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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

- subacute (28 d repated dose toxicity study) repeated dose toxicity study oral (gavage), rat (Wistar) m/f (OECD TG 407; GLP; RL1), dose levels: 0, 50, 150, 1000 mg/kg bw/day; NOAEL >/= 1000 mg a.i./kg bw/day; no effects on reproductive organs; read-across: Amphopropionate C8

Link to relevant study records

Reference

Endpoint:

reproductive toxicity, other

Remarks:

Repeated Dose 28-Day Oral Toxicity in Rodents

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation:
- Fasting period before study: no
- Housing: in groups of 5 per sex in Macrolon cages (M IV type, height 18 cm; during overnight activity monitoring individual housing in
Mlll type; height 15 cm) with sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): pelleted rodent diet (SM R/M-Z from SSNlFF Spezialdiaten GmbH, Soest, Germany), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 - 22.7°C
- Humidity (%): 30 - 74%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered undiluted.

Details on mating procedure:

animals were not mated

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days a week

Details on study schedule:

animals were not mated

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

other: yes, from day 15 onwards Milli-Q water was used

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of a 5-day dose range finding study
- Rationale for animal assignment: By computer-generated random algorithm according to body weight

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/kg body weight/day

WATER CONSUMPTION: subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OTHER: presented in more detail in section "repeated dose toxicty"
HAEMATOLOGY, CLINICAL CHEMISTRY, NEUROBEHAVIOURAL EXAMINATION

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

n.a.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes, all animals assigned to the study
ORGAN WEIGHTS: Yes, presented in more detail in section "repeated dose toxicity"
HISTOPATHOLOGY: Yes, presented in more detail in section "repeated dose toxicity"

Postmortem examinations (offspring):

n.a.

Reproductive indices:

n.a.

Offspring viability indices:

n.a.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity noted over the 28-day observation period.
Salivation as observed among all animals at 1000 mg/kg bw/day and three males at 150 mg/kg bw/day was considered to be of no toxicological significance taking into account the nature and minor severity of this finding. Instead, salivation was considered to be a physiological response to taste and/or irritancy of the test substance upon oral administration.
Alopecia of the forelegs of one male at 50 mg/kg bw/day was a sign within the range of findings encountered in this type of study and was of no toxicological relevance. No clinical signs were noted among control animals, and females at 50 and 150 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically significant changes in body weights and body weight gain of treated animals were noted.
The slightly lower body weight and body weight gain of males at 1000 mg/kg bw/day in the first week of treatment (achieving a level of statistical significance for body weights) was considered to be of no toxicological significance. The change was of a temporary and slight nature (i.e. within the range considered normal for rats of this age and strain).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was similar between treated and control animals.

Food efficiency:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic findings recorded which could be attributed to treatment with the test substance.
All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

OTHER FINDINGS (PARENTAL ANIMALS)
further results are presented in more detail in section "repeated dose toxicity"

Dose descriptor:

NOAEL

Remarks:

fertility paramaters

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects up to and including the highest administered dose

Critical effects observed:

no

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Reproductive effects observed:

no

Conclusions:

On the basis of the results obtained in this study, the dose level of 1000 mg/kg bw/day was considered the NOAEL for general toxicity and for fertility parameters.

Executive summary:

In this Repeated Dose, 28-day Oral Toxicity Study performed according to OECD guideline 407 (1995) and EU Method B.7 (1996) Amphopropionate C8  (50.6 % a.i) was administered to groups of 5 male and 5 female Wistar rats by oral gavage at dose levels of 10, 150 and 1000 mg/kg bw/day for 28 days. The control group animals received MilliQ water from day 15 onwards.

All animals survived to study termination. No test substance-related findings were detected or observed in clinical examinations, body weights, food consumption values, neurobehaviour, haematology, clinical biochemistry and clinical pathology evaluations. During the anatomical pathology examinations, no test substance-related findings were observed or detected during macroscopic examinations, organ weight evaluations or microscopic examinations. In specific, no effects were noted on reproductive organs examined in this study.

There is no evidence for specific target organ toxicity in this study.

The NOAEL is ≥ 1000 mg/kg bw/day in this study.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

OECD guideline study, GLP, RL1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No experimental data are available for the target substance Amphopropionates C12-18. However, relevant and reliable data from an oral subacute repeated dose toxicity study with additional focus on reproductive organs conducted with the closely related source substance Amphopropionate C8 are available. A justification for read-across is given below.

 

In a Repeated Dose, 28-day Oral Toxicity Study performed according to OECD guideline 407 (1995) and EU Method B.7 (1996) Amphopropionate C8  (50.6 % a.i) was administered to groups of 5 male and 5 female Wistar rats by oral gavage at dose levels of 250, 500 and 1000 mg test material/kg bw/day for 28 days. The control group animals received MilliQ water from day 15 onwards.

All animals survived to study termination. No test substance-related findings were detected or observed in clinical examinations, body weights, food consumption values, neurobehaviour, haematology, clinical biochemistry and clinical pathology evaluations. During the anatomical pathology examinations, no test substance-related findings were observed or detected during macroscopic examinations, organ weight evaluations or microscopic examinations. In specific, no effects were noted on reproductive organs examined in this study. There is no evidence for specific target organ toxicity in this study. The NOAEL is ≥ 1000 mg/kg bw/day in this study.

 

In accordance with Annex IX, 8.7.3, column 2 of the REACH Regulation (EC) No 1907/2006, the performance of an Extended One-Generation Reproductive Toxicity Study shall be proposed, if the available repeated dose toxicity studies indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity.

The substance is of low systemic toxicity as indicated by the 28 d NOAEL of 1000 mg/kg bw/d obtained with a closely related source substance. No indication of any systemic toxicity relevant in view of a potential health risk for humans was found, including effects to reproductive organs. The substance does not have genotoxic properties as proven in the full data set on the substance itself or closely related source substances.

 

Supporting data are available for Acrylic acid, demonstrating that the presence of small amounts (0 – 1%) of acrylic acid in the target substance is of no toxicological relevance:

According to EU RAR (2002), in "oral reproductive toxicity studies (rats) no effects on reproductive function (fertility) were observed.” And a “NOAEL/fertility of 460 mg/kg bw/d was derived from a guideline 2-generation study in rats”.

 

No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

 

Justification for read-across

For details on substance identity and detailed toxicological profiles, please refer also to the general justification for read-across given at the beginning of the CSR and attached as pdf document to IUCLID section 13.

This read-across approach is justified based on structural similarities and a comparable toxicological profile based on the available data. The target and source substances contain the same functional groups. Thus a common mode of action can be assumed.

 

Structural similarity and functional groups

The target substance Amphopropionates C12-18 is manufactured from fatty acids (C12-18, C18unsatd.) and aminoethylethanolamie (AEEA) to form 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-C17 odd-numbered, C17unsatd. alkyl) derivs. This is further reacted with 2-propenoic acid in the presence of sodium hydroxide (alternatively, sodium 2-propenoate can be used) and water. The molar relation between 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-C17, C17unsatd. alkyl) derivs. and 2-propenoic acid is somewhat below 1:1. Most of the excess 2-propenoic acid is stripped off by distillation. However, a small amount remains in the aqueous solution.

 

The source substance Amphopropionate C8 is manufactured from capric acid and aminoethylethanolamie (AEEA) to form 1-(2-Hydroxyethyl)-2-Heptylimidazoline. Excess AEEA is removed from the reaction mixture by distillation at elevated temperature. In a further step 2-propenoic acid is added to form Amphopropionate C8. Most of the excess 2-propenoic acid is stripped off by distillation. However, a small amount remains in the aqueous solution.

 

Differences

Chain length:

The source substance Amphopropionate C8 contains shorter C chains, whereas the major C chain in the target substance is C12.

In general the absorption declines with increasing alkyl chain length (Ramirez et al. 2001). Therefore the source substances with the shorter alkyl chain lengths are assumed to represent a worst-case scenario due to higher absorption rates than the target substance.

 

Degree of unsaturation:

In contrast to the source substance Amphopropionate C8, the target substance Amphopropionates C12-18 contains some amounts of unsaturated C18 chains.

An increase in the degree of unsaturation may lead to a slightly higher irritation potential (HERA, 2002; Stillman, 1975; Aungst, 1989). Apart from that, fatty acids irrespective of their degree of unsaturation are in general non-toxic. Irritation studies are available for the target substance itself, thus, for other endpoints, this difference in composition is of no toxicological relevance.

Presence of residual acrylic acid:

The target substance Amphopropionates C12-18 as well as the source substance Amphopropionate C8 may contain some small amounts of residual acrylic acid. Supporting data from acrylic acid are available, demonstrating that this difference in the low concentration ranges is of no toxicological relevance.

 

Comparison of reproduction toxicity data

	Target substance	Source substance
	Amphopropionates C12-18	Amphopropionate C8
Repeated dose toxicity	No data, read-across	sup_RA_Toxicity to reproduction: 64265-45-8_8.7.1_Evonik_2008_OECD407   OECD TG 407, rat (Wistar), oral: gavage   28 d NOAEL = 1000 mg a.i./kg bw/d; no toxicologically significant changes   1 (reliable without restriction), GLP

 

In the repeated dose toxicity studies performed according to OECD Guideline 407 up to and including the limit dose level of 1000 mg/kg bw/d, no indication of any effects of the substance to reproductive organs were observed. No effects on organ weights (epididymides, testes, ovaries, uterus) were noted. And no macroscopic or microscopic lesions were observed at the reproductive organs (cervix, clitoral gland, ovaries, uterus, vagina, epididymides, prostate, testes). The NOAEL (fertility parameters) for Amphopropionate C8 was established at of 1000 mg/kg bw/day.

 

Supporting data are available for acrylic acid: no effects on reproductive function were observed in a 2-generation study. Thus, the presence of small amounts of acrylic acid in the target substance is not of toxicological relevance at the max. level of 1% of acrylic acid in the target substance.

 

Quality of the experimental data of the analogues:

The available data are adequate and sufficiently reliable to justify the read-across approach.

The study was conducted according to OECD Guideline 407 and is reliable without restrictions (RL1).

The test materials used in the respective studies represent the source substance as described in the hypothesis in terms of substance identity and minor constituents.

Overall, the study results are adequate for the purpose of classification and labelling and risk assessment.

 

Conclusion

The structural similarities between the source and the target substances presented above and in more detail in the general justification for read-across support the read-across hypothesis. Adequate and reliable scientific information indicates that the source and target substances have similar toxicity profiles. Taking into account the overall low toxic activity of the substances, particularly with regard to the absence of adverse effects on reproductive organs or tissues, fertility-specific effects are highly unlikely. 

Thus, the absence of toxicity to reproductive organs observed in the sub-acute toxicity study conducted with the source substance Amphopropionate C8 is considered to be also relevant for the target substance Amphopropionates C12 -18.

 

References

Aungst, 1989. Structure/Effect Studies of Fatty Acid Isomers as Skin Penetration Enhancers and Skin Irritants. Pharmaceutical Research, March 1989, Volume 6, Issue 3, pp 244-247

 

EU RAR, 2002: European Union, Risk Assessment Report: Acrylic acid, CAS No: 79-10-7, Risk Assessment. European Union Risk Assessment Report, 1st Priority List, Vol. 2

 

HERA, 2002: Fatty Acid Salts – Human Health Risk Assessment

 

Ramírez M, Amate L, Gil A. Absorption and distribution of dietary fatty acids from different sources. Early Human Development 2001 Nov; 65 Suppl:S95-S101

 

Stillman et al., 1975. Relative irritancy of free fatty acids of different chain length. Contact Dermatitis. 1975;1(2):65-9

Effects on developmental toxicity

Description of key information

NOAEL = 300 mg/kg bw/d (maternal/developmental toxicity; based on mortality and signs of severe toxicity at the high dose level of 1000 mg/kg/day); rat, oral(gavage), OECD TG 414

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

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

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

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).

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.

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.

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

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

mortality

Abnormalities:

no effects observed

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.

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

Abnormalities:

no effects observed

Developmental effects observed:

no

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.

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

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.

Justification for classification or non-classification

There are no indications for a classification for toxicity to reproduction or developmental toxicity and teratogenicity at this time according to Regulation (EC) No 1272/2008.