Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-17 and C17 unsatd. alkyl) derivs. and sodium hydroxide and 2-propenoic acid

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 March 2020 - 05 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

ß-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-(C12-C18 and C18unsatd. acyl) derivs., monosodium salts

Cas Number:

93820-52-1

Molecular formula:

C12 fatty acid based: C19H37N2NaO4 - C18 fatty acids based: C23H45N2NaO4

IUPAC Name:

ß-Alanine, N-(2-aminoethyl)-N-(2-hydroxyethyl)-, N-(C12-C18 and C18unsatd. acyl) derivs., monosodium salts

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

dihydrogen oxide

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: females: 11 weeks (212.4 - 230.6 g), males: 13 weeks
- Housing: Before and after mating: up to 5 of one sex to a cage in clear polysulfone cages measuring 59.5x38x20 cm; During mating: 1 male to 1 female in clear polysulfone cages measuring 42.5x26.6x18.5 cm
- Diet (e.g. ad libitum): commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei 4, 20019 Settimo Milanese (MI), Italy), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20 air
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

softened by reverse osmosis

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was dissolved in the vehicle. The preparations were made at up to 7 day intervals (concentrations of 10, 30 and 100 mg/mL). Concentrations were calculated and expressed in terms of test item corrected for purity. During preparation and storage, the development of foam was avoided.

dose volume: 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis has been performed in a separate study in order to validate the analytical method and the preparation procedure and to verify the stability of the preparations (ERBC Study no. A3674). The preparation are stable for 28 hours at room temperature and 8 days at 2-8°C.
Samples of the preparations prepared onWeek 1 and Last week were analysed to check the concentration. Chemical analysis was carried out by the Analytical Chemistry Department.
Results of the analyses were within the acceptability limits stated of the laboratory for concentration of solutions (90-110%)

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Day 5 through Day 19 post coitum

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels have been selected in consultation with the Sponsor, based on a previous preliminary, non GLP compliant study (ERBC Study No. Y0450).

Examinations

Maternal examinations:

MORTALITY: Yes
- Time schedule: twice daily


CLINICAL SIGNS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: All surviving animals were weighed on Days 0, 3, 5, 8, 11, 14, 17 and 20 post coitum. Body weight for Group 4 animals was recorded and tabulated up to the day of necropsy.

FOOD CONSUMPTION: Yes
- Food consumption of surviving animals was measured on Days 3, 5, 8, 11, 14, 17 and 20 post coitum starting from Day 0 post coitum. Food consumption for Group 4 animals was recorded and tabulated up to the day of necropsy.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 (animals from Groups 1 to 3); animals from Group 4 were killed between Days 8 and 15 post coitum
- Organs examined: liver, thyroid

OTHER:
thyroid hormone determination (T3, T4 and TSH): colection of blood samples on Day 20 post coitum

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not obtained from animals found dead or killed during the study)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number and sex of dead foetuses

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes
- Anogenital distance of all live rodent pups: Yes

Structural deviations were classified as follows:
Malformations: Major abnormalities that are rare and/or affect the survival or health of the species under investigation.
Anomalies: Minor abnormalities that are detected relatively frequently.
Variants: A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by analysis of variance (one-way Anova). Inter-group differences were assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data (verified by Bartlett’s test). Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of theWilliams test. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5.

Indices:

Pre-implantation loss was calculated as a percentage from the formula:
Pre impl. Loss%= (no. of corpora lutea−no. of implantations) / (no. of corpora lutea)
×100
Post-implantation loss was calculated as a percentage from the formula:
Post impl. Loss%= (no. of implantations−no. of live foetuses) / (no. of implantations) ×100
Total implantation loss was calculated as a percentage from the formula:
Total impl. Loss%= (no. of corpora lutea−no. of live foetuses) / (no. of corpora lutea) ×100
Sex ratios of the foetuses were calculated as the percentage of males.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related clinical signs were noted in animals receiving 1000 mg/kg/day and included: decreased activity (5/25), kyphosis (21/25), piloerection (21/25), dyspnoea (3/25), rales (1/25) and salivation (2/25).
Kyphosis in one female and piloerection and salivation in 2 females were also seen in Group 3 females, receiving 300 mg/kg/day.
In one female, kyphosis was observed at 100 mg/kg.

Mortality:

mortality observed, treatment-related

Description (incidence):

Twenty-six cases of premature death occurred during the study. All the females (25) of the high dose group (Group 4) were sacrificed for humane reasons or found dead from Day 8 to Day 15 post coitum, whereas one female of the low dose group (no. X1350089) was found dead on Day 8 post coitum.
The macro- and microscopic observations noted, mainly in the large intestinal tract consisting of distension with gas, were considered treatment-related factors leading to poor health condition or death of these females.
The death of the low dose female on Day 8 post coitum could probably be ascribed to a misdosing.
All females mated and were found pregnant at necropsy, with the exception of one control female. The number of females with live foetuses on Day 20 post coitum was: 24 in the control and low dose groups and 25 in the mid-dose group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No differences in body weight and body weight gain were seen between control, low, and mid-dose females. In high-dose animals, a mean body weight loss of 4.6 % at gestation day 8 as compared to gestation day 5 was observed, corresponding to a reduced mean body weight of 9.3 % vs control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No treatment related differences were noted in food consumption between control, low, and mid-dose females. In high dose females, mean food consumption at gestation day 8 was reduced by 48 % as compared to controls.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The statistical significant decrease recorded in absolute weight gain of female of Group 3 was low in magnitude, thus considered of not toxicological relevance. Terminal bodyweight and gravid uterus weight of surviving treated females were comparable to the controls.
No relevant changes were observed in the absolute and relative liver and thyroid weights of surviving treated females, when compared to control data.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled deaths
The most relevant changes observed at post mortem examination in most of high dose female rats consisted of distention with gas of caecum (17/25) and colon (16/25), and/or distention with gas of ileum (3/25) and jejunum(3/25). At microscopic observation, in general no relevant changes were noted in the above mentioned organs.
At necropsy, in addition to the above mentioned findings, in a single instance one high dose female showed (no. X1350157) distension with gas of the stomach, while at histopathology, mild epithelial hyperplasia of the non glandular region of the stomach associated with submucosal chronic inflammation was noted; another high dose female (no. X1350155) showed also oedematous thymus with multiple red pinpoint areas which correlated microscopically with cortical lymphocytolysis of the thymus.
The most relevant changes observed at post mortem of the low dose female (no. X1350089) were foamy contents in trachea, multiple pale pinpoint areas of lungs and distention with gas of caecum, whereas at microscopic observations the most relevant changes were interstitial chronic inflammation, alveolar oedema and aggregation of alveolar macrophages in the lungs. The factor contributory to the death of this animal could be attributed to misdosing.

Final sacrifice
Females that completed the treatment period did not show relevant macroscopic changes. No treatment-related changes were noted at microscopic examination in thyroid as well as on all abnormalities. The sporadic lesions, reported in control and treated animals of the low and mid-dose groups were considered to be an expression of spontaneous and/or incidental pathology, commonly seen in this species.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

mortality

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

Small foetuses (< 2.7 g) were present in low (4/344) and mid-dose groups (1/376) without a dose relationship.
Since this incidence was very low and also the body weights of these fetuses were very close to the threshold value of 2.7g, the finding was considered unrelated to the test item.
No others abnormalities were detected.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Malformation (pubis unossified) was observed in one foetus of Group 2. The finding was considered incidental.
The other alterations (anomalies and variations) were noted both in control and treated groups with a similar incidence.

Visceral malformations:

no effects observed

Description (incidence and severity):

Malformations (extremely enlarged ureter in association with kidney pelvic dilatation extreme) were observed in one foetus of Group 1 and in one foetus of Group 2. These findings were considered incidental. The other alterations (anomalies and variations) were noted both in control and treated groups with a similar incidence.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: mortality in maternal animals

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

FATE OF FEMALES – GROUP INCIDENCE

 

	Control	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
Initial group size	25	25	25	25
Not pregnant	1	0	0	4
Found dead	0	1	0	4
Humane kill	0	0	0	21
With live foetuses at gestation Day 20	24	24	25	0

 

Further tables are attached as pdf below.

Applicant's summary and conclusion

Conclusions:

Due to the unexpected high mortality in Group 4 (1000 mg/kg/day), with four females found dead between days 8 and 11 of gestation, and also considering the treatment-related clinical signs and the reduction in body weight and food consumption in the remaining treated females of this group, the treatment of Group 4 was stopped and the females were sacrificed between days 8 and 15 of gestation.
Consequently, the study was completed with two treatment groups (100 and 300mg/kg/day) and one control group.
The macro- and microscopic observations noted, mainly in the large intestinal tract consisting of distension with gas, were considered treatment-related factors leading to poor health condition or death of these females.
One female of the low dose group was found dead on Day 8 post coitum.
The death of this female could be probably ascribed to a misdosing.
Treatment related clinical signs were noted in females of Group 4 and included: decreased activity, kyphosis, dyspnoea, piloerection and salivation. Kyphosis in one female and piloerection and salivation in few females were also seen in Group 3 females, receiving 300 mg/kg/day. No signs were detected in females receiving 100 mg/kg/day, with the exception of kyphosis in one female in one occasion.
Body weight, body weight gain and food consumption of low and mid-dose groups (100 and 300 mg/kg/day) were unaffected by treatment.
No changes were recorded between control and low and mid-dose groups in the serum levels of thyroide hormones (TSH, T3 andT4).
No differences of toxicological relevance were observed between control, low and mid-dose groups in terminal body weight, gravid uterus weight, litter data (number of implantation sites, corpora lutea, implantation losses, uterine deaths, viable foetuses, mean foetal weight and sex ratios), liver and thyroid weights and macroscopic observation of treated females when compared to controls.
No treatment-related differences were noted in the mean values of anogenital distance of foetuses of both sexes maternally exposed at 100 and 300 mg/kg/day compared to the control group.
No treatment-related changes were noted at the gross pathology examination of females that complete the treatment period, as well as at the microscopic evaluation of the thyroid gland and all abnormalities. No treatment-related findings were noted at external, skeletal and visceral examination.

On the basis of the results obtained in this study, mortality and signs of severe toxicity were noted at the high dose level of 1000 mg/kg bw/day. The dose level of 300mg/kg/day could be considered the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity.

Executive summary:

The effects of Amphopropionates C12-18 were investigated, after oral administration, in female Sprague Dawley rats during pregnancy and embryo-foetal development in a prenatal developmental toxicity study according to OECD Guideline 414 (25 June 2018).

Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each.

The original plan for this study was to investigate doses of 100, 300 and 1000 mg a.i./kg/day with a dose volume of 10 mL/kg body weight, during the gestation period from Day 5 through Day 19 post coitum. Control females received the vehicle (softened water) at the same dose volume during the same treatment period.

However, due to the unexpected high mortality in Group 4 (1000 mg/kg/day), with four females found dead between Days 8 and 11 of gestation, treatment of the remaining females was stopped and all remaining animals in Group 4 were sacrificed between Days 8 and 15 of gestation. Consequently, the study was completed with two treatment groups (100 and 300 mg/kg/day) and one control group.

Body weight, daily clinical signs and food consumption were recorded during the in vivo phase. All surviving females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination. Blood collection for hormone determination and liver and thyroid weights were performed on Day 20 post coitum from all females. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, liver and uterus weights, foetal weight and sex were recorded. All foetuses were examined for external abnormalities. The anogenital distance (AGD) in all live foetuses was recorded.

Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.

 

Mortality and fate of females

All females of the high dose group (Group 4) were sacrificed for humane reasons or found dead from Day 8 to Day 15 post coitum. The macro- and microscopic observations noted, mainly in the large intestinal tract consisting of distension with gas, were considered treatment-related factors leading to poor health condition or death of these females.

One female of the low dose group was found dead on Day 8 post coitum. The death of this female could be probably ascribed to a misdosing.

All females mated and were found pregnant at necropsy, with the exception of one control female.

The number of females with live foetuses on Day 20 post coitum was: 24 in the control and low dose groups and 25 in the mid-dose group.

 

Clinical signs

Treatment related clinical signs were noted in animals receiving 1000 mg/kg/day and included: decreased activity, kyphosis, dyspnoea, piloerection and salivation.

Kyphosis in one female and piloerection and salivation in few females were also seen in Group 3 females, receiving 300 mg/kg/day.

No signs were detected in females receiving 100 mg/kg/day.

 

 Maternal body weight and body weight gain

No differences in body weight and body weight gain were seen between control, low, and mid-dose females.

 

Food consumption

No treatment related differences were noted in food consumption between control, low, and mid-dose females.

 

Thyroid hormone determination

No changes were recorded in the determination of T3, T4 and TSH between control, low, and mid-dose females.

 

Terminal body weight, uterus weight and absolute weight gain

No differences of toxicological relevance were recorded in terminal body weight, gravid uterus weight and absolute weight gain of surviving treated females compared to the controls.

 

Organ weight

No relevant changes were observed in the absolute and relative liver and thyroid weights of surviving treated females, when compared to control data.

 

Litter data and sex ratios

No differences in litter data or sex ratios were observed in females of low and mid-dose group compared to the controls.

 

Anogenital distance

No treatment-related differences were noted in the mean values of the anogenital distance of foetuses of both sexes of low and mid-dose groups compared to the control group.

 

Macroscopic and microscopic examinations

Unscheduled deaths

The macro- and microscopic observations noted mainly in the large intestinal tract, consisting of distension with gas, were considered treatment-related factors leading to poor health condition or death of high dose treated females. The factor contributory to the death of the low dose female could be attributed to misdosing.

 

Final sacrifice

Females that completed the treatment period did not show relevant macroscopic changes. No treatment-related changes were noted at microscopic examination in thyroid as well as on all abnormalities.

 

External examinations of foetuses

Few small foetuses (< 2.7 g) were present in low and mid-dose groups without a dose relationship and with body weights very close to the threshold value of 2.7g. Thus the finding was considered unrelated to the test item.

 

Skeletal examinations of foetuses

No alterations considered treatment related were detected at skeletal examination of foetuses.

 

Visceral examinations of foetuses

No alterations considered treatment related were detected at visceral examination of foetuses.

 

Conclusion

On the basis of the results obtained in this study, mortality and signs of severe toxicity were noted at the high dose level of 1000 mg/kg/day. The dose level of 300 mg/kg/day could be considered the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity.