Fatty acids, C18 unsat, reaction products with diethylenetriamine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jan 2021 - 18 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met. Any deviations are detailed (table 01). Following consideration, none were considered to affect the integrity of the study or evaluation of results.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Remarks:

Reference to Dose Range Finding study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

yes

Remarks:

The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study. Detailed within 'Any other information on materials and methods incl. tables'.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

yes

Remarks:

The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study. Detailed within 'Any other information on materials and methods incl. tables'.

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

yes

Remarks:

The deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study. Detailed within 'Any other information on materials and methods incl. tables'.

Principles of method if other than guideline:

Guideline study conducted to GLP.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

Fatty acids, C18 unsat, reaction products with diethylenetriamine

EC Number:

629-715-1

Cas Number:

1226892-43-8

Molecular formula:

UVCB substance - not applicable

IUPAC Name:

Fatty acids, C18 unsat, reaction products with diethylenetriamine

Test material form:

liquid

Details on test material:

- Chemical Name: Fatty acids, C18 unsat, reaction products with diethylenetriamine
- CAS: 1226892-43-8 / EC: 629-715-1
- Lot/Batch No: 1865159
- Clear slightly viscous amber liquid
- pH: 10-12 At a concentration of 75%
- Specific gravity / density: 0.926 at 20°C
- Production Date: July 2019
- Best before Date. July 2022
- The substance is a 100% UVCB.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light container flushed with nitrogen
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability for at least 24 hours at room temperature under normal laboratory light conditions, for at least 8 days in the refrigerator and for at least 3 weeks in the freezer, is confirmed over the concentration range 1 to 200 mg/mL (solutions), Test Facility Study No. 20257162.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: test item handling using amber glassware or wrap containers in aluminum-foil
TREATMENT OF TEST MATERIAL PRIOR TO TESTING: None

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light container flushed with nitrogen
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability for at least 24 hours at room temperature under normal laboratory light conditions, for at least 8 days in the refrigerator and for at least 3 weeks in the freezer, is confirmed over the concentration range 1 to 200 mg/mL (solutions), Test Facility Study No. 20257162.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: test item handling using amber glassware or wrap containers in aluminum-foil

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: None

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Age at study initiation: 18-20 weeks old
- Weight at study initiation: 2873 – 4158 g females
- Fasting period before study: None
- Housing: labelled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm)
- Diet (e.g. ad libitum): Restricted access to pelleted diet for rabbits (KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from Kliba NAFAG Granovit AG, Kaiseraugst, Switzerland). From post-coitum Days 14-17 onwards (for the different subgroups, respectively), celery was daily provided to all animals in order to stimulate food consumption.
- Water (e.g. ad libitum): Municipal tap water ad libitum
- Acclimation period: 5 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-19 ˚C
- Humidity (%): 49-52 %
- Air changes (per hr):
- Photoperiod (12 hrs dark / 12 hrs light):

IN-LIFE DATES: From: 01 Mar 2021 To: 26 Mar 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

Merck, Darmstadt, Germany | Batch: Progly12 and Progly14 | specific gravity: 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 7 to Day 28 post-coitum, inclusive. The dose volume for each animal were based on the most recent body weight measurement. The doses were given using a plastic catheter attached to a plastic disposable syringe.
The dosing formulations were stirred continuously during dose administration.
Dose pot identification via Provantis were used as additional check to verify the dosing procedure according to Standard Operating Procedures.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Propylene glycol (Merck, Darmstadt, Germany). Test item selection confirmed and justified during tolerability and dose-range finding studies.
- Concentration in vehicle: see experimental design (table 3)
- Amount of vehicle (if gavage): dose volume for each animal based on the most recent body weight measurement. See experimental design (table 3)
- Lot/batch no. (if required): Progly12 and Progly14
- Purity: N/A

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The objective of the analytical study was to determine the accuracy of preparation and homogeneity of the test item in formulations. Accuracy and homogeneity were determined for formulations prepared for use during treatment in Week 1.
The chemical analyses were conducted as specified below. Samples and remaining formulation were protected from light using amber glassware.
Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 10 mL (Group 1 and Group 2),
20 mL (Group 3) or 50 mL (Group 4). For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations.
The volumetric flasks were filled up to the mark with methanol. The solutions of Groups 2-4 were further diluted with methanol by a factor of 10000 (Group 2 and Group 3) or 15000 (Group 4) to obtain concentrations within the calibration range.
Accuracy: No test item was detected in the Group 1 formulation.
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were after correction for the mean recovery of the corresponding QC samples, in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 7 to Day 28 post-coitum, inclusive.

Frequency of treatment:

Daily gavage

Duration of test:

24. Feb - 26 Mar 2021 (29 days per animal, staggered arrival 24-26 Feb)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of the dose range finder, and in an attempt to produce graded responses to the test item.
- Rationale for animal assignment (if not random): N/A
- Fasting period before blood sampling for (rat) dam thyroid hormones: N/A
- Time of day for (rat) dam blood sampling: N/A
- Other: N/A

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/Moribundity Checks At least twice daily throughout the study.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily, starting on Day 7 post-coitum up to the day prior to necropsy. During the dosing period, this observation will be performed postdose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum. In addition, data on body weight Day 0 post-coitum (i.e. the day of mating) were provided by the Supplier (non-GLP) and included in the report.

FOOD CONSUMPTION: YEs
- Food consumption of animals were measured daily from Day 3 post-coitum onwards.


WATER CONSUMPTION: Yes
- Regular basis throughout the study. Water consumption was monitored by visual inspection of the water bottles.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29 post-coitum
- Organs examined:

OTHER: N/A

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
- Other: The number and distribution of live and dead fetuses.

Blood sampling:

N/A

Fetal examinations:

- External examinations: Yes: [ all per litter ]
- Soft tissue examinations: Yes: [ all per litter ]
- Skeletal examinations: Yes: [ half per litter ]
- Head examinations: Yes: [ half per litter ]
- Anogenital distance of all live rodent pups: N/A

Statistics:

STATISTICAL ANALYSIS : All statistical analyses were performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated below according to sex and occasion. See additional information on materials and methods.

Indices:

Litter Variables
Sex Ratio (% males): (No. male fetuses x 100) / Total no. of fetuses |
Pre-Implantation Loss (%): ((No. of corpora lutea – no. of implants) x 100) / No. of corpora lutea |
PostImplantation Loss (%): ((No. of implants – no. of live fetuses) x 100) / No. of implants |
Litter % of Fetuses with Abnormalities: (No. of fetuses in litter with a given finding x 100) / No. of fetuses in litter examined

Historical control data:

Yes, HCD held by laboratory.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Note: Individual findings of females that died preterm are given under Mortality. Decreased feces production was observed for most females in all groups, including control. This finding was considered to be unrelated to treatment with the test item, as it occurred in both treated and control animals at comparable incidence.
Erected fur was incidentally observed in one or two females surviving until scheduled necropsy in the control group and at 5, 15 and 50 mg/kg/day, respectively.
Other clinical observations recorded (i.e. scabs, salivation, loss of fur, swollen or discolored forepaw, hunched posture) occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

A total of 13 females did not survive until scheduled necropsy: two females at 5 mg/kg/day, two at 15 mg/kg/day and nine at 50 mg/kg/day.
Of these premature deaths, five at 50 mg/kg/day (Female Nos. 68, 70, 71, 73 and 82) and one at 15 mg/kg/day (Female No. 49) were considered to be related to treatment with the test item. All females showed strongly reduced or no food intake, along with body weight loss (5-10%) and reduced feces output. Other clinical signs included erected fur, pallor, thin appearance, abnormal feces (pale, mucoid, small in size) and/or anorexia. See table 04. For both females at 5 mg/kg/day (Nos. 37 and 40) and all other females at 50 mg/kg/day (Nos. 67, 74, 79 and 81), the cause of death was not clear, but was likely to be secondary to other influences such as the gavage procedure rather than to the test item. For all of these females, labored breathing (with abnormal sounds), salivation, erected fur and/or pallor were noted (shortly) after dosing. For some of these females, red fluid was noted on the dosing tube (data taken from study daybook). No other (relevant) signs or changes in food intake and body weight were noted for these animals. Macroscopic findings included an enlarged gallbladder, small and large intestines and/or colon distended with gas, thoracic cavity filled with dark, brown watery, gritty fluid or frothy, white content in the trachea, multifocal dark, red foci or pale discoloration in the lungs, pale heart, irregular surface, gelatinous content, mucoid white content, a multifocal crater and/or black multifocal foci in the stomach (fundus wall), accessory lobe (liver), cysts in the oviduct, ectopic splenic tissue, and/or pale discoloration of the kidney).
Finally, Female No. 56 at 15 mg/kg/day was sacrificed in extremis on post-coitum Day 12 which was considered unrelated to treatment with the test item, as findings/observations commenced prior to start of treatment. As such it was concluded that the animal was not able to acclimatize to the test facility within the standard five days. Food intake of this animal was absent from Day 3 post-coitum, resulting in a body weight loss of 13% on Day 12 (compared to Day 0). In-life, reduced feces production, erected fur and a swollen forepaw were noted for this female. At necropsy, dark, black multifocal foci in the stomach (fundus) and ectopic splenic tissue were present. See Table 04.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Note: Individual findings of females that died preterm are given in Section 'Mortality'. At 50 mg/kg/day, slight mean body weight loss (-1% and up to 10% for individual animals) was observed between post-coitum Days 7-9 followed by a reduced body weight gain between post-coitum Days 9-24 (not statistically significant), when compared to the concurrent control group. This was mainly attributed to Female Nos. 68, 70, 71 and 73, that were sacrificed in this period (see Mortality & table 04). Normal body weight gain was noted over post-coitum Days 24-29. This resulted in a reduced absolute mean body weight between post- coitum Days 18-24 (up to 6-7% lower than control). At the end of treatment (on post-coitum Day 29), mean body weight was 5% lower than control (not statistically significant).
At 15 mg/kg/day, a slight reduced body weight gain was noted between post-coitum Days 7-9 (no statistical significance achieved), which recovered to control levels between post-coitum Days 9-29. Therefore, this was considered to be of no toxicological significance.
No changes in body weight and body weight gain were noted at 5 mg/kg/day.
Mean body weight loss (corrected for the weight of gravid uterus), was observed in all groups including controls, without a dose-related trend and was therefore considered to be unaffected by treatment with the test item. See Table 05, and Figure 1 of attached results.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Note: Individual findings of females that died preterm are given in Section 'Mortality'.
Mean food consumption was decreased at 50 mg/kg/day (up to 30% lower, when compared to the concurrent control group) from post-coitum Days 7 onwards (statistical significance achieved between post-coitum Days 7-12 only), which recovered to similar levels over post-coitum Days 18-29 when compared to control.
At 15 mg/kg/day, slightly lower mean food consumption was observed at the start of treatment between post-coitum Days 7-9 (11% of control; no statistical significance achieved) with complete recovery thereafter. Therefore, this was considered of no toxicological relevance.
Note: From post-coitum Days 14, 15 16 or 17 onwards, daily celery was offered to all females in addition to the standard food to stimulate appetite. For some females, recovery of food consumption was noted. See Table 06, and Figure 2 of attached results.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Note: Individual findings of females that died preterm are given in Section Mortality. Macroscopic observations at scheduled necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.
A crater in the stomach (fundus) was noted in 2/22, 5/22, 2/22 and 3/22 females of the control, 5, 15 and 50 mg/kg/day groups, respectively.
Other findings noted among control and treated animals are occasionally seen among rabbits used in these types of study and in the absence of correlated microscopic findings they were considered changes of no toxicological significance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

At microscopic examination mild erosions in the stomach (fundus) were recorded for a single female of the control, 5 and 15 mg/kg/day groups, respectively. This was regarded the microscopic correlate to the craters in the fundus recorded at necropsy. One additional erosion was found in the cardia of the stomach of one female at 15 mg/kg/day.
No microscopic correlates could be found for the recorded craters in the fundus of the remaining animals.
The remainder of the recorded microscopic findings in the stomach were within the range of background pathology encountered in female rabbits subjected to a Prenatal Developmental Toxicity Study. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

The lower mean number of implantation sites at 5, 15 and 50 mg/kg/day was mainly attributed to relatively high values in the control group when compared to historical control data. As values remained well within the range of historical control data, these changes were considered to be unrelated to treatment with the test item.
Mean pre-implantation loss was higher at 50 mg/kg/day when compared with mean concurrent control values (15.82 vs. 5.47% in the control group). As treatment was initiated after the implantation period was completed, this was considered to be unrelated to treatment with the test item.
The number of corpora lutea, post-implantation loss and early and late resorptions were comparable between all groups and within the range of normal biological variation. See table 07 - 08.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The number of corpora lutea, post-implantation loss and early and late resorptions were comparable between all groups and within the range of normal biological variation.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The number of corpora lutea, post-implantation loss and early and late resorptions were comparable between all groups and within the range of normal biological variation.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

All females, including those that were sacrificed or died preterm were found to be pregnant, except for Female No. 16 (control group), Nos. 29, 36 and 42 (5 mg/kg/day), Nos. 47, 51 and 66 (15 mg/kg/day) and Nos. 78 and 81 (50 mg/kg/day). Excluding of females that were sacrificed/died prior to scheduled necropsy and the non-pregnant females resulted in a total of 21, 17, 17 and 12 pregnant females with viable litters for evaluation in the control, 5, 15 and 50 mg/kg/day groups, respectively.
Note: Although a minimum of 16 litters is required for evaluation of developmental data according to the guidelines, the 12 litters available at 50 mg/kg/day were included for evaluation in this study. As such, results at 50 mg/kg/day should be interpreted with caution.
See table 07 - 09.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

mortality

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no test item-related effect on fetal body weights (both sexes) noted following treatment up to 50 mg/kg/day. Fetal weight was comparable between the control and test groups and within the range of biological variation. See table 09.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

See table 08.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 50 mg/kg/day. The apparent lower number of male fetuses at 5 mg/kg/day was considered to be unrelated to treatment with the test item as this finding was not dose-related. See table 08.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Mean litter sizes were 10.7, 9.2, 9.2 and 8.6 live fetuses/litter for the control, 5, 15 and
50 mg/kg/day groups, respectively. The lower mean litter size at 5, 15 and 50 mg/kg/day was
mainly attributed to relatively high values in the control group when compared to historical control data . As values remained well within the range of historical control data and no clear dose-related response was noted, these changes were considered unrelated to treatment with the test item. See table 08 - 09.

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were observed in two fetuses. One fetus at 50 mg/kg/day was noted with a small, open eye and absent pinna and one fetus at 15 mg/kg/day was found with a hyperflexed hindlimb. Due to the lack of a dose-related response and single observations in different dose groups, the recorded external malformations were considered chance findings and not related to treatment with the test item.
No external variations were observed in any groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in the skull, ribs and vertebra. The most frequently observed skeletal malformations were supernumerary lumbar vertebra in 4 (4) and 2 (2) fetuses (litters) of the control and 5 mg/kg/day groups, respectively. Due to the occurrence of these findings in the control group, they were considered incidental and not related to treatment with the test item.
All other skeletal malformations and variations occurred in the absence of a dose-related trend and/or were also found in the control group and considered not related to treatment with the test item.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations were observed in 1 (1), 7 (4), 1 (1) and 3 (2) fetuses (litters) of the control, 5, 15 and 50 mg/kg/day groups, respectively. The malformations principally concerned the heart and major vessels, but also the kidney and brain.
Of the visceral malformations observed, retroesophageal subclavian arteries were seen across different groups. As this finding was noted also in the control group and no dose-related response was observed, this was considered to be unrelated to treatment with the test item. Additionally, four fetuses in one litter (Litter No. 25) at 5 mg/kg/day were observed with tetralogy of fallot and two fetuses in one litter (Litter No. 76) at 50 mg/kg/day were observed with malpositioned kidneys. As each malformation was identified within one litter only and no dose-related response was observed, these findings were considered a familiar cause and not related to treatment with the test item.
The remaining visceral malformations affecting the heart and major vessels were noted in one single fetus at 5 mg/kg/day (Litter No. 39, Fetus R9). Therefore, these findings were considered to be chance findings.
Visceral variations occurred across a variety of structures, the low incidences and group distribution of these findings did not indicate any test item-related effects.

Details on embryotoxic / teratogenic effects:

Summaries of Fetal Abnormalities by Finding and by Classification are included in attachment.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: based on data from 12 litters (due to systemic toxicity leading to maternal mortalities).

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 04: Test Item-Related Premature Decedents

Group	Female No.	Day of No. Sacrifice	Findings
Group 3: 15 mg/kg/day	49	Sacrificed in extremis on Day 19 PC	-  Extremely low FC for 9consecutive days -  BW loss of 6% compared to BW Day 9 PC -  Decreased feces output from Day 10 PC onwards, pale feces output on Day 17-19 PC, erected fur on Day 18 and 19 and anorexia on Day 18 (recorded by the veterinarian). -  Dark, red discoloration of the stomach (fundus) at necropsy -  Gravid with 12 live embryos
Group 4: 50 mg/kg/day	68	Sacrificed in extremis on Day 24 PC	-  Extremely low/absent FC for 8 consecutive days -  BW loss of 5% compared to BW Day 7 PC -  Decreased feces output from Day 9 PC onwards, absent feces output on Days 23 and 24, pale feces on Days 22-24 and erected fur on Days 20-24 PC -  No macroscopic findings at necropsy -  Gravid with 6 live fetuses and 5 late resorptions examined
	70	Sacrificed in extremis on Day 20 PC	-  Extremely low/absent FC for 7 consecutive days -  BW loss of 7%compared to BW Day 7 PC -  Decreased feces output from Day 13 PC onwards, pale feces on Days 19 and 20 PC and erected fur on Day 20 PC -  Enlarged gallbladder and ectopic splenic tissue at necropsy -  Gravid with 12 live embryos
	71	Sacrificed in extremis on Day 20 PC	- Extremely low/absent FC for 7 consecutive days - BW loss of 10% compared to BW Day 7 PC •                -  Decreased feces output from Day 9 PC onwards, mucoid feces and reduced in size on Day 20 PC, erected fur from Day 15 PC onwards and thin and pallor on Days 19 and 20 PC •                -  Enlarged gallbladder and a watery content in the cecum and colon at necropsy •                -  Gravid with 11 live embryos
	73	Sacrificed in extremis on Day 24 PC	- Extremely low/absent FC for 8 consecutive days - BW loss of 6%comparedtoBWDay7PC - Decreased feces output on Day 11 PC and from Day 14 PC onwards, absent feces on Days 21-24, erected fur and thin appearance on Day 24 PC - A prominent lobular architecture of the liver and mucoid white content in the wall of the stomach at necropsy - Gravid with 9 live fetuses
	82	Sacrificed in extremis on Day 20 PC	- Extremely low/absent FC for 8 consecutive days - BWlossof5%comparedtoBWDay7PC - Decreased feces output from Day 10 PC onwards, pale feces reduced in size on Day 20 PC and erected fur on Days 18-20 PC - No macroscopic findings at necropsy - Gravid with 8 live embryos and 1 early resorption

PC = post-coitum, FC = food consumption, BW = body weight.

 

Table 05: Summary of Body Weight Gains (g): Gestation

Bodyweight Gain (Interval)		Day(s) Relative to Mating (Litter: A)
Sex: Female		7 → 9 [G]	9 → 12 [G1]	12 → 15 [G]	15 → 18 [G1]	18 → 21 [G]	21 → 24 [G]	24 → 27 [G]	27 → 29	7 → 29
0 mg/kg/day Group 1	Mean SD N	39.0 46.2 21	32.0 34.1 21	48.6 49.7 21	42.2 83.2 21	49.3 39.0 21	62.6 41.3 21	35.7 61.7 21	31.4 41.1 21	340.9 162.9 21
5 mg/kg/day Group 1	Mean SD N	29.1 43.4 19	71.1 64.6 19	93.9 65.7 18	28.2 46.7 18	39.1 45.5 18	48.1 53.5 17	61.9 53.3 17	49.4 49.1 17	415.7 198.5 17
15 mg/kg/day Group 1	Mean SD N	3.7 74.5 19	45.5 56.7 19	73.5 62.9 18	8.4 58.2 18	48.1 54.4 17	55.4 50.5 17	17.0 62.3 17	57.6 37.8 17	346.8 172.5 17
50 mg/kg/day Group 1	Mean SD N	-23.1** 63.9 20	21.0 90.6 20	38.9 90.5 20	14.9 92.2 18	28.6 78.3 15	46.9 57.9 15	60.2 65.8 13	49.1 54.5 12	326.6 193.7 12

[G] - Anova & Dunnett: ** = p ≤ 0.01

[G1] - Kruskal-Wallis & Dunn

 

Table 06: Summary of Food Consumption: Gestation

Food Mean Daily Consumption (g/animal/day)		Day(s) Relative to Mating (Litter: A)
Sex: Female		7 → 9 [G]	9 → 12 [G1]	12 → 15 [G]	15 → 18 [G1]	18 → 21 [G]	21 → 24 [G]	24 → 27 [G]	27 → 29	7 → 29
0 mg/kg/day Group 1	Mean SD N	133.14 26.09 21	126.90 21.44 21	80.24 43.04 21	96.10 39.71 21	115.63 28.42 21	100.59 24.12 21	86.30 27.27 21	89.07 36.02 21	104.18 20.40 21
5 mg/kg/day Group 1	Mean SD N % Diff	138.03 18.90 19 3.67	132.18 22.47 19 4.15	101.33 43.63 19 26.29	121.07 28.66 18 25.99	133.15 20.75 18 15.15	108.08 28.48 17 -2.27	93.41 27.86 17 8.24	92.00 29.44 17 3.29	114.41 20.25 17 9.84
15 mg/kg/day Group 1	Mean SD N % Diff	188.13 40.38 19 -11.27	113.46 44.89 19 -10.60	99.65 45.03 18 24.19	97.70 53.39 18 1.67	116.82 31.79 17 1.03	114.39 26.03 17 3.44	90.57 34.24 17 4.94	94.68 28.10 17 6.29	109.30 26.00 17 4.93
50 mg/kg/day Group 1	Mean SD N % Diff	100.78** 25.07 20 -30.09	88.72** 34.12 20 -30.09	66.02 34.86 20 -17.72	69.59 46.08 18 -27.58	107.51 50.61 15 -7.03	106.33 45.09 15 -3.85	95.03 27.23 13 10.11	89.17 36.10 12 0.11	99.62 17.03 12 -4.37

[G] - Anova & Dunnett: ** = p ≤ 0.01

[G1] - Kruskal-Wallis & Dunn

 

Table 07: Summary of Maternal Performance and Mortality

		0 mg/kg/day Group 1	5 mg/kg/day Group 1	15 mg/kg/day Group 1	50 mg/kg/day Group 1
Group Size - Females		22	22	22	22
Number of Females Pregnant [f]	N+ve %	21 95.5	19 86.4	19 86.4	20 90.9
Female with Live Fetuses [f]	N+ve %	21 100.0	18 94.7	19 100.0	19 95.0
Total Resorptions [f]	N+ve %	0 0.0	0 0.0	0 0.0	0 0.0
Female with all Nonviable [f]	N+ve %	0 0.0	1 5.3	0 0.0	1 5.0
Terminal Euthanasia [f]	N+ve %	22 100.0	20 90.9	20 90.9	13** 59.1**
Unscheduled Death/Euthanasia [f]	N+ve %	0 0.0	2 9.1	2 9.1	9** 40.9**
Found Dead [f]	N+ve %	0 0.0	1 4.5	0 0.0	1 4.5
Unscheduled Euthanasia [f]	N+ve %	0 0.0	1 4.5	2 9.1	8** 36.4**
Delivered [f]	N+ve %	0 0.0	0 0.0	0 0.0	0 0.0

[f] - Fisher's Exact: ** = p ≤ 0.01

 

Table 08: Summary of Ovarian and Uterine Examinations and Litter Observations

Days relative to mating		0 mg/kg/day Group 1	5 mg/kg/day Group 2	15 mg/kg/day Group 3	50 mg/kg/day Group 4
Female with live fetuses	N +ve %	21 100	17 100.0	17 100.0	12 100.0
Number of corpora lutea (k)	Mean SD N % diff	12.0 1.9 21 -	11.2 2.7 17 -6.0	11.1 2.0 17 -7.5	11.6 3.3 12 -3.1
Number of Implantations (k)	Mean SD N % diff	11.2 1.6 21 -	10.1 2.4 17 -10.5	9.9 2.4 17 -12.1	9.4 2.6 12 -16.2
Pre-implantation loss (%) (k)	Mean SD N % diff	5.47 7.63 21 -	9.29 14.07 17 70.04	10.74 11.69 17 96.52	15.82 23.75 12 189.41
Total number of fetuses (k)	Mean SD N % diff	10.7 1.5 21 -	9.2 2.4 17 -13.4	9.2 2.0 17 -13.4	8.7 3.0 12 -18.8
Number of libe fetuses	Mean SD N % diff	10.7 1.5 21 -	9.2 2.4 17 -13.4	9.2 2.0 17 -13;4	8.6 2.9 12 -19.5
Number of dead fetuses (k)	Mean SD N % diff	0.0 0.0 21 -	0.0 0.0 17 -	0.0 0.0 17 -	0.1 0.3 12 -
Number of early resorptions (k)	Mean SD N % diff	0.3 0.7 21 -	0.5 0.8 17 58.8	0.5 0.9 17 41.2	0.6 1.2 12 75.0
Number of late resorptions (k)	Mean SD N % diff	0.2 0.4 21 -	0.3 0.8 17 23.5	0.2 0.7 17 -25.9	0.2 0.4 12 -30.0
Total Number of Resorptions [k]	Mean SD N % diff	0.6 0.7 21 -	0.8 1.2 17 44.1	0.6 1.1 17 13.2	0.8 1.5 12 31.3
Post-implantation Loss (%) [k]	Mean SD N % diff	4.92 5.92 21 -	8.12 12.70 17 64.86	5.70 9.60 17 15.81	9.61 14.43 12 95.12
Number of Live Male Fetuses [k]	Mean SD N % diff	5.6 1.4 21 -	3.9* 2.0 17 -29.9	4.9 2.2 17 -12.1	4.3 1.9 12 -24.4
Number of Live Female Fetuses [k]	Mean SD N % diff	5.0 1.9 21 -	5.3 2.8 17 5.9	4.3 1.4 17 -14.1	4.3 1.7 12 -13.3
Live Male Fetus/Litter (%) [k]	Mean SD N % diff	53.35 14.30 21 -	44.74 23.81 17 -16.15	51.95 17.25 17 -2.63	47.82 12.53 12 -10.36
Live Female Fetuses/Litter (%) [k]	Mean SD N % diff	46.21 14.76 21 -	55.26 23.81 17 19.58	48.05 17.26 17 3.97	51.34 13.01 12 11.10
Mean Fetal Weight males (g) [G]	Mean SD N % diff	38.37 3.61 21 -	39.40 6.70 17 2.67	39.74 5.16 17 3.57	41.17 5.37 12 7.29

[k] - Kruskal-Wallis & Dunn: * = p ≤ 0.05

[G] - Anova & Dunnett

 

Table 09: Summary of Ovarian and Uterine Examinations and Litter Observations

Sex: Female Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	5 mg/kg/day Group 2	15 mg/kg/day Group 3	50 mg/kg/day Group 4
Mean Fetal Weight females (g) [G]         Mean Fetal Weight all (g) [G]	Mean SD N %Diff   Mean SD N %Diff	37.36 4.18 21 - 3   37.89 3.76 21 -	38.42 3.91 16 2.82   39.34 4.99 17 3.81	40.00 5.02 17 7.06   39.91 4.73 17 5.33	39.46 4.39 12 5.61   40.23 4.60 12 6.16

Applicant's summary and conclusion

Conclusions:

Based on the results of this prenatal developmental toxicity study in time-mated female New Zealand White rabbits, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Fatty acids, C18 (unsaturated), reaction products with diethylenetriamine were established:
Maternal NOAEL: 15 mg/kg/day (based on test item-related mortality, due to reduced food intake and lower body weight gain at
50 mg/kg/day). | Developmental NOAEL: At least 50 mg/kg/day (based on data from 12 litters).

Executive summary:

Objective

The objectives of this study were to determine the potential of Fatty acids, C18 (unsaturated), reaction products with diethylenetriamine (the test item) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female New Zealand White rabbits from Day 7 to 28 post- coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

 

Study Design

The dose levels in this study were selected to be 0, 5, 15, 50 mg/kg/day, based on the results of the Dose Range Finder (Test Facility Reference No. 20257164). The study design is outlined within table 04.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, macroscopic examination, uterine contents, corpora lutea, implantation sites and pre- and post-implantation loss.

In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations.

Formulation analyses confirmed that formulations of test item in Propylene glycol were prepared accurately and homogenously.

 

Maternal Toxicity

Treatment with the test item resulted in a significant level of maternal toxicity at 50 mg/kg/day.

At this high dose level, five females were euthanized in extremis between post-coitum Days 17 and 24. These females showed severely reduced or no food consumption for at least 7 consecutive days and a significant body weight loss (5-10% compared to body weight at start of treatment) during the treatment period. Clinical signs of toxicity noted among these females included erected fur, thin appearance, pallor, and/or pale or mucoid feces, feces reduced in size and amount.

At 15 mg/kg/day, one female was sacrificed in extremis on Day 19, based on similar findings as observed for the animals at 50 mg/kg/day (reduced or absent food consumption, body weight loss and reduced, pale feces production, erected fur and anorexia). Due to this single occurrence at 15 mg/kg/day, which incidentally also occurs in controls, this sacrifice was not considered to be toxicologically relevant.

In addition, two females at 5 mg/kg/day, one female at 15 mg/kg/day and three females at 50 mg/kg/day died or were sacrificed in extremis between post-coitum Days 13 and 23, due to severe breathing issues and quick deterioration of condition. For some of these animals, red fluid was noted on the dosing tube and at necropsy macroscopic observations related to the dosing issues were observed for most of the females. Therefore, the cause of death for these females was considered to be gavage procedure-related, rather than test item-related.

Finally, the preterm sacrifice of one female at 15 mg/kg/day was considered unrelated to treatment with the test item, as the findings that led to early sacrifice were already present prior to start of treatment (reduced feces production, erected fur and a swollen forepaw).

Overall, a lower mean food consumption (up to 30%) was noted in females at 50 mg/kg/day between post-coitum Days 7-18 (statistically significantly over Days 7-12 post-coitum), which appeared to recover to control levels over post-coitum Days 18-29. As a result, body weight loss (up to 10% for individual animals) was noted for most females treated at 50 mg/kg/day between post-coitum Days 7-9 followed by a reduced body weight gain between post-coitum Days 9-24 in individual females. As a result, five of these females were euthanized in extremis between post-coitum Days 17-24. In pregnant females surviving until scheduled necropsy, normal body weight gain was noted from post-coitum Day 9 (n=8) or 12 (n=4) onwards. Despite the apparent recovery, mean body weight was 5% lower than control on post-coitum Day 29. Given the magnitude of the observed effects on body weight gain and food consumption, resulting in sacrifice of five females at 50 mg/kg/day, these effects were considered adverse.

Findings for the stomach included macroscopic craters in the fundus and were noted in 2/22, 5/22, 2/22 and 3/22 females of the control, 5, 15 and 50 mg/kg/day groups, respectively. At microscopic examination of the control group and the 5 and 15 mg/kg/day treated groups, focal mild erosions in the fundus were found in a single animal, this was regarded to be the microscopic correlate for the craters. In most rabbits there was no microscopic correlate. These erosions were not seen in the rabbits at 50 mg/kg/day and not regarded a test item-effect. As such, they are suggested to be mainly related to the gavage procedure (tip of gavage touching fundus on insertion).

After exclusion of the females sacrificed/died prior to scheduled necropsy (No. 40 at 5 mg/kg/day, Nos. 49 and 56 at 15 mg/kg/day and Nos. 67, 68, 70, 71, 73, 74, 79, 81 and 82 at 50 mg/kg/day) and the non-pregnant females (No. 16 of control, Nos. 29, 36 and 42 at 5 mg/kg/day, Nos. 47, 51 and 66 at 15 mg/kg/day and No. 78 at 50 mg/kg/day), a total of 21, 17, 17 and 12 pregnant females were available with viable litters for developmental evaluation in the control, 5, 15 and 50 mg/kg/day groups, respectively.

The number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, and pre- and post-implantation loss were considered not to be affected by treatment with the test item up to 50 mg/kg/day.

 

Developmental Toxicity

Note: The total number of litters with viable fetuses available for evaluation was 21, 17, 17 and 12 in the control, 5, 15 and 50 mg/kg/day groups, respectively. Although a minimum of 16 litters is required for evaluation of developmental data according to the guidelines, for completeness of information the 12 litters available at 50 mg/kg/day were included for evaluation in this study. As such, the results at 50 mg/kg/day should be interpreted with caution.

No test item-related changes were noted up to 50 mg/kg/day in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female New Zealand White rabbits, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for the test item were established:

Maternal NOAEL: 15 mg/kg/day (based on test item-related mortality, due to reduced food intake and lower body weight gain at

50 mg/kg/day).
Developmental NOAEL: At least 50 mg/kg/day (based on data from 12 litters).