sex/group were selected to continue on their respective treatment group's diet (one/sex/litter when possible). The remainder were sacrificed and discarded without pathological examination. Clinical observations, mortality, body weights, and food consumption of the F1 offspring were recorded during a 90-day feeding phase. F1 offspring were given ophthalmological examinations at the beginning and end of the 90-day feeding period. Haematology, clinical chemistry and urine analyses were conducted after approximately 45 and 90 days of feeding.
Postmortem examinations (parental animals):
Not conducted.
Postmortem examinations (offspring):
At the end of the 90-day feeding period, all surviving rats were sacrificed and given a gross pathological examination. All tissues from the control and high-concentration groups were examined microscopically. Lungs, liver, kidneys, and organs with lesions in the low- and intermediate-concentration groups were also examined microscopically.
Statistics:
For the P premating, gestation, and lactation, and F1 feeding phases, body weights, body weight gains, clinical laboratory measurements, and organ weights were analyzed by a one-way analysis of variance. When the test for differences among test group means (F test) was significant, pairwise comparisons between test and control groups were made with the Dunnett's test. Incidence of clinical observations was evaluated by the Fisher's Exact test with a Bonferroni correction and the Cochran-Armitage test for trend. Homogeneity of variances of organ weights and clinical laboratory data were analyzed with the Bartlett's test (alpha = 0.005). When the results of Bartlett's test were significant (variance was not homogeneous), the Mann-Whitney U test was employed instead of Dunnett's test for comparison of means (alpha = 0.05). Comparisons of offspring numbers and weights were made with the Mann-Whitney U test and Jonckheere's test for trend. Incidences of clinical observations in offspring were evaluated by Fisher's Exact test with a Bonferroni correction and the Cochran-Armitage test for trend. Indices of reproductive performance were also evaluated by Fisher's Exact test and the Cochran-Armitage test for trend.
Reproductive indices:
Mating index, fertility index, gestation index.
Offspring viability indices:
Percent pups born alive, viability index, lactation index, litter survival, average number of pups/litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were increased in any dose group (or controls).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences from controls in mean body weight or weight gain or food consumption.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant differences from controls in mean body weight or weight gain or food consumption.
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: Variablility in the mean daily intake values over time and between sexes is related to the normal differences in body weight and food consumption relative to age and sex.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No significant differences between controls and TIPA treated groups.

Details on results (P0)

In the parent rats, there were no significant differences between groups in clinical observations, body weights, and body weight gains during the premating period or in maternal rats during gestation or lactation. No parental rats died during the premating, mating, gestation, or lactation period. Food consumption and food efficiency of parental rats were similar between groups. There were no differences in reproductive parameters (mating index, fertility index and gestation length), attributed to the administration of TIPA

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 7 500 other: ppm (602 mg/kg bw/day for males; 693 mg/kg bw/day for females)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects were observed up to the highest dose tested (602 mg/kg bw/day for males; 693 mg/kg bw/day for females)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No significant differences between the controls and TIPA treated groups.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences between the controls and TIPA treated groups.
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences between the controls and TIPA treated groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant differences between the controls and TIPA treated groups.
Histopathological findings:
no effects observed
Description (incidence and severity):
No significant differences between the controls and TIPA treated groups.

Details on results (F1)

There were no differences attributed to administration of TIPA in gestation length, the number of litters produced (fertility index), or any other indices of reproductive performance. No treatment-related effects on F1 pups from any TIPA-treated groups were observed in the number born, survival, body weights, or clinical signs.

In addition, in the 90-day feeding phase of the F1 rats, there were no TIPA-related changes in clinical signs, body weights, body weight gains, of food consumption. The mean daily intake of TIPA over the entire 90-day feeding period was 0, 39.7, 160, and 609 mg/kg/day. For females at the same dose group the mean daily intake ws 0, 43.7,182, and 700 mg/kg/day over the same period.

No changes in the clinical chemistry evaluations, urine analyses, ophthalmological examinations, organ weights, or pathological evaluations conducted on F1 rats were considered compound-related or biologically significant.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 7 500 other: ppm (609 mg/kg bw/day for males; 700 mg/kg bw/day for females
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects were observed up to the highest dose tested (609 mg/kg bw/day for males; 700 mg/kg bw/day for females)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a FDA guideline study to GLP, groups of 25 parental rats were fed TIPA for 5 weeks prior to pairing and throughout mating, gestation and lactation. The offspring were continued on the diet for 90-days. Both generations were fed at doses of 0, 500, 2000 or 7500 ppm. Since no significant or treatment related effects were observed in the parental rats or offspring at any dose, the no-observed-adverse-effect level (NOAEL) was considered to be 7500 ppm (609 mg/kg bw/day for male and 700 mg/kg bw/day for females), the highest dose tested.
Executive summary:

In a FDA guideline study to GLP, the reproductive toxicity of TIPA in rats was assesed. Groups of 25 parental rats were continually fed TIPA for five weeks prior to pairing and during mating, gestation and lactation at doses of 0, 500, 2000 or 7500 ppm. Clinical signs of toxicity, mortality and body weights of parental rats were recorded throughout. Food consumption and food efficiency was also recorded during the pre-mating period. Once the F1 generation was produced the parental rats were sacrificed without pathological examination.

Clinical signs, mortality and group body weights of the offspring were recorded prior to weaning. At weaning, twenty rats/sex/dose were selected to continue as the F1 generation and were fed TIPA for a further 90 days at the same doses. Clinical signs, mortality, body weights and food consumption of the offspring were recorded during the 90-day feeding period. Clinical chemistry and urine analyses were conducted after approximately 45 and 90 days of feeding. At the end of the feeding period, pathological examinations were carried out on all surviving rats.

There were no significant differences in clinical signs, body weights, food consumption and efficiency between controls and dose groups, during the pre-mating period or in parental rats during gestation and lactation. There were no differences in the reproductive performance and no treatment related effects observed on F1 pups in any TIPA treated groups. In the 90-day feeding period there were no TIPA related changes in clinical signs, body weights or food consumption. Any changes observed in the clinical chemistry evaluations, urine analyses, organ weights, ophthalmological examinations or pathological evaluations were not considered to be biologically significant or related to TIPA administration.

From these results, a no-observed-adverse-effect level (NOAEL) was determined to be 7500 ppm (609 mg/kg bw/day for male and 700 mg/kg bw/day for females), the highest dose tested.