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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1988
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: Toxicity to reproduction (fertility and developmental toxicity / teratogenicity)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
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)
Principles of method if other than guideline:
- Principle of test: In utero exposure and feeding study in rats
- Short description of test conditions: The parental (P) rats (25 rats/sex/group) were continuously fed TIPA in diet for five weeks prior to mating and during mating, gestation and lactation. Among offspring of the P rats (= F1, 20/sex/group) were fed TIPA for 90-days after weaning. Prior to weaning, these rats were exposed to TIPA in utero during pregnancy of the P rats, in maternal milk during lactation, and in any diet they consumed prior to weaning. Both generations were fed diets that contained 0, 500, 2,000, or 7,500 ppm of TIPA.
GLP compliance:
yes
Limit test:
no
Justification for study design:
According to REACH Annex XI, section 1 and in line with the ECHA Guidance R.7a: Endpoint specific guidance (v6.0, July 2017) and R.6 QSARs and grouping of chemicals (May 2008), a testing (EOGRTS) does not appear scientifically necessary, because there is sufficient weight of evidence from available toxicological data for TIPA and structural similar substances (DIPA and MIPA), leading to the conclusion, that TIPA does not have an adverse effect on reproduction (fertility) and the available data are adequate to support a robust risk assessment.
- see justification attached to IUCLID section 13.2

SPECIFICATION OF STUDY DESIGN FOR ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: 5 weeks
- Basis for dose level selection: selected after consultation with the FDA (for further details please refer to 'Details on study design' below)
- Exclusion of extension of Cohort 1B
- Termination time for F2: no F2 generation was produced
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B
- Exclusion of developmental immunotoxicity Cohort 3
- Route of administration: oral by feeding

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Purity: 98.76%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York
- Age: (P) 22 days old upon arrival and 42 days old at study start
- Weight at study initiation: (P) Males: 189.6-193.4 g; Females: 133.0-138.3 g
- Housing:
- during acclimation: 3 animals/cage, sexes separate, in stainless steel, wire-mesh cages suspended above Upjohn Deotized Animal Cage Boards (DACB) or R2 Reemay-backed cage boards
- after grouping: 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: housed on separate cage racks
- Diet: ad libitum, Irradiated Purina Certified Rodent Chow #5002 (IPCRC) diet
- Water: ad libitum, tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2
- Humidity (%): 50±10
- Air changes (per hr): not specified
- Photoperiod: 12-hour light/12-hour dark
IN-LIFE DATES: From: 03-09-1987 To: 09-08-1987

Administration / exposure

Route of administration:
oral: feed
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- 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.
- Rate of preparation of diet: prepared weekly
- Mixing appropriate amounts with: distilled water (200 mL) and mixed on a magnetic stirrer until dissolved; added to Irradiated Purina Certified Rodent Chow (IPCRC) diet afterwards
- Storage temperature of food: refrigerated until used
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of seven days for the first approach; seven days for the second approach
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: 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:
The parental generation was 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
Dose / conc.:
500 ppm (nominal)
Remarks:
in diet (equivalent to 39.7 and 43.7 mg/kg bw/day for males and females of the F1 generation, respectively)
Dose / conc.:
2 000 ppm (nominal)
Remarks:
in diet (equivalent to 160 and 182 mg/kg bw/day for males and females of the F1 generation, respectively)
Dose / conc.:
7 500 ppm (nominal)
Remarks:
in diet (equivalent to 609 and 700 mg/kg bw/day for males and females of the F1 generation, respectively)
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 1,350 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).

- Fasting period before blood sampling for clinical biochemistry:
Two days prior to collection of blood samples for clinical evaluation, ten rats randomly selected from each group were placed in metabolism cages. The day before blood collection, these rats were fasted for approximately 16 hours. Urine was collected from each rat during this period. At the conclusion of this period, blood samples were collected from the orbital sinus of each rat while the rat was under light carbon dioxide anesthesia.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: conducted at least once daily throughout the study
- Cage-site examinations to detect moribund or dead rats and abnormal behavior and appearance among rats

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: during the premating phase of the study
- each rat was individually handled at each weighing and carefully examined for abnormal behavior and appearance

BODY WEIGHT: Yes
- Time schedule for examinations: once a week during the five-week premating phase of the study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each test group determined during the premating phase: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in F1 male parental generations:
- testis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
- no
Postmortem examinations (parental animals):
None
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 111 (21 days p.p. weaning + 90 days feeding phase) days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

HISTOPATHOLOGY / ORGAN WEIGTHS
- The following tissues indicated were prepared for microscopic examination and weighed, respectively:
Bone marrow, Lymph nodes (mandibular and mesenteric), Spleen, Aorta (thoracic), Heart, Salivary glands, Esophagus, Stomach, Liver (two sections), Pancreas, Small intestine (duodenum, jejunum, and ileum), Large intestine (cecum, colon, and rectum), Kidneys, Bladder, Pituitary, Thyroid - Parathyroid, Adrenals; Males: Prostate, Testes, Epididymides, Seminal vesicles, Females: Mammary gland, Ovaries, Uterus, Vagina; Brain (three coronal sections), Spinal cord, Peripheral nerve (sciatic), Bone (femur and sternum), Eyes, Exorbital lacrimal glands, Harderian glands, All gross lesions
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:
Further details given in other information.
Mating index, fertility index, gestation index
Offspring viability indices:
Further details given in other information.
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):
In the parent rats, there were no significant differences between groups in clinical observations during the premating period or in maternal rats during gestation or lactation.
Mortality:
no mortality observed
Description (incidence):
No rats died in the premating period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In the parent rats, there were no significant differences between groups in body weights and body weight gains during the premating period or in maternal rats during gestation or lactation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption and food efficiency of parental rats were similar between groups. Variability in the values over time and between sexes was related to the normal differences in body weight and food consumption relative to age and sex.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
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.

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
7 500 ppm (nominal)
Based on:
test mat.
Remarks:
equivalent to 602 mg/kg bw/day for males; 693 mg/kg bw/day for females
Sex:
male/female
Remarks on result:
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)

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related effects on F1 pups from any TIPA-treated groups were observed in clinical signs.
In the 90-day feeding phase of the F1 rats, there were no TIPA-related changes in clinical signs.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment-related effects on F1 pups from any TIPA-treated groups were observed in the number born or survival.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related, significant effects on F1 pups from any TIPA-treated groups were observed in body weights.
In the 90-day feeding phase of the F1 rats, there were no TIPA-related changes in body weights or body weight gains.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In the 90-day feeding phase of the F1 rats, there were no TIPA-related changes in food consumption. The mean daily intake of TIPA over the entire 90-day feeding period was 0, 39.7, 160, and 609 mg/kg bw/day. For females at the same dose group the mean daily intake ws 0, 43.7,182, and 700 mg/kg bw/day over the same period.
Food efficiency:
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No changes in the ophthalmological examinations conducted on F1 rats were considered compound-related or biologically significant.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No changes in haematology on F1 rats was considered to be biologically significant or compound related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No changes in the clinical chemistry evaluations conducted on F1 rats were considered compound-related or biologically significant.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No changes in the urine analyses conducted on F1 rats were considered compound-related or biologically significant.
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No changes in the organ weights conducted on F1 rats were considered compound-related or biologically significant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No changes in the pathological evaluations conducted on F1 rats were considered compound-related or biologically significant.
Histopathological findings:
effects observed, non-treatment-related
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
7 500 ppm (nominal)
Based on:
test mat.
Remarks:
(609 mg/kg bw/day for males; 700 mg/kg bw/day for females)
Sex:
male/female
Remarks on result:
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)

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Mean absolute organ weights (g) from male F1 rats

GROUP

CONC.(PPM)

LIVER

KIDNEYS

HEART

ADRENALS

BRAIN

TESTES

I-1

0

20.327 (2.457)

3.679 (0.388)

1.584 (0.120)

0.066 (0.011)

2.129 (0.191)

3.513 (0.231)

III-1

500

21.192 (2.372)

3.821 (0.478)

1.577 (0.148)

0.059 (0.012)

2.114 (0.133)

3.484 (0.231)

V-1

2000

22.022 (3.142)

3.900 (0.391)

1.688 (0.175)

0.066 (0.012)

2.200 (0.151)

3.617 (0.315)

VII-1

7500

21.743 (3.262)

3.924 (0.498)

1.626 (0.167)

0.067 (0.019)

2.181 (0. 143)

3.529 (0.417)

 

Table 2: Mean relative organ weights (% of body weight) from male F1 rats

GROUP

CONC.(PPM)

LIVER

KIDNEYS

HEART

ADRENALS

BRAIN

TESTES

I-1

0

3.7428 (.2441)

0.6792 (.0524)

0.2932 (.0235)

0.0122 (.0019)

0.3958 (.0501)

0.6518 (.0642)

III-1

500

3.8119 (.3235)

0.6874 (.0705)

0.2841 (.0237)

0.0106 (.0021)

0.3818 (.0326)

0.6284 (.0426)

V-1

2000

3.7627 (.3125)

0.6715 (.0746)

0.2898 (.0224)

0.0114 (.0021)

0.3794 (.0381)

0.6251 (.0822)

VII-1

7500

3.8580 (.3470)

0.6987 (.0640)

0.2902 (.0264)

0.0119 (.0035)

0.3902 (.0327)

0.6302 (.0722)

Table 3: Mean absolute organ weights (g) from female F1 rats

GROUP

CONC.

(PPM)

LIVER

KIDNEYS

HEART

ADRENALS

BRAIN

II-1

0

9.623(1.428)

2.212 (0.232)

1.023 (0.125)

0.076 (0.010)

1.995 (0.088)

IV-1

500

9.586(1.455)

2.167 (0.266)

1.029(0.112)

0.073 (0.012)

1.984 (0.064)

VI-1

2000

9.979(1.485)

2.301 (0.201)

1.073 (0.097)

0.078 (0.014)

1.969 (0.075)

VIII-1

7500

9.596(1.242)

2.255 (0.216)

1.027 (O.097)

0.075 (0.014)

1.930 (0.185)

Table 4: Mean absolute organ weights (% of body weight) from female F1 rats

GROUP

CONC.

(PPM)

LIVER

KIDNEYS

HEART

ADRENALS

BRAIN

II-1

0

3.3253 (.2382)

0.7685 (.0638)

0.3552 (.0349)

0.0265 (.0048)

0.6982(.0758)

IV-1

500

3.3637(.2409)

0.7627 (.0521)

0.3631 (.0300)

0.0256 (.0031)

0.7051 (.0715)

VI-1

2000

3.2420(.3182)

0.7539 (.0889)

0.3503 (.0284)

0.0255 (.0045)

0.6458(.0654)

VIII-1

7500

3.4011 (.2820)

0.8009 (.0562)

0.3659 (.0381)

0.0269 (.0053)

0.6888 (.0824)

Standard deviation in paranthesis

+ - Significantly different (P<0.05) from control group by LSD (least standard deviation)

# - Significantly different (P<0.05) from control group LSD and Dunnet's test

Applicant's summary and conclusion

Conclusions:
The two highest levels produced mean daily intake comparable to those at which pathological changes of the kidneys and liver were reported in a drinking water study by another laboratory. However, in the present study no effects were seen at 7,500 ppm. The no-observable-effect level (NOEL) for this study was 7,500 ppm since no effects were observed at any dietary concentration in parental rats or in offspring rats prior to or after weaning.
Executive summary:

The reported feeding study in Crl:CDBR rats describes a combination of a sub-chronic and in utero study, i.e. F1 offspring rats were fed triisopropanolamine (TIPA) for 90-days after exposure prior to weaning / during pregnancy.

Prior to weaning, these F1 rats were exposed to TIPA in utero, in maternal milk during lactation, and in any diet they consumed. The P rats had been continuously fed their group's assigned concentration of TIPA for five weeks prior to mating and during mating, gestation and lactation. Twenty-five rats/sex/group were used for the P generation. Among offspring of the P rats, twenty/sex/group were selected to continue as the F1 generation. Both generations were fed diets that contained 0, 500, 2,000, or 7,500 ppm of TIPA.

Clinical signs, mortality, and body weights of parental rats were recorded throughout the premating period and for females continued to be recorded during the gestation and lactation periods. Food consumption and mean daily intake of TIPA were determined during the premating period for both sexes and for females during gestation. After production of the F1 generation was complete, P rats were sacrificed and discarded without pathological examination. 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. Clinical chemistry and urine analyses were conducted after approximately 45 and 90 days of feeding.

At the end of the 90-day feeding period, all surviving rats were sacrificed and given a gross pathological examination. 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.

In the parent rats, there were no significant differences between groups in clinical observations, body weights, and 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 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 the 90-day feeding phase of the F1 rats, there were no TIPA-related changes in clinical signs, body weights, body weight gains, or food consumption. One death occurred in the high-concentration group, but is not considered compound related. The mean daily intake of TIPA over the entire

90-day feeding period was 0, 39.7, 160, and 609 mg/kg/day for the control, low-, intermediate-, and high-concentration male groups, respectively. For females at the same concentrations the mean daily intake was 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.

The no-observable-effect-level (NOEL) on this study was 7,500 ppm, the highest level tested.