Propanol, iminobis-, N-(C16-18 and C18-unsatd. alkyl) derivs.

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Oct 2021 - 17 Dec 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol

EC Number:

695-977-9

Cas Number:

1309955-79-0

Molecular formula:

Not applicable UVCB

IUPAC Name:

2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol

Test material form:

liquid

Details on test material:

Chemical name: 2-[(2-hydroxypropyl)(C16-18 sat. C18 unsat. alkyl)amino]propan-1-ol
EC no.: 695-977-9

To the best of knowledge, the sample used is representative to the boundary composition shared and agreed by each registrant

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 10-14 weeks old
- Weight at study initiation: 187 – 269 g
- Fasting period before study: not applicable
- Housing: Individually in polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: ad libitum, SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany. Results of analysis for nutritional components and environmental contaminants were provided by the Supplier. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water: ad libitum, municipal tap water. Periodic analysis of the water was performed. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: 5-6 days
- Justification of Test System and Number of Animals: At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models, which do not use live animals, currently do not exist. The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for developmental toxicity testing by regulatory agencies. The test facility has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of developmental toxicants. The total number of animals to be used in this study is considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.


ENVIRONMENTAL CONDITIONS
- Temperature: 21°C (actual daily mean)
- Humidity: 39-70% (actual daily mean)
- Air changes: At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light):12 h light /12 h dark

IN-LIFE DATES: From: 18 Oct 2021 To: 05 Nov 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Remarks:

Specific Gravity: 0.91

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least weekly. Formulations were divided into aliquots and stored in the refrigerator (4°C) until use. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment were made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 0, 25, 75, 93.75 mg/mL
- Amount of vehicle: 4 mL/kg
- Lot/batch no.: not reported
- Specific Gravity: 0.91

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During week 1 of treatment dose formulation samples were collected for analysis.

For concentration analysis, samples from the middle part of the dosing container of all dose groups were taken (2 x approximately 500 mg). For concentration, all mean sample concentration results within or equal to ± 10% (solutions) of theoretical concentration were considered acceptable.

For homogeneity analysis, samples from the top, middle and bottom parts of the dosing container of groups 2 and 4 (low- and high-dose groups) were taken (2 x approximately 500 mg). Samples were stored at a temperature set to maintain 18-22°C. For homogeneity, a relative standard deviation (RSD) of concentrations of ≤10% for each group was considered acceptable.

Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20279388) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Details on mating procedure:

Untreated females were mated at the Supplier and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).

Duration of treatment / exposure:

Day 6 to Day 20 post-coitum

Frequency of treatment:

once daily

Duration of test:

from Day 2 to Day 21 (scheduled euthanasia)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2 - Low dose group

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3 - Mid dose group

Doses / concentrations 3

Dose / conc.:

375 mg/kg bw/day (actual dose received)

Remarks:

Group 4 - High dose group

No. of animals per sex per dose:

22 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on information provided by the Sponsor and the results of a Dose Range Finding Study with oral exposure of Tallow amine propoxylate in male rats (Test Facility Study No. 20279383), and in an attempt to produce graded responses to the test item. In the Dose Range Finding Study, 3 male rats per group were treated with 150, 200, 250, 300, 450 and 600 mg/kg bw/day. At 600 mg/kg bw/day, all 3 males were sacrificed in extremis on Day 12 of treatment with signs of erected fur, hunched posture, decreased activity, pale tail and salivation. Body weight loss (up to 18%) and reduced food consumption were noted. Macroscopic examination showed decreased adipose tissue in 3/3 males and dark red discoloration of the adrenals in 1/3 males. At 450 mg/kg bw/day, no mortality occurred. Clinical signs that were noted included erected fur, hunched posture and salivation on several days. Reduced body weight gain and food consumption were noted during Day 1-14 of treatment. Absolute mean body weight was reduced by 22% and 15% compared to treatment at 150 and 300 mg/kg bw/day, respectively. Macroscopic examination revealed no abnormalities; relative kidney and liver weights were slightly decreased. At 300 mg/kg bw/day, all animals were noted with erected fur, hunched posture and salivation on selective days only. Reduced body weight gain and food consumption was noted during Day 1-4 of treatment, which remained slightly lower between Days 4-14 of treatment. Macroscopic examination revealed no abnormalities; relative kidney and liver weights were slightly decreased. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

- Fasting period before blood sampling for (rat) dam thyroid hormones: Animals were not fasted

- Time of day for (rat) dam blood sampling: Sampled between 07.00 and 09.00 from the jugular vein on necropsy day

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was checked at least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency was at least once daily. Cageside observations were performed twice daily starting on Day 6 post-coitum up to the day prior to necropsy. Observations were made directly pre-dose and 1 to 2 hours postdose.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 2, 6, 15 and 21 post-coitum

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule for examinations: Over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 post-coitum using carbon dioxide inhalation. All animals (including animals found dead or sacrificed before planned necropsy and females with delivery prior to necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.

OTHER:
- Tissue Collection: The thyroid gland and macroscopic abnormalities were collected from all animals and preserved in 10% buffered formalin.
- Microscopic evaluation: Thyroid gland of all animals was examined microscopically.

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: Number and distribution of live and dead fetuses. Distribution of early and late resorptions.

Blood sampling:

- Plasma: No
- Serum: Yes
- Volume collected: 1 mL
- Serum was collected for T3, T4 and TSH analysis

Fetal examinations:

- External examinations: Yes: all per litter (for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum, late resorptions and fetuses of dams not surviving until Day 21 post-coitum)

- Soft tissue examinations: Yes: half per litter (for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum)

- Skeletal examinations: Yes: half per litter (for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum)

- Head examinations: Yes: half per litter(for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum)

- Anogenital distance of all live rodent pups: Yes, for viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum

- Internal and external sex determination: Yes: all per litter (for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum)

- Body weight: Yes: all per litter (for viable and non-viable fetuses of dam surviving until scheduled necropsy on Day 21 post-coitum)

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons have been conducted using two sided tests and will be reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

- Parametric/Non-Parametric: Levene’s test was used to assess the homogeneity of group variances. The groups was compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons was conducted using Dunnett’s or Dunn’s test, respectively.

- Non-Parametric: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.

The data corresponding to a response variable of interest and to a related covariate was submitted to an analysis of covariance (ANCOVA), including only groups with at least three non missing paired values and if found to be significant, then pairwise comparisons was conducted using Dunnett’s test.

- Incidence: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Indices:

- Body Weight Gains: Calculated for the following intervals: Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21 and 6 to 21 post-coitum.

- Corrected Body Weight Gain: Body weight on Day 21 post-coitum - body weight on Day 6 post-coitum - gravid uterus weight.

- Overall Food Consumption: Calculated between each scheduled interval (individual data only) and as specified above for body weight gains. Summarization and statistical analysis intervals reflect the same intervals as the body weight gains.

- Pregnancy Rate (%): No. of pregnant females x 100 / No. of mated females

- Organ Weight Relative to Body Weight: Calculated against body weight recorded on Day 21 post-coitum

- Male Fetuses (%): No. of male fetuses x 100 / No. of fetuses

- Female Fetuses (%): No. of female fetuses x 100 / No. of fetuses

- Pre-Implantation Loss (%): No. of corpora lutea – No. of implantations x 100 / No. of corpora lutea

- Post-Implantation Loss (%): No. of implantations – No. of live fetuses x 100 / No. of implantations

- Litter % of Fetuses with Abnormalities: No. of fetuses in litter with a given finding x 100 / No. of fetuses in litter examined

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, erected fur and/or a hunched posture was noted for all females on most days of the Dosing Period. It should be noted that on most days, these post-dose findings were continuously observed pre-dose on the following day. In addition, 2/9 females had black discoloration of their feces on post-coitum Days 13 and 14.

At 300 mg/kg bw/day, all females presented with erected fur on several occasions during the Dosing Period and a hunched posture was noted for 6/19 females on 1-5 days. These postdose findings were continuously observed pre-dose on the following day. No discoloration of the feces was observed at this dose level.

At 100 mg/kg bw/day, erected fur was noted for 4/22 females on 1-3 days. For comparison, a single female in the control group was noted with erected fur on a single day. Additionally, black feces was noted for 8/22 females of the 100 mg/kg/day dose group on 1-3 days.

Salivation seen after dosing among 2/22, 17/19 and 9/9 females of the 100, 300 and 375 mg/kg bw/day dose groups mostly from the second week of treatment onwards was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

Incidental findings that were noted included fur loss, and scabbing, red discoloration of the skin and/or slight swelling of several parts of the body. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered not to be signs of toxicological relevance.

Mortality:

mortality observed, treatment-related

Description (incidence):

In total, 16 females did not survive until scheduled necropsy, of which 13 females were treated at 375 mg/kg bw/day and 3 females were treated at 300 mg/kg bw/day. Details on these females are given in the text table below. At 100 mg/kg bw/day, no mortality occurred.

Test item-related mortality at 375 mg/kg bw/day:
Eleven females were euthanized in extremis based on body weight loss (between 9-14% compared to their highest body weights). Food consumption was moderately to severely reduced for all females. Relevant clinical signs included hunched posture, erected fur, decreased activity, reduced production and/or black or orange discoloration of the feces, and/or incidentally salivation. Macroscopic findings that were noted at necropsy included yellow, gelatinous content of the stomach and a large part of the intestine (i.e. duodenum, jejunum, ileum; for one female, and cecum for four females, enlargement of the adrenal glands for four females, black discoloration of the right adrenal gland for one female, and a small thymus for two females. Three female were without necropsy findings. All females were pregnant with live fetuses.
Another two females at 375 mg/kg bw/day were euthanized in extremis, based on clinical signs including decreased activity, closed eyes, hunched posture, erected fur, black and/or orange discolored feces and/or incidentally salivation. Food consumption was severely reduced for both females and body weight loss (4% compared to their highest body weights) was noted. Macroscopic findings that were noted at necropsy included enlargement of the adrenal glands and a small thymus for both females. Both females were pregnant with live fetuses.

Test item-related mortality at 300 mg/kg bw/day:
Three females were euthanized in extremis based on severe body weight loss (between 10-12% compared to their highest body weights). Food consumption was moderately to severely reduced for all females. Clinical signs included hunched posture, erected fur, black or orange discolored feces and/or incidentally salivation. The only macroscopic finding at necropsy was enlargement of the adrenal glands for 2/3 females. These two females were pregnant with live fetuses, the third female was non-pregnant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, body weight gain was lower throughout the Dosing Period, resulting in a 20% lower mean body weight compared to control, at the end of the Dosing Period.

At 300 mg/kg bw/day, a lower mean body weight gain was observed throughout the Dosing Period (statistically significant between Days 9-12 and 18-21 post-coitum), resulting in 13% lower mean body weight compared to control at the end of the Dosing Period (not statistically significant until Day 13 post-coitum).

At 100 mg/kg bw/day, mean body weight gain was in the same range as control during the observation period. The apparent slightly lower mean body weight was caused by the fact that animals allocated to the low dose group coincidentally had a mean body weight that was 3% lower than control (not statistically significant).

At 300 and 375 mg/kg bw/day, gravid uterus weight showed a decreasing trend with increasing dose concentrations (6% and 17% lower than control, respectively; not statistically significant). It should be noted that due to high toxicity at 375 mg/kg bw/day, data from only nine females were available for evaluation at this highest dose tested. Gravid uterus weight at 100 mg/kg bw/day was in the same range as control.
Note: One female at 100 mg/kg bw/day) and one at 300 mg/kg bw/day were noted with low gravid uterus weight due to small litters (3 and 5 live fetuses, respectively).

Body weight loss when corrected for gravid uterus weight was noted at 300 and 375 mg/kg bw/day (-6 grams and -22 grams). At the individual level, 11/18 females at 300 mg/kg bw/day and all 9 females at 375 mg/kg bw/day were noted with body weight loss (up to 32 and 38%, respectively). For comparison, none of the females in the control and 100 mg/kg bw/day groups showed body weight loss after correction for gravid uterus weight. At 100 mg/kg bw/day, body weight corrected for gravid uterus weight was in the same range as control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 300 and 375 mg/kg bw/day, food consumption was lower from start of dosing onwards, reaching a maximal difference of 39 and 62% below control, respectively, between Days 18-21 post-coitum.

At 100 mg/kg bw/day, food consumption was slightly lower than control (up to 8% between Days 9-12 post-coitum; not statistically significant).

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, lower mean serum levels of T3, T4 and TSH were noted (0.50, 0.51 and 0.57x of control mean, respectively). The mean T3 and T4 levels were below the lower limit of the available historical control range. The mean TSH value (0.1397 mU/L; not statistically significantly lower than control) was at the lower limit of the available historical control range.

At 300 mg/kg bw/day, lower mean serum levels of T3 and T4 were noted (0.60 and 0.66x of control mean, respectively). The mean T3 and T4 levels (and individual serum levels of 14/19 and 12/19 females, respectively) were below the lower limit of the available historical control range. The mean TSH level remained within the normal range of biological variation.

The individual values show a very wide and overlapping distribution over all dose groups. Considering the magnitude of the differences between the groups averages with values below or at the lower limit of the available historical control range and an apparent dose-related response, this decrease was considered test item-related. However, the biological consequences of thyroid hormone changes are not assessed within this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed. As such, this finding was not taken into account when determining the maternal NOAEL.

At 100 mg/kg bw/day, the mean T3, T4 and TSH levels remained within the normal range of biological variation.

Note 1: A single female at 100 mg/kg bw/day was not gravid and therefore excluded from the data tables.

Note 2: The 375 mg/kg bw/day group contained only 9 females.

[- Historical control data for pregnant Wistar Han rats (period 2020-2021): T3 (ng/mL) mean: 0.439; P5 - P95: 0.280 – 0.616 (n = 263); T4 (ng/mL) mean: 23.2; P5 - P95: 16.3 – 35.2 (n = 154)]
[- Historical control data for pregnant Wistar Han rats (period 2016-2020): TSH (mU/L) mean: 0.353; P5 - P95: 0.127 – 0.699 (n = 373)].

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean thyroid gland weight (absolute) was lower compared to control in all treatment groups, but without a dose-related response and great overlap between the groups (reaching statistical significance at 300 mg/kg bw/day only). The relative thyroid gland weight was in the same range as control for all treatment groups.

Note 1: A single female at 100 mg/kg bw/day was not gravid and therefore excluded from the data tables.
Note 2: The 375 mg/kg bw/day group contains only 9 females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 375 mg/kg bw/day, a small thymus was noted for 1/9 females surviving until scheduled necropsy. It should be noted that the same finding was observed in 4/13 females in this high dose group that were euthanized preterm (see mortality section). In addition, in the 13 females that were euthanized in extremis at 375 mg/kg bw/day, test-item related enlargement and/or black discoloration of the adrenal glands (6/13 females), reduction in thymus size (4/13 females) and yellow gelatinous content in different parts of the intestine (4/13 females) was observed.

At 300 mg/kg bw/day macroscopic evaluation revealed test-item related enlargement of the adrenal glands for 2/3 females that were euthanized in extremis.

Remaining findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain.

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean numbers of pre- and post-implantation loss in the control, 100 and 300 mg/kg bw/day groups were similar and in the range of normal biological variation.

At 375 mg/kg bw/day the mean numbers of pre- and post-implantation loss were in the range of normal biological variation.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean numbers of early and late resorptions in the control, 100 and 300 mg/kg bw/day groups were similar and in the range of normal biological variation.

At 375 mg/kg bw/day the mean numbers of early and late resorptions were in the range of normal biological variation.

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

All females, including those that were necropsied preterm (see section mortality), were pregnant, except for a single female at 100 mg/kg bw/day and 300 mg/kg bw/day respectively. Excluding non pregnant females and females that did not survive until scheduled necropsy, the number of females with viable litters for evaluation was 22, 21, 19 and 9 females in the control, 100, 300 and 375 mg/kg bw/day groups, respectively.

Other effects:

no effects observed

Description (incidence and severity):

The mean numbers of corpora lutea in the control, 100 and 300 mg/kg bw/day groups were similar and in the range of normal biological variation.

At 375 mg/kg bw/day the mean numbers of corpora lutea were in the range of normal biological variation.

Details on maternal toxic effects:

As according to the guideline, a minimum of 16 females with implantations surviving until scheduled necropsy is required for an adequate evaluation of developmental data, the 9 litters available at 375 mg/kg bw/day were considered not sufficient for a robust and valid evaluation of developmental data. Therefore, the developmental data from the 375 mg/kg bw/day group were not used for any conclusions and were reported separately.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

mortality

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day, male, female and combined fetal body weight was 8, 9 and 8% lower than control, respectively, and below the historical control range of the Test Facility.

At 100 mg/kg bw/day, male, female and combined fetal body weight remained within the normal range of biological variation.

At 375 mg/kg bw/day, male, female and combined fetal weight was 15, 16 and 14% lower than control, respectively, and below the historical control range of the Test Facility.

[Historical control data for fetal body weights of Wistar Han rats (period 2016-2020): Males (g): mean: 5.4; P5 - P95: 5.2 – 5.5 (n= 6774); Females (g): mean: 5.1; P5 - P95: 4.9 – 5.3 (n= 7024); Combined (g): mean: 5.3; P5 - P95: 5.0 – 5.4 (n= 13798)]

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male : female ratio of control, 100 and 300 mg/kg bw/day groups was within the normal range of biological variation, with great overlap between all groups.

At 375 mg/kg bw/day, the male:female ratio was within the normal range of biological variation.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Mean litter size of the 100 and 300 mg/kg bw/day groups was in the same range as the control group.

At 375 mg/kg bw/day, the mean litter size was in the same range as the control group.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Fetal anogenital distance (both sexes; absolute and normalized for fetal weight) of the 100 and 300 mg/kg bw/day groups was in the same range as the control group.

At 375 mg/kg bw/day, fetal anogenital distance (both sexes; absolute) was slightly lower than control (not statistically significant). The mean fetal anogenital distance normalized for fetal weight was in the same range as control.

External malformations:

no effects observed

Description (incidence and severity):

No external malformations and variations were observed in the control, 100 and 300 mg/kg bw/day group.

At 375 mg/kg bw/day, the fetuses of the females surviving until scheduled necropsy did not show any external malformations or variations.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two fetuses in the 100 mg/kg bw/day group had a skeletal malformation. The first fetus presented bent limb bones (radius, ulna and femur) and the second fetus had an extra lumbar vertebra.

Regarding skeletal variations, at 100 mg/kg bw/day, the fetal incidence of incomplete ossification of both parietals was significantly increased. This variation occurred in five low-dose fetuses from five litters.

Note: At 300 mg/kg bw/day, there were no fetuses observed with misaligned ilia, resulting in a significantly decreased incidence of this finding compared to the control group.
At 375 mg/kg bw/day, the fetuses of the females surviving until scheduled necropsy did not show any skeletal malformations. Skeletal variations were observed in 33(9) fetuses (litters).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral malformations were observed in a single fetus of the control group with situs inversus and fused lung lobes.

Visceral variations in the control, 100 and/or 300 mg/kg bw/day groups were limited to supernumerary liver lobes, absent innominate artery and convoluted or dilated ureters.
At 375 mg/kg bw/day, the fetuses of the females surviving until scheduled necropsy did not show any visceral malformations. Visceral variations were observed in 8(3) fetuses (litters). Five fetuses from a single female were noted with absent renal papilla and distended urinary bladder of which three also had related dilated and/or convoluted ureters.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw (total dose)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Any other information on results incl. tables

Dose Formulation Analyses

1. Accuracy
   The concentrations analyzed in the formulation of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean sample concentration results were within or equal to 90-110% of target concentration). No test item was detected in the Group 1 formulation.


2. Homogeneity
   The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%)

Table 1 - Accuracy and Homogeneity Test (Week1)

Group	Sample position	Concentration [mg/mL]		Recovery [%]		Coefficient of variation [%]
		Target	Analyzed	Individual	Mean
1	50% height	0	n.d.	n.a.	n.a.	n.a.
		0	n.d.	n.a.
2	90% height	25.0	26.0	104	101	2.7
		25.0	25.8	103
	50% height	25.0	24.8	99
		25.0	24.7	99
	10% height	25.0	26.1	104
		25.0	24.6	98
3	50% height	75.0	81.1	108	110	n.a.
		75.0	84.2	112
4	90% height	93.75	106	113	105	4.8
		93.75	96.3	103
	50% height	93.75	96.5	103
		93.75	95.9	102
	10% height	93.75	93.7	100
		93.75	103	109

n.d. Not detected.

n.a. Not applicable.

Table 2: Overview of maternal and foetal findings

Dose level (mg/kg bw/day)	0	100	300	375
Pregnant/total dams	22/22	21/22	21/22	22/22
-early resorptions -late resorptions (% per litter)	11.8 0.7	18.52 0	9.35 0	13.94 0
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses	0	0	0	0
Pre-implantation loss (number and percent)	19 (6.56 %)	12 (7.22 %)	18 (7.48 %)	10 (7.83 %)
Post-implantation loss (number and percent)	16 (6.26 %)	13 (6.17 %)	10 (3.94 %)	7 (6.20 %)
Body weight on day 21	341.2	329.4	298.1**	272.2**
Body weight gain day 6-21 (%)	31.74	31.87	23.45	16.42
Gravid uterine weight (g)	79.51	79.47	74.63	66.17
Mean live offspring (number)	11.4	11.2	11.5	11.0
Live offspring (percent)	100 %	100 %	100 %	100 %
Mean fetal/pup body weight males (g)	5.391	5.276	4.965**	4.594**
Mean fetal body weight females (g)	5.112	5.049	4.669**	4.277**
Mean fetal body weight (sexes combined) (g)	5.233	5.192	4.814**	4.475**
Malformations (including runts) number and percent of fetuses per litter	1 (1.14 %)	2 (2.38 %)	0 (0 %)	0 (0 %)
Variations (% per litter) -external -soft tissue -skeletal	0 6.55 72.08	0 10.48 76.89	0 6.89 51.60	0 15.56 70.21

 

 

Table 3: Summary of Body Weight Gains (g): Gestation

Sex: Female		Day(s) Relative to Mating (Litter: A)
		6 → 9 [G]	9 → 12 [G]	12 → 15 [G]	15 → 18 [G]	18 → 21 [G]	6 → 21 [G1]
0	Mean	6.6	16.1	16.0	30.7	39.0	108.3
mg/kg/day	SD	4.7	4.6	5.4	5.0	7.8	15.9
Group 1	N	22	22	22	22	22	22
100	Mean	6.9	15.4	15.1	30.6	37.0	105.0
mg/kg/day	SD	2.9	3.0	4.8	7.4	8.1	17.7
Group 2	N	21	21	21	21	21	21
300	Mean	3.0	12.3*	8.8	23.6	16.1**	69.9**
mg/kg/day	SD	4.5	5.4	10.7	13.8	12.6	20.5
Group 3	N	21	21	21	21	19	19
375	Mean	-0.4**	5.5**	1.9**	8.6**	1.0**	44.7**
mg/kg/day	SD	6.6	11.7	8.5	17.0	11.9	15.3
Group 4	N	22	22	16	14	9	9

 

Table 4: Summary of Food Consumption: Gestation. Food Mean Daily Consumption (g/animal/day)

Sex: Female		Day(s) Relative to Mating (Litter: A)
		6 → 9 [G]	9 → 12 [G1]	12 → 15 [G1]	15 → 18 [G1]	18 → 21 [G1]	6 → 21 [G]
0	Mean	19.26	20.91	20.94	22.79	22.06	21.19
mg/kg/day	SD	2.30	2.48	2.05	1.72	1.40	1.49
Group 1	N	22	22	22	22	22	22
100	Mean	18.79	19.22	19.92	21.37	20.90	20.04
mg/kg/day	SD	2.04	2.22	1.76	1.91	2.50	1.52
Group 2	N	21	21	21	21	21	21
	%Diff	-2.41	-8.07	-4.87	-6.24	-5.24	-5.43
300	Mean	14.79 **	15.84 **	15.56 **	15.65 **	13.35 **	15.59 **
mg/kg/day	SD	2.83	3.42	4.23	5.44	3.47	2.09
Group 3	N	21	21	21	21	19	19
	%Diff	-23.18	-24.24	-25.71	-31.32	-39.48	-26.42
375	Mean	12.97 **	13.05 **	12.79 **	10.52 **	8.33 **	14.17 **
mg/kg/day	SD	2.89	5.59	5.40	6.71	4.21	2.02
Group 4	N	22	22	16	14	9	9
	%Diff	-32.65	-37.61	-38.91	-53.82	-62.23	-33.13

 

 

Table 5: Maternal performance and Mortality

Sex: Female   Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	375 mg/kg/day Group 4
Group Size - Females		22	22	22	22
Number of Females Pregnant [f]	N+ve	22	21	21	22
	%	100.0	95.5	95.5	100.0
Female with Live Fetuses [f]	N+ve	22	21	21	22
	%	100.0	100.0	100.0	100.0
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	0
	%	0.0	0.0	0.0	0.0
Terminal Euthanasia [f]	N+ve	22	22	19	9**
	%	100.0	100.0	86.4	40.9**
Unscheduled Death/Euthanasia [f]	N+ve	0	0	3	13 **
	%	0.0	0.0	13.6	59.1**
Unscheduled Euthanasia [f]	N+ve	0	0	3	13 **
	%	0.0	0.0	13.6	59.1**

 

Table 6: Summary of Ovarian and Uterine Examinations and Litter Observations

Sex: Female   Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	375 mg/kg/day Group 4
Female with Live Fetuses	N+ve	22	21	19	9
	%	100.0	100.0	100.0	100.0
Number of Corpora Lutea [k]	Mean SD	13.0 2.1	12.7 1.3	13.0 2.3	12.9 1.5
	N	22	21	19	9
	%Diff	-	-1.9	0.4	-0.5
Number of Implantations [k]	Mean SD	12.1 2.5	11.8 2.2	12.1 2.5	11.8 1.7
	N	22	21	19	9
	%Diff	-	-2.3	-0.3	-2.6
Pre-implantation Loss (%) [k]	Mean SD	6.56 12.96	7.22 13.94	7.48 11.36	7.83 13.99
	N	22	21	19	9
	%Diff	-	10.16	14.16	19.44
Total Number of Fetuses [k]	Mean SD	11.4 2.7	11.2 2.7	11.5 2.3	11.0 1.6
	N	22	21	19	9
	%Diff	-	-1.1	1.4	-3.2
Number of Live Fetuses [k]	Mean SD	11.4 2.7	11.2 2.6	11.5 2.3	11.0 1.6
	N	22	21	19	9
	%Diff	-	-1.5	1.4	-3.2
Number of Dead Fetuses [k]	Mean SD	0.0 0.0	0.0 0.0	0.0 0.0	0.0 0.0
	N	22	21	19	9
	%Diff	-	-	-	-
Number of Early Resorptions [k]	Mean SD	0.7 0.8	0.6 1.2	0.5 0.7	0.8 1.0
	N	22	21	19	9
	%Diff	-	-9.2	-22.8	14.1
Number of Late Resorptions [k]	Mean SD	0.0 0.2	0.0 0.0	0.0 0.0	0.0 0.0
	N	22	21	19	9
	%Diff	-	-100.0	-100.0	-100.0
Total Number of Resorptions [k]	Mean SD	0.7 0.9	0.6 1.2	0.5 0.7	0.8 1.0
	N	22	21	19	9
	%Diff	-	-14.9	-27.6	6.9
Post-implantation Loss (%) [k]	Mean SD	6.26 7.94	6.17 12.81	3.94 5.01	6.20 7.71
	N	22	21	19	9
	%Diff	-	-1.36	-37.04	-0.99
Number of Live Male Fetuses [k]	Mean SD	4.9 2.0	5.9 2.2	5.6 1.3	6.3 2.2
	N	22	21	19	9
	%Diff	-	21.4	14.7	30.2
Number of Live Female Fetuses [k]	Mean SD	6.5 2.0	5.3 2.4	5.9 2.1	4.7 1.7
	N	22	21	19	9
	%Diff	-	-18.7	-8.5	-28.2
Live Male Fetus/Litter (%) [k]	Mean SD	42.24 12.19	54.79 20.24	49.63 11.93	56.98 16.51
	N	22	21	19	9
	%Diff	-	29.71	17.49	34.89
Live Female Fetuses/Litter (%) [k]	Mean SD	57.76 12.19	45.21 20.24	50.37 11.93	43.02 16.51
	N	22	21	19	9
	%Diff	-	-21.73	-12.79	-25.52
Mean Fetal Weight males (g) [G]	Mean SD	5.391 0.279	5.276 0.338	4.965 ** 0.330	4.594 ** 0.379
	N	22	21	19	9
	%Diff	-	-2.129	-7.901	-14.780
Mean Fetal Weight females (g) [G]	Mean SD	5.112 0.267	5.049 0.303	4.669 ** 0.314	4.277 ** 0.460
	N	22	20	19	9
	%Diff	-	-1.223	-8.653	-16.320
Mean Fetal Weight all (g) [G]	Mean SD	5.233 0.254	5.192 0.314	4.814 ** 0.313	4.475 ** 0.379
	N	22	21	19	9
	%Diff	-	-0.780	-8.005	-14.478
Mean Fetal AGD males (mm) [G1]	Mean SD	3.00 0.24	2.98 0.26	2.92 0.27	2.79 0.25
	N	22	21	19	9
	%Diff	-	-0.68	-2.56	-6.94
Mean Fetal AGD females (mm) [G1]	Mean SD	1.27 0.16	1.33 0.15	1.27 0.13	1.24 0.18
	N	22	20	19	9
	%Diff	-	4.74	0.31	-2.21
Mean Normalized Fetal AGD m [G]	Mean SD	1.713 0.137	1.713 0.137	1.717 0.166	1.683 0.164
	N	22	21	19	9
	%Diff	-	0.029	0.230	-1.730
Mean Normalized Fetal AGD f [G]	Mean SD	0.735 0.092	0.773 0.085	0.761 0.082	0.765 0.117
	N	22	20	19	9
	%Diff	-	5.163	3.466	4.099

 

Table 7: Fetal Abnormalities by classification

Exam Type: Visceral Body     Number of Fetuses Examined: Number of Fetuses Evaluated: Number of Litters Examined: Number of Litters Evaluated:	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	375 mg/kg/day Group 4
	124 251 22 22	116 235 21 21	110 219 19 19	51 99 9 9
Variation Number of Fetuses Litter % of Fetuses [k] Number of Litters Malformation Number of Fetuses Litter % of Fetuses [k] Number of Litters All classifications Number of Fetuses Litter % of Fetuses [k] Number of Litters	8 6.55 6   1 1.14 1   9 7.69 7	13 10.48 7   0 0.00 0   13 10.48 7	8 6.89 7   0 0.00 0   8 6.89 7	8 15.56 3   0 0.00 0   8 15.56 3
Exam Type: Skeletal     Number of Fetuses Examined: Number of Fetuses Evaluated: Number of Litters Examined: Number of Litters Evaluated:	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	375 mg/kg/day Group 4
	126 251 22 22	119 235 21 21	109 219 19 19	48 99 9 9
Variation Number of Fetuses Litter % of Fetuses [k] Number of Litters Malformation Number of Fetuses Litter % of Fetuses [k] Number of Litters All classifications Number of Fetuses Litter % of Fetuses [k] Number of Litters	91 72.08 22   0 0.00 0   91 72.08 22	92 76.89 21   2 2.38 2   92 76.89 21	55 51.60 17   0 0.00 0   55 51.60 17	33 70.21 9   0 0.00 0   33 70.21 9

Applicant's summary and conclusion

Conclusions:

Based on mortality (13/22 at 375 mg; 3/22 at 300 mg), clinical signs, reduced food consumption and body weight gain, body weight loss after correcting for gravid uterus weight, on macroscopic findings of the adrenal glands and intestine (at 300 and 375 mg/kg bw/day) and the thymus (at 375 mg/kg bw/day), the maternal No Observed Adverse Effect Level (NOAEL) for Tallow amine propoxylate was established as being 100 mg/kg bw/day.

The developmental NOAEL for Tallow amine propoxylate was established as being 100 mg/kg bw/day, based on decreased fetal body weights at 300 and 375 mg/kg bw/day as only effects.

The developmental data from the 375 mg/kg bw/day group was not used for any conclusions as with 9 evaluable litters the minimum required number of 16 litters for a robust and valid evaluation of the developmental data was not reached.

Executive summary:

The objectives of this study were to determine the potential of Tallow amine propoxylate 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 Wistar Han rats from Days 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The study was performed according to GLP principles and in agreement with the OECD TG 414.

The dose levels in this study were selected to be 0, 100, 300, 375 mg/kg bw/day, based on information provided by the Sponsor and the results of the Dose Range Finding study.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. Formulation analyses confirmed that formulations of test item in peanut oil were prepared accurately and homogenously.

 

Maternal Findings
At 100 mg/kg bw/day, no mortality occurred, and no test item-related findings were noted.

At 300 mg/kg bw/day, three females were euthanized in extremis between post-coitum Days 14 and 19. These females were noted with body weight loss, strongly reduced food consumption and/or clinical signs indicative of poor general condition prior to death. Macroscopic evaluation revealed enlargement of the adrenal glands for 2/3 females. All these findings were considered test item-related.
At 300 mg/kg bw/day, for females that survived until scheduled necropsy, the following, considered test item-related findings were present:
Erected fur and hunched posture were noted in combination with strongly reduced food consumption, reduced body weight gain and body weight loss after correction for gravid uterus weight. Together, based on their severity, these findings were considered adverse. Non-adverse lower gravid uterus weight was noted. This decrease was considered to be related to the observed lower fetal weights (see below).

At 300 mg/kg bw/day, mean serum levels of T3 and T4 were lower compared to control. The individual values show a very wide and overlapping distribution over all dose groups, and microscopical evaluation of the thyroid glands showed no remarkable findings. However, considering that group average values were below the lower limit of the available historical control range and an apparent dose-related response, this decrease was considered test itemrelated. However, the biological consequences of thyroid hormone changes are not assessed within this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed. As such, this finding was not considered when determining the maternal NOAEL.

At 375 mg/kg bw/day, 13 females were euthanized in extremis between post-coitum Days 12 and 19. For these females, body weight loss, severely reduced food consumption and/or clinical signs indicative of poor general condition were observed prior to death. Macroscopic evaluation revealed enlargement and/or black discoloration of the adrenal glands, reduction in thymus size and yellow gelatinous content in different parts of the intestine. All these findings were considered test item-related.
At 375 mg/kg bw/day, for females that survived until scheduled necropsy, the following, considered test item-related findings were present:
Erected fur, hunched posture, and black and/or orange discolored feces were noted in combination with strongly reduced food consumption, reduced body weight gain and body weight loss after correction for gravid uterus weight. For a single female, a small thymus was noted. As these findings were in line with the findings for females that were euthanized in extremis, and based on their severity, they were considered adverse. Non-adverse lower gravid uterus weight was noted. This decrease was considered to be related to the observed lower fetal weights (see below).
At 375 mg/kg bw/day, mean serum levels of T3, T4 and TSH were lower compared to control. The individual values show a very wide and overlapping distribution over all dose groups, and microscopical evaluation of the thyroid glands showed no remarkable findings. However, considering the magnitude of the differences between the groups averages with values below or at the lower limit of the available historical control range and an apparent dose-related response, this decrease was considered test item-related. However, the biological consequences of thyroid hormone changes are not assessed within this type of study and therefore possible adversity of the effects on thyroid hormones could not be assessed. As such, this finding was not taken into account when determining the maternal NOAEL.
No test item-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e. thyroid gland weights, microscopic evaluation of the thyroid gland, uterine contents, corpora lutea, implantation sites and pre- and post-implantation loss).

Fetal Findings
Note: According to the guidelines, a minimum of 16 females with implantations at scheduled necropsy is required in each group for an adequate evaluation. As in the high dose group (375 mg/kg/day) only 9 females were available for evaluation, the available data was considered not sufficient for a robust and valid evaluation of the developmental data.
At 100 mg/kg bw/day, no test item-related findings were noted.
At 300 mg/kg bw/day, the following test item-related findings were present: Mean fetal body weights (male, female and total) were decreased compared to control, with mean values below the historical control range. Given the magnitude of the effect, this finding was considered adverse.
At 375 mg/kg bw/day, the following test item-related findings were present: Mean fetal body weights (male, female and total) were decreased compared to control and were below the available historical control data.

No test item-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, anogenital distance, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats the maternal No Observed Adverse Effect Level (NOAEL) for Tallow amine propoxylate was established as being 100 mg/kg bw/day. This was based on mortality,
clinical signs, reduced food consumption and body weight gain, body weight loss after correcting for gravid uterus weight, on macroscopic findings of the adrenal glands and intestine (at 300 and 375 mg/kg/day) and the thymus (at 375 mg/kg/day). The developmental NOAEL for Tallow amine propoxylate was established as being 100 mg/kg/day, based on decreased fetal body weights at 300 mg/kg/day.

 

Note: The developmental data from the 375 mg/kg bw/day group was not used for any conclusions as with 9 evaluable litters the minimum required number of 16 litters for a robust and valid evaluation of the developmental data was not reached.