1-(N,N-bis(2-hydroxyethyl)amino)propan-2-ol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

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

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.

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.

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

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.

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: 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.

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

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)

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.

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.

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)

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Good-quality, reliable, GLP-compliant, guideline reproductive toxicity study on the closely-related TIPA (Mullin, 1988), which meets the REACH tonnage-driven data requirements. No adverse effects on fertility were seen at any dose (NOAEL > 600 mg/kg bw/day) in the reproductive toxicity study (Mullins, 1988).

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No adverse effects on the reproductive organs or tissues (including examination of the prostate, seminal vesicles, testes, epididymides, mammary gland, ovaries, uterus and vagina) were seen in the available 28-day study with DEIPA, or in the 90-day study with TIPA. Therefore, an Extended One-Generation Reproductive Toxicity Study (EOGRTS) is not required at Annex IX.

In a FDA guideline study to GLP, the reproductive toxicity of the closely-related material TIPA in rats was assessed. Groups of 25 parental rats were continually fed TIPA for 5 wk 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 (Mullin, 1988).

Short description of key information:
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 the highest tested dose of 7500 ppm (609 mg/kg bw/day for males and 700 mg/kg bw/day for females).

Justification for selection of Effect on fertility via oral route:
Good-quality, GLP-compliant (reliability 2) guideline reproductive toxicity study on TIPA.

Effects on developmental toxicity

Description of key information

In a good-quality guideline study to GLP, gavage administration of DEIPA to time-mated CD rats on GD 6-20 did not produce any clinical signs of toxicity, although treatment at the highest dose (1000 mg/kg bw/day) resulted in maternal toxicity as evidenced by increased kidney weights. Reproductive parameters were not altered and no signs of malformations were seen at up to 1000 mg/kg bw/day. Fetal examinations indicated increases in skeletal variations, exhibited as delayed ossification of several skull bones in fetuses at 300 and 1000 mg/kg bw/day. Based on these data, the no-observed-effect level (NOEL) for maternal toxicity was 300 mg/kg bw/day and the NOEL for fetotoxicity was 100 mg/kg bw/day (Marty and Zablotny, 2001).

 

In a good quality guideline study, conducted to GLP, groups of 24 time-mated female New Zealand white rabbits were administered DEIPA by gavage at dose levels of 0, 100, 300, or 1000 mg/kg bw/day on days 6-28 of gestation. Animals were killed on gestation day 29 and examined for gross pathological alterations. Gravid uterine weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead foetuses. All foetuses were weighed and sexed, and examined for external, visceral and (in 50%) skeletal alterations. No clinical signs of toxicity were seen, reproductive parameters were not altered, and there were no treatment-related malformations or other developmental toxicity observed in the foetuses at any dose of DEIPA. On this basis, the no-observed-adverse-effect levels (NOAELs) for maternal toxicity and developmental toxicity were 1000 mg/kg bw/day, the highest dose tested.

 

 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 October 2020 - 04 January 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

July 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Sasol, Marl, Germany; Batch no. 200162527.
- Purity: 99.6%.
- Appearance: clear liquid.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature, in the dark.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS, UK.
- Age at study initiation: 17 - 22 weeks.
- Weight at study initiation: 2.30 - 4.17 kg.
- Fasting period before study: No.
- Housing: One animal (female) per cage.
- Diet: Commercial diet, 200 g/animal/day. "A small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly."
- Water: Tap water ad libitum.
- Acclimation period: Five days before commencement of treatment (days 1 - 5 after mating).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15 - 21 °C.
- Humidity (%): 45 - 70%.
- Air changes (per hr): Not specified. Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 14 hours light / 10 hours dark.

IN-LIFE DATES:
From: Approx. July 2020 (based on age on arrival at testing facility).
To: 24 - 27 November 2020.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Water was added to pre-weighed test material and mixed (for at least 40 minutes) to obtain a homogenous solution at the highest concentration. Lower concentrations were prepared by serial dilution.

VEHICLE
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in the first and last preparation were analyzed for achieved concentration of the test item.

Details on mating procedure:

Animals were received at the test facility having already been successfully mated. Natural mating with New Zealand white bucks of established fertility at the supplier’s facility. Males and females were not closely related.

Duration of treatment / exposure:

Days 6 - 28 (inclusive) after mating (i.e. 23 days total).

Frequency of treatment:

Once daily.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels selected for this study, were based on the results from a Combined Pilot and Preliminary Study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: At least twice daily.
- Specific cage side observations not listed in study report.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: At least twice daily.

BODY WEIGHT: Yes.
- Time schedule for examinations: On arrival (day 1 after mating), day 3 after mating, and daily on days 6 - 29 after mating.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 29 after mating.
- Organs examined: A full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes.
Examinations included:
- Gravid uterus weight: Yes (including cervix and ovaries).
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of early resorptions: Yes.
- Number of late resorptions: Yes.
- Other: Number of foetuses (live and dead).

Fetal examinations:

- External examinations: Yes [all per litter].
- Soft tissue examinations: Yes [all per litter].
- Skeletal examinations: Yes [all per litter].
- Head examinations: Yes [50% per litter].
- Anogenital distance of all live rodent pups: Not applicable.

Statistics:

The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
Placental, litter and fetal weights

A parametric analysis, using the t-test or F1 approximate test, was performed if Bartlett's test was not significant at the 1% level.

A non-parametric analysis, using Kruskal-Wallis' test, Wilcoxon rank sum tests, or the H1 approximate test, was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.

Historical control data:

Historical control data from 14 studies conducted from January 2020 are included, using New Zealand white rabbits from the same supplier (Envigo RMS) as used in the present study.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal in each of the vehicle control, mid-dose and high-dose groups was killed early for welfare reasons.

In the case of the control and mid-dose animals, the litters had resorbed. "The poor clinical condition of [the mid-dose animal] was attributed to the loss of the litter and not attributable to treatment with DEIPA."

The high-dose animal was killed for welfare reasons on suspicion that the pregnancy had resorbed or aborted. Upon examination, the death of the animal was due to a twisted caecum, and not attributable to treatment with DEIPA.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See Table 4.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was 18% lower than control in the high-dose group. See Table 1.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

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 specified

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

not specified

Description (incidence and severity):

Data not available in study report.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

See Table 3.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

not specified

Description (incidence and severity):

Data on stillbirths and/or dead foetuses not available in study report.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Data, including on early deliveries, not available in study report.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

11 animals were not pregnant: 3 in the control group, 2 in the low-dose group, 4 in the mid-dose group and 2 in the high-dose group. See Table 2.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Total litter weight was marginally reduced in the high-dose group, attributed to the marginally smaller litter size.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

"Marginally smaller" litter size at the high dose. See Table 5.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

See Table 5.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

See Table 6.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

See Table 7.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There was 1 incidence each of lumbar scoliosis, lumbar hemivertebra, bent clavicle and pointing backwards hindpaw(s). See Table 7.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were 2 incidences each of dilated ascending aorta and narrow pulmonary trunk, and 1 each of acephalostomia, truncus arteriosus and septal defect. See Table 7.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of incompletely ossified/unossified epiphyses and metacarpals/phalanges at all treated doses exceeded the concurrent and historical controls ranges. These are a transient stage in foetal development and indicative of foetal immaturity, and therefore non-adverse. See Table 8.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Developmental effects observed:

no

Table 1 - Food consumption during gestation days 6-29

Group	Nominal dose (mg/kg bw/day)	Mean food consumption (g/animal/day)	Standard Deviation
1	0	102	14.6
2	100	100	11.7
3	300	97	14.0
4	1000	84**	14.7

** p < 0.01

 

Table 2 - Number of pregnancies

Group	Nominal dose (mg/kg bw/day)	Number of treated animals	Number of animals that died during study	Number of non-pregnant dams	Number of pregnant dams
1	0	24	1	3	20
2	100	24	0	2	22
3	300	24	1	4	19
4	1000	24	1	2	21

 

Table 3 - Pre- and post-implantation loss - litter data

Group	Nominal dose (mg/kg bw/day)	Number of pregnant dams	Resorptions			Pre-implantation loss (%)	Post-implantation loss (%)
			Early	Late	Total
1	0	20	0.6	0.1	0.7	10.0	8.7
			SD 0.94	SD 0.31	SD 0.92	SD 12.08	SD 11.87
2	100	22	0.7	0.4	1.0	15.3	12.9
			SD 1.36	SD 0.49	SD 1.36	SD 22.51	SD 15.66
3	300	19	0.6	0.4	0.9	8.8	10.3
			SD 1.02	SD 0.76	SD 1.18	SD 12.23	SD 12.05
4	1000	21	0.5	0.3	0.8	13.3	10.8
			SD 0.98	SD 0.64	SD 1.21	SD 18.31	SD 16.00

 

Table 4 - Body and gravid uterine weights

		Group mean values (kg)
Group	Nominal dose (mg/kg bw/day)	Body weight, day 6	Terminal body weight	Body weight change	Gravid uterine weight	Adjusted terminal body weight	Adjusted body weight change
1	0	3.13	3.42	0.29	0.420	3.00	-0.13
		SD 0.415	SD 0.343	SD 0.133	SD 0.0997	SD 0.343	SD 0.154
2	100	3.19	3.45	0.25	0.396	3.05	-0.14
		SD 0.374	SD 0.318	SD 0.145	SD 0.1325	SD 0.222	SD 0.175
3	300	3.13	3.41	0.28	0.408	3.00	-0.13
		SD 0.321	SD 0.301	SD 0.115	SD 0.0718	SD 0.278	SD 0.102
4	1000	3.08	3.36	0.28	0.366	2.99	-0.08
		SD 0.323	SD 0.307	SD 0.174	SD 0.0990	SD 0.274	SD 0.132

 

Table 5 - Live offspring

Group	Nominal dose (mg/kg bw/day)	Mean number of live young			Sex ratio (% male)
		Male	Female	Total
1	0	3.9	3.6	7.5	50.3
		SD 2.13	SD 1.27	SD 2.37	SD 14.27
2	100	4.2	2.8	7.0	62.8
		SD 2.18	SD 2.14	SD 2.60	SD 25.75
3	300	3.1	4.2	7.3	41.5
		SD 1.66	SD 1.65	SD 1.53	SD 21.33
4	1000	3.0	3.7	6.7	43.7
		SD 1.70	SD 1.76	SD 2.19	SD 19.50

 

Table 6 - Litter and foetal weights

Group	Nominal dose (mg/kg bw/day)	Mean total litter weight (g)	Mean foetal weights (g)
			Male	Female	Combined
1	0	279.5	39.2	37.5	38.4
		SD 71.98	SD 6.26	SD 5.97	SD 5.68
2	100	260.6	38.0	36.7	38.0
		SD 96.02	SD 6.86	SD 6.03	SD 5.99
3	300	268.4	36.6	37.3	37.2
		SD 55.61	SD 4.93	SD 4.68	SD 4.71
4	1000	242.9	36.2	36.2	36.6
		SD 74.07	SD 6.06	SD 4.80	SD 4.40

 

Table 7 - Foetal examinations - major abnormality findings

Group			1	2	3	4
Nominal dose (mg/kg bw/day)			0	100	300	1000
Litters examined			20	22	19	21
Total number of litters affected			1	0	0	2
Foetuses examined			150	154	139	141
Total number of foetuses affected			3	0	0	4
Number of foetuses with major abnormalities
Skeletal	Head	Acephalostomia	0	0	0	1
	Cervical/Thoracic	Thoracic scoliosis	1	0	0	0
		Dorsoventral distortion of sternum	1	0	0	0
	Lubmar (and abdominal)/ sacral/ caudal	Sacral/caudal spina bifida	1	0	0	0
		Lumbar scoliosis	0	0	0	1
		Lumbar hemivertebra	0	0	0	1
	Appendicular	Bent clavicle	0	0	0	1
		Pointing backwards hindpaw	0	0	0	1
Visceral	Cervical/Thoracic	Dilated ascending aorta/aortic arch	0	0	0	2
		Dorsally displaced pulmonary trunk	1	0	0	0
		Truncus arteriosus	0	0	0	1
		Membranous ventricular septal defect	1	0	0	1
		Muscular ventricular septal defect	0	0	0	1
		Fistula ascending aorta/pulmonary trunk	1	0	0	0
		Narrow/marked pulmonary trunk	0	0	0	2

 

Table 8 - Foetal examinations - minor abnormality findings

Group		1	2	3	4
Nominal dose (mg/kg bw/day)		0	100	300	1000
Litters examined		20	22	19	21
Foetuses examined		150	154	139	141
Number of foetuses with minor abnormalities
Skeletal	Cranial abnormality - unossified area(s)	0	0	1	0
	Cranial abnormality - sutural bone(s)	1	0	0	0
	Cranial abnormality - bent cornu(a) of hyoid	0	8	3	3
	Vertebral abnormality - thoracic	1	0	0	1
	Vertebral abnormality - lumbar	1	0	0	1
	Rib abnormality - short without costal cartilage	1	0	0	0
	Rib abnormality - misaligned	1	0	0	0
	Rib abnormality - partially fused	1	0	0	0
	Rib abnormality - distally thickened	1	0	0	0
	Rib abnormality - branched with additional costal cartilage	1	0	0	0
	Sternebrae - fused/partially fused	3	3	1	4
	Sternebrae - misaligned hemicentres	0	2	1	0
	Sternebrae - supernumerary site	0	1	0	0
	Sternebrae - wide	1	0	0	0
	Sternebrae - misshapen	2	0	0	0
	Sternebrae - branched 6th	0	1	0	0
	Costal cartilage - partially fused	2	0	0	0
	Costal cartilage - misaligned	0	3	1	2
	Costal cartilage - branched	1	1	0	0
	Costal cartilage - additional	1	0	0	0
	Costal cartilage - 7th not connected to sternum	0	2	0	0
	Appendicular abnormality - absent 1st digit forepaw(s)	0	0	0	1
	Cervical rib abnormality - short supernumerary	1	9	7	2
	Cervical rib abnormality - full supernumerary	1	0	0	0
	Number of 13th ribs - short supernumerary	44	34	37	45
	Number of 13th ribs - full supernumerary	35	32	22	57
	Number of 13th ribs - total	67	61	54	89
	Thoracolumbar vertebrae - 18	1	0	1	0
	Thoracolumbar vertebrae - 20	7	12	4	19
	Pelvic girdle - unilateral cranial shift	2	0	0	0
	Pelvic girdle - unilateral caudal shift	6	2	0	4
Delayed/incomplete ossification	Cranial - large posterior fontanelle	1	0	0	1
	Sternebrae - 5th	20	23	39	24
	Sternebrae - 1st-4th, 6th	9	5	11	13
	Sternebrae - total	28	26	46	32
	Vertebrae - cervical	3	0	0	2
	Vertebrae - thoracic	1	0	0	0
	Appendicular - pubes	0	2	0	1
	Appendicular - epiphyses	11	20	10	14
	Appendicular - talus	1	1	0	0
	Appendicular - metacarpals/phalanges	9	16	15	17
	Appendicular - metatarsals/phalanges	2	7	1	4
Increased ossification	Cranial - partially fused jugal to maxilla	1	1	0	0
	Vertebrae - long lumbar transverse processes	1	0	0	0
Visceral	Eyes - folded retina	3	2	0	0
	Brain - dilated interventricular foramen	1	0	0	0
	Head - supernumerary minor upper incisor	0	1	0	0
	Lungs - abnormal lobation	1	0	0	0
	Gall bladder - bilobed	0	0	1	0
	Ovary - cyst(s)	0	0	1	0
	Head - tongue present	0	0	0	1
	Limb(s) - flexure forepaw(s)	1	1	0	3
	Tail - curled	0	0	0	1
	Tail - kinked	0	2	0	0

Conclusions:

In a good-quality guideline study to GLP, gavage administration of DEIPA at doses up to 1000 mg/kg bw/day (nominal) to time-mated rabbits on GD 6-28 (inclusive) did not produce any clinical signs of toxicity. Reproductive parameters were not altered and no signs of treatment-related malformations were seen. On this basis, the study NOAELs for both maternal and developmental toxicity were 1000 mg/kg bw/day (nominal), the highest dose tested.

Executive summary:

In a good quality guideline study, conducted to GLP, groups of 24 time-mated female New Zealand white rabbits were administered DEIPA by gavage at dose levels of 0, 100, 300, or 1000 mg/kg bw/day (nominal) on days 6-28 of gestation. Animals were killed on gestation day 29 and examined for gross pathological alterations. Gravid uterine weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead foetuses. All foetuses were weighed and sexed, and examined for external, visceral and (in 50%) skeletal alterations.

 

No clinical signs of toxicity were seen, reproductive parameters were not altered, and there were no treatment-related malformations or other developmental toxicity observed in the foetuses at any dose of DEIPA. On this basis, the no-observed-adverse-effect levels (NOAELs) for maternal toxicity and developmental toxicity were 1000 mg/kg bw/day (nominal), the highest dose tested.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Nov 2000 to 14 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guidelne study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)IGS BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (CRL, Portage, MI)
- Age at study initiation: 10-11 weeks (sexually mature adults)
- Weight at study initiation: Approximately 200-250 g
- Fasting period before study: None
- Housing: One animal per cage in stainless steel cages
- Diet (e.g. ad libitum): LabDiet(R) Certified Rodent Diet #5002 (PMI Nutrition International, St Louis, MO) in meal form, ad libitum
- Water (e.g. ad libitum): Municipal water, ad libitum
- Acclimation period: Approximately 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 40-70
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12 hr / 12 hr

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DEIPA was administered as an aqueous solution such that a dose volume of 4 ml/kg bw yielded the target dose. Dose solutions were adjusted for purity. Dose volumes were adjusted daily based on individual body weights. Solutions were prepared periodically based on stability data and were stirred continuously.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of all dosing suspensions from the first mix were initiated prior to the start of dosing. The dose samples were diluted, the DEIPA derivatized, extracted into solvent and the extracts were analyzed by GC/MS.

Since the test material was in a solution, no homogeneity analyses were indicated.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- M/F ratio per cage: Sexually mature virgin females were naturally mated with males of the same strain (one male:one female). Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males' cages.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

Administration on days 6-20 of gestation

Frequency of treatment:

Once daily

Duration of test:

From Day 0 of gestation to necropsy on day 21 of gestation

No. of animals per sex per dose:

25 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a preliminary screening study [see Supporting study - Zablotny et al., 2001]. The high dose of 1000 mg/kg bw/day represented a limit dose as defined in the study guideline. The lower dose levels were selected to provide dose response data for any toxicity that may be observed among the high-dose group rats.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least daily
- Cage side observations included: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behaviour, moribundity, mortality, and the availability of feed and water

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily. Careful, hand-held evaluation of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), unusual swelling or masses, and animal behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0; GD 3 and daily during the dosing period; and at necropsy GD 21

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Stomach, liver, kidneys were dissected and incised and any obvious gross pathologic alterations were noted. Weight of liver, kidneys, gravid uterus were recorded. Stomach and representative sections of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin, although microscopic examination of tissues was not conducted.

OTHER: Maternal necropsy included examination of external tissues and all orifices. Skin was reflected from the carcass, the thoracic and abdominal cavities were opened, and viscera examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

Statistics:

Maternal body weights, body weight gains, organ weights (absolute and relative), fetal body weights and feed consumption were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha=0.05, analysis by Dunnett's test (alpha=0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha=0.05; Hollander and Wolfe, 1973) with Bonferroni's correction (Miller 1966) was performed, respectively. Frequency of pre-implantation loss, post-implantation loss, resorptions per litter and resorptions per fetal population, and fetal alterations were analyzed using a censored Wilcoxon test (alpha=0.05; Haseman and Hoel, 1974) with Bonferroni's correction. The number of corpora lutea and implantations, and number of viable fetuses/litter were evaluated using a nonparametric ANOVA (alpha=0.05) followed by the Wilcoxon Rank-Sum test (alpha=0.05) with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (alpha=0.05; Siegel, 1956) with Bonferroni’s correction. Fetal sex ratios were analyzed using a binomial distribution test. Non-pregnant females were excluded from the appropriate analyses. Statistical outliers were identified, using a sequential method (alpha=0.02; Grubbs, 1969), and excluded if justified by sound scientific reasons. Both Dunnett’s test and Bonferroni’s correction correct for multiple comparisons to the control group to keep the experiment-wise alpha at 0.05. Both were reported at the experiment-wise alpha level.

Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) could be greater than the nominal alpha levels. Therefore, final interpretation of data considered statistical analyses along with other factors, e.g dose-response, biological consistency.

Indices:

Pre-implantation loss = (No. corpora lutea-implantations / No. corpora lutea) x 100

Post-implantation loss = (No. implantations-live born pups / No. implantations) x 100

Historical control data:

Final interpretation of data considered statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Treatment at the highest dose (1000 mg/kg bw/day) resulted in maternal toxicity as evidenced by increased absolute (10.2% increase) and relative (7.0% increase) kidney weights. Kidney weights were unaffected at doses of 100 or 300 mg/kg bw/day. Neither liver weights, body weights, body weight gains nor feed consumption were adversly affected by DEIPA treatment. There were no deaths and no treatment-related clinical signs of toxicity at any dose.

Dose descriptor:

NOEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal examinations indicated that DEIPA induced increases in skeletal variations, exhibited as delayed ossification of several skull bones in fetuses at the 300 and 1000 mg/kg bw/day dose levels.

In the 1000 mg/kg bw/day group, increased incidences of delayed ossification of the parietals, interparietals, and occipitals were statistically identified and were well outside the range of historical controls. In addition, the incidence of delayed ossification of the frontal bones, which are adjacent to the parietals, was well outside the range of historical controls and was considered to be a treatment-related finding. Slight numerical increases in the incidence of delayed ossification of the zygomatic and thoracic centra at 1000 mg/kg bw/day were not statistically identified, were within the range of historic control values, and therefore, were not considered to be toxicologically significant.


In the 300 mg/kg bw/day group, treatment-related skeletal variations were limited to statistically identified increases in the incidence of delayed ossification of the parietals and interparietals.

Although the incidence of delayed ossification of the interparietal and occiptal bones appeared to be slightly increased at 100 mg/kg bw/day, these increases were not statistically identified. Furthermore, the incidence of delayed ossification of the interparietal bone was within the range of historical control values, and delayed ossification of the occipital bone lacked a dose-response relationship. Thus, the skull variations observed at 100 mg/kg bw/day were considered to be incidental.

With regard to malformations, there were no statistically identifed differences in any of the treatment groups when compared to controls. All malformations observed in foetuses from dams administered DEIPA either occurred at low frequency and/or were not dose related.

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

DEIPA: Terminal Body Weights, Kidney weights

Parameter	Dose Level (mg/kg bw/day)
	0	100	300	1000
Final Body Weight (g)	385.0	391.4	392.0	398.4
Absolute Kidney Weight (g)	2.026	1.997	2.066	2.233*
Relative Kidney Weight (g/100)	0.525	0.512	0.525	0.562*

*Statistically different from control mean by Dunnett’s test, alpha-0.05. Bold type indicates effects judged to be treatment-related.

DEIPA: Incidence of Delayed Ossification (D.O.) of Selected Bones

Parameter	Dose Level (mg/kg bw/day)
	Unit	0	100	300	1000
D.O., frontal	F	0.8 (1)^a	0	1.3 (2)	5.4 (8)
	L	4.3 (1)	0	8.0 (2)	12.0 (3)
D.O., parietals	F	0.8 (1)	0.7 (1)	5.9 (9)	12.8 (19)
	L	4.3 (1)	4.0 (1)	28.0 (7)*	40.0 (10)*
D.O., interparietal	F	2.4 (3)	4.7 (7)	16.4 (25)	26.4 (39)
	L	8.7 (2)	20.0 (5)	44.0 (11)*	60.0 (15)*
D.O., occipital	F	0	4.1 (6)	2.6 (4)	6.1 (9)
	L	0	8.0 (2)	8.0 (2)	20.0 (5)*
D.O., zygomatic	F	0.8 (1)	0.7 (1)	0.7 (1)	2.0 (3)
	L	4.3 (1)	4.0 (1)	4.0 (1)	12.0 (3)
D.O., thoracic centra	F	0.8 (1)	0	0.7 (1)	2.0 (3)
	L	4.3 (1)	0	4.0 (1)	12.0 (3)

F = Fetuses; L = Litters

^a= Percent Affected (Number Affected)

*Statistically identified as different from the control mean by Wilcoxon’s test, alpha-0.05. Bold type indicates effects judged to be treatment-related.

Conclusions:

In a good-quality gudeline study to GLP, gavage administration to time-mated CD rats on GD 6-20 did not produce any clinical signs of toxicity, although treatment at the highest dose (1000 mg/kg bw/day) resulted in maternal toxicity as evidenced by increased kidney weights. Reproductive parameters were not altered and no signs of malformations were seen at up to 1000 mg/kg bw/day. Fetal examinations indicated increases in skeletal variations, exhibited as delayed ossification of several skull bones in fetuses at 300 and 1000 mg/kg bw/day. Based on these data, the no-observed-effect level (NOEL) for maternal toxicity was 300 mg/kg bw/day. The NOEL for fetotoxicity was 100 mg/kg bw/day.

Executive summary:

In a good quality guideline study, conducted to GLP, groups of 25 time-mated female CD rats were administered DEIPA by gavage at target dose levels of 0, 100, 300, or 1000 mg/kg bw/day on days 6-20 of gestation. Animals were killed on gestation day 21 and examined for gross pathological alterations. Liver, kidneys and gravid uterine weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead fetuses. All fetuses were weighed and sexed, then one-half were examined for either visceral or skeletal alterations, respectively.

No clinical signs of toxicity were seen, although treatment at the highest dose (1000 mg/kg bw/day) resulted in maternal toxicity as evidenced by increased absolute and relative kidney weights. Kidney weights were unaffected at doses of 100 or 300 mg/kg bw/day. Neither liver weights, body weights, body weight gains nor feed consumption were affected by DEIPA treatment. Reproductive parameters were not altered at doses up to 1000 mg/kg bw/day. Fetal examinations indicated increased skeletal variations, exhibited as delayed ossification of several skull bones in fetuses at 300 and 1000 mg/kg bw/day [see discussion below]. There was no indication of embryo/fetal effects at 100 mg/kg bw/day and no evidence of increased malformations at any dose of DEIPA. Based on these data, the no-observed-effect level (NOEL) for maternal toxicity was 300 mg/kg bw/day. The NOEL for fetotoxicity was 100 mg/kg bw/day.

Delayed (or incomplete) ossification is one of the most common skeletal variations encountered in regulatory guideline developmental toxicity studies. The affected bones form the calvarium (frontals, parietals, interparietal, occipital) which ossify rapidly during the late gestation phase and variable ossification of these late ossifying bones is normal. Delays in ossification are not thought to persist – the postnatal skeleton having considerable capacity to remodel – and are thought to be more indicative of a generalised fetal growth delay that can be influenced by e.g. maternal malnutrition or reduced feed intake. A delay in ossification is not thought to have general predictive value for teratogenesis, and indeed no malformations were seen at any dose. Classification as a reproductive toxin is not deemed necessary in cases where the only effects recorded in experimental animal studies are considered to be of low or minimal toxicological significance. Guidance indicates that such effects include small changes in the proportions of common variants such as are observed in skeletal examinations. On this basis, the reported increased incidence of skeletal variations, indicative of a minor delay in skeletal ossification, and thought to be a transient effect that is completely reversible postnatally, does not justify classification of DEIPA for toxicity to reproduction.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable and good quality guideline developmental toxicity studies in both rats (Marty and Zablotny, 2001) and rabbits (Leggatt, 2021) are available, along with the results of a screening developmental toxicity study in rats (Zablotny et al., 2001).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Two good quality guideline studies, conducted to GLP, are available on the developmental toxicity potential of DEIPA. In the first, groups of 25 time-mated female CD rats were administered DEIPA by gavage at target dose levels of 0, 100, 300, or 1000 mg/kg bw/day on days 6-20 of gestation. Animals were killed on gestation day 21 and examined for gross pathological alterations. Liver, kidneys and gravid uterine weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead foetuses. All foetuses were weighed and sexed, then one-half were examined for either visceral or skeletal alterations, respectively. No clinical signs of toxicity were seen, although treatment at the highest dose (1000 mg/kg bw/day) resulted in maternal toxicity as evidenced by increased absolute and relative kidney weights. Kidney weights were unaffected at doses of 100 or 300 mg/kg bw/day. Neither liver weights, body weights, body weight gains nor feed consumption were affected by DEIPA treatment. Reproductive parameters were not altered at doses up to 1000 mg/kg bw/day. Foetal examinations indicated increased skeletal variations, exhibited as delayed ossification of several skull bones in foetuses at 300 and 1000 mg/kg bw/day. There was no indication of embryo/foetal effects at 100 mg/kg bw/day and no evidence of increased malformations at any dose of DEIPA. Based on these data, the no-observed-effect level (NOEL) for maternal toxicity in rats was 300 mg/kg bw/day. The NOEL for rat foetotoxicity was 100 mg/kg bw/day (Marty and Zablotny, 2001).

 

In the second study, groups of 24 time-mated female New Zealand white rabbits were administered DEIPA by gavage at dose levels of 0, 100, 300, or 1000 mg/kg bw/day on days 6-28 of gestation. Animals were killed on gestation day 29 and examined for gross pathological alterations. Gravid uterine weights were recorded, along with the number of corpora lutea, implantations, resorptions, and live/dead foetuses. All foetuses were weighed and sexed, and examined for external, visceral and (in 50%) skeletal alterations. No clinical signs of toxicity were seen, reproductive parameters were not altered, and there were no treatment-related malformations or other developmental toxicity observed in the foetuses at any dose of DEIPA. On this basis, the no-observed-adverse-effect levels (NOAELs) for maternal toxicity and developmental toxicity in rabbits were 1000 mg/kg bw/day (nominal), the highest dose tested.

 

In support, no developmental effects on the offspring were seen in the reproduction study on TIPA (Mullin, 1988).

Justification for classification or non-classification

Examinations of foetuses of rats administered DEIPA at 300 and 1000 mg/kg bw/day, doses that did not cause maternal toxicity, showed increased skeletal variations, exhibited as delayed ossification of several skull bones.

 

 

Delayed (or incomplete) ossification is one of the most common skeletal variations encountered in regulatory guideline developmental toxicity studies. The affected bones form the calvarium (frontals, parietals, interparietal, occipital) which ossify rapidly during the late gestation phase and variable ossification of these late ossifying bones is normal. Delays in ossification are not thought to persist – the postnatal skeleton having considerable capacity to remodel – and are thought to be more indicative of a generalised foetal growth delay that can be influenced by e.g. maternal malnutrition or reduced feed intake. A delay in ossification is not thought to have general predictive value for teratogenesis, and indeed no malformations were seen at any dose in the rat study with DEIPA.

 

 

Classification as a reproductive toxin is not deemed necessary in cases where the only effects recorded in experimental animal studies are considered to be of low or minimal toxicological significance. Guidance indicates that such effects include small changes in the proportions of common variants such as are observed in skeletal examinations. On this basis, the reported increased incidence of skeletal variations, indicative of a minor delay in skeletal ossification and thought to be a transient effect that is completely reversible postnatally, does not justify classification of DEIPA for toxicity to reproduction.

 

 

The non-classification of DEIPA is supported by the pre-natal developmental toxicity study in rabbits, in which no adverse effects were seen up to the highest treated (nominal) dose of 1000 mg/kg bw/day.