N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

Endpoint summary

Description of key information

NOAEL (Systemic toxicity) = >= 1000 mg/kg bw/day; OECD 422; Anon. (2018)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29.07.16

Deviations:

yes

Remarks:

Deviations were considered to have not affected the integrity or validity of the study results.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.
- Stability under test conditions: stable for 12 days at room temperature (20 ± 5 ºC) and in the dark.
- Solubility and stability of the test substance in the solvent/vehicle:Validation done at 10-100 mg/mL demonstrates that formulations were stable for 12 days at room temperature (20 ± 5 ºC) and in the dark.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Small amounts of vehicle were added and mixed with the test item using a pestle. Any lumps were broken up at this point, resulting in a suspension. The suspension was transferred to a suitable container and mixed with a shear mixer until homogenization. The formulations prepared at three different concentrations were analyzed twice over the course of the study to verify their correct preparation - Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Dose volume 10 mL/kg/day
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Hannover Wistar Rat (HsdHan®:WIST)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V. Kreuzelweg 53, 5961 NM Horst, Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: Males: 277-342 g & Females: 189-236 g.
- Fasting period before study: no
- Housing: Cages with standard, granulated, S8-15 sawdust bedding (J. Rettenmaier & Söhne). Premating (maximum 5 animals/cage) Makrolon type-IV cages. Mating (one male and one female/cage) Makrolon type-III cages Postmating, gestation and lactation (individual) Makrolon type-III cages
-Diet: Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum (supplied by Envigo RMS, S.L.).Pelleted standard Teklad 2018C rat/mouse maintenance diet (supplied by Envigo RMS, S.L.) ad libitum, for lactating females and pups (until sacrifice).
- Water (e.g. ad libitum): Tap water in bottles ad libitum
- Acclimation period: Five days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:
See above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 ºC
- Humidity (%): 30 - 60 %
- Air changes (per hr): 21 air changes per hour
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES:
From: 5 days acclimatisation until necropsy

Route of administration:

oral: gavage

Details on route of administration:

Oral, by gastric gavage.

Vehicle:

other: 1% carboxymethylcellulose sodium salt (medium viscosity) in distillated water

Remarks:

1% carboxymethylcellulose sodium salt

Details on oral exposure:

Method Oral, by gastric gavage. For each dose group the order of administration was all males first and then all females. Each day of treatment the starting order was alternated between males and females. It was stated in the raw data.
Frequency Once daily
- F0 males: Two weeks prior to mating start until the day before sacrifice (during week 6 of treatment). They were then sacrificed.
- F0 females: Two weeks prior to mating start until day 13/15 of lactation, including the day before sacrifice.
- F1: Potential indirect exposure in utero and through the milk during lactation

Dose levels The amount of test item to be administered was calculated according to the most recent body weight recorded.
Group 1: 0 mg/kg/day
Group 2: 100 mg/kg/day
Group 3: 350 mg/kg/day
Group 4: 1000 mg/kg/day
Administration volume 10 mL/kg
Duration of treatment period 5-8 weeks. 
Female no. 96 at 0 mg/kg/day and female no. 80 at 1000 mg/kg/day were administered for 9 and 10 consecutive weeks, respectively, as they got pregnant later than expected.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before commencement of treatment, an analytical method (TL25JD/APMT1) was validated in the present study. Formulations were prepared at two concentration levels (target concentrations: 10 and 100 mg/mL). The validation parameters and acceptance criteria were met.

Duration of treatment / exposure:

5-8 weeks.
Female no. 96 at 0 mg/kg/day and female no. 80 at 1000 mg/kg/day were administered for 9 and 10 consecutive weeks, respectively, as they got pregnant later than expected.

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

350 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10

Control animals:

other:

Details on study design:

Method Oral, by gastric gavage. For each dose group the order of administration was all males first and then all females. Each day of treatment the starting order was alternated between males and females. It was stated in the raw data.
Frequency Once daily
-F0 males: Two weeks prior to mating start until the day before sacrifice (during week 6 of treatment). They were then sacrificed.
-F0 females: Two weeks prior to mating start until day 13/15 of lactation, including the day before sacrifice.
-F1: Potential indirect exposure in utero and through the milk during lactation
Dose levels The amount of test item to be administered was calculated according to the most recent body weight recorded.
Group 1: 0 mg/kg/day
Group 2: 100 mg/kg/day
Group 3: 350 mg/kg/day
Group 4: 1000 mg/kg/day
Administration volume 10 mL/kg
Duration of treatment period 5-8 weeks. 
Female no. 96 at 0 mg/kg/day and female no. 80 at 1000 mg/kg/day were administered for 9 and 10 consecutive weeks, respectively, as they got pregnant later than expected.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- pre-test: Twice
- treatment to lactation: weekly

BODY WEIGHT: Yes
- pre-test: Weekly
- treatment to lactation: on day one then weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- pre-test: weekly
- treatment to lactation: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes but only visual observation (not drinking water study)
- Not examine

SENSORY REACTIVITY & GRIP STRENGTH:
- Week 5 (before dosing)
- Week 5 (before dosing)

MOTOR ACTIVITY:
- Week 5 (before dosing)
- Days 8-9 of lactation (before dosing)

HAEMATOLOGY: Yes
- At termination

BLOOD CHEMISTRY: Yes
- At termination

URINALYSIS: Yes
- Not examine

IMMUNOLOGY: No
- Not examine

OTHER: Yes (see below);

ESTROUS CYCLE: Yes
- Dry smears - taken for 15 days before pairing (reproductive feamles only).
- Wet smears - taken for 14 days before treatment (all females); females that failed to exhibit 4-5 day cycles were not allocated to the reproductive group.
- after pairing until mating (reproductive females only).
- four days before scheduled termination (all females except premature decedents).
- Females showed no evidence of mating: following completion of pairing period female was separated from the male and vaginal smearing continued for up to five days or until the first estrous smear was seen. If a female shows an estrous smear during this period, she was killed and subject to macroscopic examination.

THYROID HORMONE ANALYSIS: Yes
- Time schedule: - Day 4 of age (F1 offspring, two females per litter (where possible) - no pups were eliminated when total litter size dropped below ten/litter).
- one pup for T3/T4 (serum)
- one pup for TSH (plasma)
- Day 13 of age (F1 offspring, two males and two females per litter (where possible)
- two for T3/T4 (serum): where possible one male and one female
- two for TSH (plasma): where possible one male and one female
- Termination (All Toxicity and Recovery phase F0 males and all Reproductive phase F0 females surviving to scheduled termination).

TERMINAL INVESTIGATIONS: yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Clinical signs:

no effects observed

Description (incidence and severity):

Although little or no milk was observed in the stomach in some pups within the first days of lactation, the majority of the offspring survived the first days of the phase. One female (no. 74-2F) at 350 mg/kg/day showed little milk in the stomach during the whole lactation period.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Although males administered at 350 and 1000 mg/kg/day revealed significantly lower values in glucose and proteins and in all test item administered groups in phosphorous with respect to Control. These differences were considered not related with treatment based on the absence of a dose-effect relationship, their magnitude (with the exception of proteins), the fact that all values are within the historical control data and that they were not present in females.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Treatment with the test item during 5-8 weeks of treatment showed higher and statistically significant mean adjusted values in males in brain and in females in liver at 1000 mg/kg/day. These differences were considered not treatment related as the values were within the historical control data and as there was no correlation with histopathology and consequently these differences were devoid of any toxicological relevancy.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Hematology and coagulation investigations on days 14-15 of lactation revealed slight changes not considered to be toxicologically relevant.
In males, after 5 weeks of treatment, statistically significant differences compared to the Controls were observed in reticulocytes and basophils (lower with respect to Control) and MCHC (higher with respect to Control). However, all of the mean values in the treated groups were within the expected Control range and the findings were considered to be related to unusual control values rather than to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There was no effect of treatment on litter size, sex ratio or offspring survival. It was, however, noted that the mean litter size in the control group was incidentally slightly larger than in the treated groups.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Key result

Critical effects observed:

no

Tables containing raw data are attached in background material.

Conclusions:

The effects of oral (gavage) administration of N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide to Wistar rats receiving 100, 350 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

No signs of evident toxicity related to clinical signs, body weights, food consumption or effects on sensory reactivity. Non-treatment related observations included, significantly higher mean forelimb values among males in all test-item administered groups,statistically significant differences compared to the Controls were observed in reticulocytes and basophils (lower with respect to Control) and MCHC (higher with respect to Control), in males significantly lower phosphorus values compared to Control in all test item administered groups and glucose and total protein values at 350 and 1000 mg/kg/day, statistically significant mean adjusted values in males in brain and in females in liver at 1000 mg/kg/day. Clinical biochemistry in males administered at 350 and 1000 mg/kg/day revealed significantly lower values in glucose and proteins and in all test item administered groups in phosphorous with respect to Control.Estrous cycles and reproductive parameters of pre-coital interval, mating performance, fertility or gestation length or index were unaffected by treatment.

There was no effect on offspring growth. There were no offspring clinical or necropsy signs indicative of a reaction to the test item. Also, there was no effect on litter size, survival indices, sex ratio, body weights, ano-genital distance or nipple areolae.
Systemic toxicity: The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day for males and females, taking into account that findings observed in clinical pathology did not affect the general well-being, growth, development or life span as well as that the findings observed in grip strength or motor activity were not corroborated with clinical signs.

Reproductive / developmental toxicity: The No Observed Effect Level (NOEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on estrous cycle, pre-coital interval, mating performance, fertility and gestation length or in the offspring on litter size, sex ratio, survival, clinical signs, body weights, ano-genital distances or macropathology.

Executive summary:

In an OECD 422, three groups of ten male and ten female rats each received the test item  at the doses of 100, 350 and 1000 mg/kg/day. Males were treated continuously for two weeks before pairing up to necropsy, after a minimum of 37 consecutive days. Females were treated continuously for two weeks before pairing, throughout pairing and gestation, and until Day 13-15 of lactation (the day before sacrifice). Females were allowed to litter and rear their offspring, and litters were killed on Day 13-15 of lactation (the day before the corresponding female was killed). F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted control group received the vehicle, 1% carboxymethylcellulose sodium salt (medium viscosity) in distilled water for irrigation.

During the study, mortality, clinical signs, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology and coagulation, blood biochemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated.

Clinical signs, behavior assessment, litter size and survival, sex ratio, body weight and macropathology were also assessed for all offspring.

Results:

The study results can be summarized as follows:

-  There were no unscheduled deaths during the study.

-  Administration of the test item at the dose levels of 100, 350 or 1000 mg/kg/day had no effects on clinical condition, body weight, food consumption or sensory reactivity. Statistically lower mean values with respect to Control, in all test item administered males groups were observed in forelimb grip strength, as well as higher mean motor activity values with respect to Control were observed in general in all test item administered groups in males and females.

-  No effects of test item were observed in pre-coital interval, mating performance or fertility and gestation length.

-  All females allocated to the study showed regular 4-day estrous cycles prior to the start and no relevant effects were observed during treatment. At termination, all reproductive phase females showed diestrus with the exception of one female at 350 mg/kg/day, which had recovered the cycle.

-  There were no differences in hematology, coagulation or organ weights considered to be toxicologically relevant. T4 analyses of samples in Main study males and F1 offspring on day 13 did not reveal any differences attributable to treatment.

-  There were no macroscopic findings that could be considered test-item-related.

-   Histopathology reveals no test-item-related changes. No treatment-related effects were detected in the reproductive organs or mammary glands.

-   There was no relevant effect on offspring survival due to

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

or on litter size, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.

Conclusion:

In conclusion, the effects of oral (gavage) administration of

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

to Wistar rats receiving 100, 350 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

Systemic toxicity:

−   The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day for males and females, taking into account that findings observed in clinical pathology did not affect the general well-being, growth, development or life span as well as that the findings observed in grip strength or motor activity were not corroborated with clinical signs.

Reproductive / developmental toxicity:

−  The No Observed Effect Level (NOEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on estrous cycle, pre-coital interval, mating performance, fertility and gestation length or in the offspring on litter size, sex ratio, survival, clinical signs, body weights, ano-genital distances or macropathology.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

13 September 2017 - 12 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day dietary study in the Crl:CD(SD) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

3 October 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Principles of method if other than guideline:

The 14d dose range-finder is based on the general provisions of the above guidelines, to allow appropriate dose setting for the OECD 422 main study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle:As no stability data is available, the test item was prepared daily and administered within five hours of preparation in 1% carboxymethylcellulose sodium salt (medium viscosity) in distillated water.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: No
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No

Species:

rat

Strain:

Wistar

Remarks:

Hannover Wistar Rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V. Kreuzelweg 53, 5961 NM Horst, Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Males: 140-170 g & Females: 140-150 g.
- Fasting period before study: no
- Housing: Same-sex groups of no more than four animals during acclimatization and three during treatment, housed in Makrolon type-IV cages with Lignocel S8-15 sawdust bedding (supplied by J. Rettenmaier & Söhne).
- Diet Pelleted standard Teklad 2014C rat/mouse maintenance diet ad libitum batch no. 011017MA (supplied by Envigo RMS, S.L.). Results of analyses for composition and contaminant analyses are included in the raw data.
- Water (e.g. ad libitum): Tap water in bottles ad libitum
- Acclimation period: Five days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: See above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 13th September 2017 To: 06th October 2017

Route of administration:

oral: gavage

Details on route of administration:

1. Weigh the required quantity of test item in a single-use container (the test item can be weighed ahead of time and stored under the corresponding conditions).
2. Transfer the test product to an appropriate size mortar and grind to a fine powder.
3. Add small amounts of vehicle and mix this using a pestle. Any agglomerate must be broken down at this stage; the final result is a suspension.
4. Pass the suspension to a volumetric vessel which will have been previously wetted with the vehicle, wash the mortar and the single-use container with the vehicle until the container is completely clean (avoid remaining traces of test item) and add this vehicle to the volumetric container to make up to the mark.
5.Transfer the suspension to a suitable container and mix using a magnetic stirrer until a homogeneous suspension is obtained.
6. Maintain formulations under agitation after preparation and during administration.

Vehicle:

other: 1% carboxymethylcellulose sodium salt (medium viscosity) in distillated water

Remarks:

Batch No. 000502958 (CMCNa); 16213410 (distilled water)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The formulations were prepared by diluting the test item with the vehicle

DOSE ADMINSITRATION: gastric gavage
- Frequency of administration: daily
- Administration volume: 10 mL/kg
- Storage temperature of food: At room temperature (10-30 ºC) and in the dark

VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% carboxymethylcellulose sodium salt (medium viscosity) in distillated water
- Concentration in vehicle: not stated
- Amount of vehicle (if gavage): not stated
- Lot/batch no. (if required): 000502958 (CMCNa); 16213410 (distilled water)
- Purity: not stated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

TBC

Duration of treatment / exposure:

14 consecutive days

Frequency of treatment:

Once daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

- Dose selection rationale: the acute oral study demonstrated that the acute lethal oral dose (LD50) of the test item was greater than 2000 mg/kg, with no mortality reported.
- Rationale for animal assignment (if not random): Body weights were reviewed by Study Management and allocation has been adjusted to reduce inter‑/intra-group variation.
- Rationale for selecting satellite groups: n/a
- Post-exposure recovery period in satellite groups: n/a
- Section schedule rationale (if not random): n/a
Oral, by gastric gavage. For each dose group the order of administration was all males first and then all females. Each day of treatment the starting order was alternated between males and females.

Positive control:

n/a

Observations and examinations performed and frequency:

AGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once at pretest and once during each treatment week and on Day 15 prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Once during pre-test, on day 1 and twice weekly during treatment and before sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Before treatment start and twice weekly during treatment period
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Three times per week from pre-test and until the day of sacrifice.

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

HAEMATOLOGY: No
- Time schedule for collection of blood: n/a
- Anaesthetic used for blood collection: n/a
- Animals fasted: n/a
- How many animals: n/a

CLINICAL CHEMISTRY: No
- Time schedule for collection of blood: n/a
- Animals fasted: No
- How many animals: n/a

URINALYSIS: No
- Time schedule for collection of urine: n/a
- Metabolism cages used for collection of urine: n/a
- Animals fasted: n/a

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a

IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/s
- Dose groups that were examined: n/a

OTHER: no

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: No

Other examinations:

No

Statistics:

No

Clinical signs:

no effects observed

Description (incidence and severity):

No adverse clinical signs were recorded during the course of the study.

Mortality:

no mortality observed

Description (incidence):

All animals survived the scheduled treatment period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No alterations in body weight were recorded during the course of the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test-item-related effects were recorded during the course of the study

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no organ weight changes in liver, kidneys or spleen that were considered to be attributable to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test-item-related macroscopic findings noted at the end of the study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

All raw data tables are attached in background material as follows:

Table 1.1. F0 Body weight - Males - group mean values

Table 1.2. F0 Body weight - Pregnant females - group mean values

Table 2.1. F0 Food consumption - Males - group mean values

Table 2.2. F0 Food consumption - Pregnant females - group mean values

Table 3.1. F0 Detailed Signs - Males - group distribution of observations

Table 3.2. Detailed Signs - Pregnant females - group distribution of observations

Table 4. FOB: Sensory activity - group mean values

Table 5. FOB: Grip Strength - group mean absolute values

Table 6. FOB: Motor Activity - group mean absolute values (beam counts)

Table 7.1. F0 Estrous cycles - group values before treatment

Table 7.2. F0 Estrous cycles - group values during treatment

Table 8. F0 Pre-coital interval - group values

Table 9. F0 Gestation length and gestation index - group values

Table 10. F0 Mating performance and fertility - group values

Table 11. F0 Stage of estrous cycle at termination - group values

Table 12. F0 Hematology and Coagulation - group values

Table 13. F0 Clinical Biochemistry - group values

Table 14.1. F0 Macropathology - Males - group distribution of findings

Table 14.2. F0 Macropathology - Pregnant females - group distribution of findings

Table 15.1. F0 Organ weights - Males - group mean absolute and adjusted values

Table 15.2. F0 Organ weights - Pregnant females - group mean absolute and adjusted values

Table 16. F1 Offspring survival indices

Table 17. F1 Litter size - group mean values

Table 18. F1 Offspring sex ratio - group mean values

Table 19. F1 Body weight - group mean values for offspring

Table 20. F1 Ano-genital distance / Nipple/areolae - group mean litter values

Conclusions:

In conclusion, treatment with the test item at 100, 500 and 1000 mg/kg/day had no definite effect on clinical condition, food & water consumption, macropathology or organ weights. There were no alterations in body weight were recorded during the course of the study. Therefore, oral gavage exposure at up to 1000 mg/kg/day would be a suitable high dose for the OECD 422 screening study.

Executive summary:

OECD 422 (2017) - The purpose of this test was to assess the systemic toxicity potential of

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

following repeated exposure to 3 males and 3 femals Hannover Wistar rats at 100, 500 and 1000 mg/kg/day for 14 consecutive days. The findings would be used to select suitable dose levels for a subsequent combined repeat dose toxicity study with reproductive/ developmental toxicity screening study (OECD 422).

A summary of adult responses to the test item are described below;

 

Mortality - No mortality was observed during the test.

 

Clinical signs - No clinical signs or dose observations related to treatment were observed during the test.

  

Bodyweight - No alterations in body weight were recorded during the course of the study.

Food consumption and efficiency - No test-item-related effects were recorded during the course of the study

Water consumption - No dose related responses were observed during the test.

   

Necropsy - No toxicologically significant effects were detected in terminal kill animals of either sex treated with the test item.

  

Organ weights - There were no organ weight changes in liver, kidneys or spleen that were considered to be attributable to treatment.

In conclusion, under the conditions of this study, the daily oral gavage administration of

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

to Hannover Wistar rats daily for 14 consecutive days at dose levels up to 1000 mg/kg/day caused no signs of evident toxicity.

Based on the results obtained, 1000 mg/kg/day should be considered as a suitable dose for a high dose level in subsequent studies

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

The endpoint is concluded based on a single key study with a Klimisch rating of 1. The database for this endpoint met all relevant data requirements under REACH for the respective tonnage band.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

It is not possible to conclude on the the mode of action of the test item since no adverse effect level was observed following subacute exposure in rats.

Additional information

In an OECD 422 (2018), oral administration (by gavage) of

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

to Hannover Wistar rats at the doses of 100, 350 and 1000 mg/kg/day for two weeks prior to mating and up to the day before sacrifice inclusive (males) or up to days 13-15 of lactation (females) was well tolerated.

No mortality was recorded during the study.

There were no signs of evident toxicity related to clinical signs, body weights, food consumption or effects on sensory reactivity. The differences observed in grip strength among males in all test-item administered groups are not considered relevant in the absence of clinical signs that could corroborate them. Regarding motor activity, the higher mean values observed in all test-item administered groups were not considered relevant given the variability observed in this parameter and as no dose-effect relationship could be established.

The differences observed in the test-item administered males in hematology (reticulocytes, basophil and MCHC) were minor, lacked dose-effect relationship and were not corroborated in the histopathology examination. These values were within those observed in males of this strain and age.

Clinical biochemistry in males administered at 350 and 1000 mg/kg/day revealed significantly lower values in glucose and proteins and in all test item administered groups in phosphorous with respect to Control.  These differences were considered not related with treatment based on the absence of a dose-effect relationship, their magnitude (with the exception of proteins), the fact that all values are within the historical control data and that they were not present in females.

There was no indication of an effect of the test item on T4 levels and there was no evidence of a test-item effect in the thyroid histopathology performed on F0 adults.

The slight differences observed in male brains and in female livers at 1000 mg/kg/day with respect to Control were devoid of toxicological relevancy, taking into account that they were within the normal values observed in rats of this strain and age and as there were no test-item related effects observed in the histopathology examination that could corroborate them.

The microscopic examination performed 5-8 weeks after treatment at 1000 mg/kg/day revealed no test-item-related changes. All the histological findings were considered incidental.

In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages.  No cell or stage-specific abnormalities were noted in males treated at 1000 mg/kg/day.

Estrous cycles and reproductive parameters of pre-coital interval, mating performance, fertility or gestation length or index were unaffected by treatment.

There was no effect on offspring growth. There were no offspring clinical or necropsy signs indicative of a reaction to the test item. Also, there was no effect on litter size, survival indices, sex ratio, body weights, ano-genital distance or nipple areolae.

In conclusion, the effects of oral (gavage) administration of

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

to Wistar rats receiving 100, 350 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

Systemic toxicity: The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day for males and females, taking into account that findings observed in clinical pathology did not affect the general well-being, growth, development or life span as well as that the findings observed in grip strength or motor activity were not corroborated with clinical signs.

Reproductive / developmental toxicity: The No Observed Effect Level (NOEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on estrous cycle, pre-coital interval, mating performance, fertility and gestation length or in the offspring on litter size, sex ratio, survival, clinical signs, body weights, ano-genital distances or macropatholog

Justification for classification or non-classification

The substance does not meet the criteria for classification under reproductive toxicity in accordance with GHS and Regulation (EC) No 1272/2008 (CLP).