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Registration Dossier
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EC number: 701-349-8 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Repeated dose toxicity: oral
Administrative data
- 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.
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- developmental toxicity
- 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:
- other: OECD 422 Guideline for testing of chemicals adopted 29.07.16: 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: table 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
- Remarks:
- Hannover Wistar Rat (HsdHan®:WIST)
- 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): Not reported.
- Photoperiod (hrs dark / hrs light): 12:12
IN-LIFE DATES: From: 5 days acclimatisation until necropsy - Route of administration:
- oral: gavage
- Vehicle:
- other: 1% carboxymethylcellulose sodium salt (medium viscosity) in distillated water
- Remarks:
- 1% carboxymethylcellulose sodium salt
- Details on exposure:
- Oral, by gastric gavage.
- 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.
- Details on mating procedure:
- Paired for mating: After minimum of 2 weeks of treatment.
Male/female ratio: 1:1
Duration of pairing: Up to 2 weeks
Daily checks for evidence of mating Ejected copulation plugs. Sperm within vaginal smear.
Day 0 of gestation: When positive evidence of mating detected.
Male/female separation: Day when mating evidence detected.
Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.
Females showing no evidence of pregnancy (female 50 at 0 mg/kg/day, females no. 62 and 91 at 100 mg/kg/day and female no. 93 at 350 mg/kg/day) were sacrificed 26 days after the last day of the mating period.
Female no. 80 (1000 mg/kg/day), initially paired with male no. 31, did not showed any signs of mating. Consequently, after 14 consecutive days, this female was paired with a second male (no. 32) in order to achieve pregnancy. - Duration of treatment / exposure:
- 5- 8 weeks
- Frequency of treatment:
- Once daily
- Duration of test:
- 5- 8 weeks
- 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: Group 1 receiving vehicle only
- 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. - Maternal examinations:
- 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
TERMINAL INVESTIGATIONS: yes - Ovaries and uterine content:
- Light microscopy qualitative evaluation of one section from each ovary was consucted.
- Fetal examinations:
- Offspring examinations:
Individual values tabulated.
Ano-genital distance presented as distance in mm – was normalized to a measure of pup size using body weight as a covariate for statistical analysis. Nipple/areolae counts presented as mean and individual numbers for males per litter and an overall group mean calculated.
Absolute and adjusted for terminal body weight: Group mean values and SD calculated. - Statistics:
- In-house statistical analysis
- Indices:
- Survival indicices was evaluated
- Historical control data:
- yes
- Clinical signs:
- no effects observed
- Dermal irritation (if dermal study):
- not examined
- 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
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- A single female at 100 mg/kg/day had incidental issue with implantation sites
- Early or late resorptions:
- not specified
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- no effects observed
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- Systemic Toxicity
- Effect level:
- >= 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Dose descriptor:
- NOEL
- Remarks:
- Reproductive toxicity
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- There was no relevant effect of treatment on mean body weights of male or female offspring on day 1 of age or on subsequent body weight measurements until day 13 of age. The statistically significant high mean values observed at 1000 mg/kg/day were not considered to be adverse. Although there was no obvious explanation for the larger mean pup weights at 1000 mg/kg/day, it was considered that this increase may have reflected the marginally smaller litter size and the slightly larger number of females with a 22 day gestation period.
- 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:
- no effects observed
- External malformations:
- no effects observed
- Skeletal malformations:
- no effects observed
- Visceral malformations:
- no effects observed
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Conclusions:
- Exposure to the test item up to 1000 mg/kg bw/day caused no evident toxicity related to clinical signs, body weights, food consumption or effects on sensory reactivity. 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.No cell or stage-specific (testes and seminiferous tubules) abnormalities were noted in treated males.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.
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:
OECD 422 (2018): A study was conducted to assess the general systemic toxic potential of the test item, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, when administered to Wistar rats by oral gavage administration for 5-8 weeks.
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 the test item 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.
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
- Reason / purpose for cross-reference:
- reference to same study
Reference
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- tudy 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:
- 29.07.2016
- Deviations:
- yes
- Remarks:
- Measurement of GGT (Gamma glutamil transferase) & LIP (Lipase) for male no. 5 & Albumin (chemical analysis) for male no. 21. T4 and T3 differs from the CoA and SDS. Male and female weighted different frequencies.
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- 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): Not reported.
- Photoperiod (hrs dark / hrs light): 12:12
IN-LIFE DATES: From: 5 days acclimatisation until necropsy - Route of administration:
- oral: gavage
- Vehicle:
- other: 1% carboxymethylcellulose sodium salt (medium viscosity) in distillated water
- Remarks:
- 1% carboxymethylcellulose sodium salt
- Details on exposure:
- Oral, by gastric gavage.
- Details on mating procedure:
- Paired for mating: After minimum of 2 weeks of treatment.
Male/female ratio: 1:1
Duration of pairing: Up to 2 weeks
Daily checks for evidence of mating: Ejected copulation plugs. Sperm within vaginal smear.
Day 0 of gestation: When positive evidence of mating detected.
Male/female separation: Day when mating evidence detected.
Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.
Females showing no evidence of pregnancy (female 50 at 0 mg/kg/day, females no. 62 and 91 at 100 mg/kg/day and female no. 93 at 350 mg/kg/day) were sacrificed 26 days after the last day of the mating period.
Female no. 80 (1000 mg/kg/day), initially paired with male no. 31, did not showed any signs of mating. Consequently, after 14 consecutive days, this female was paired with a second male (no. 32) in order to achieve pregnancy. - 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.
- Frequency of treatment:
- Once daily
- Details on study schedule:
- 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. - 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
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
BODY WEIGHT: Yes
- Time schedule for examinations: once day before treatment commenced, on the day on treatment (Day 1) and once a week thereafter (including termination). During gestation day 0, 7, 14 & 20 and lactation day 1, 4 and 13.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Weekly
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): not specified
OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations: n/a
- Dose groups that were examined: n/a
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (5 males/group) and (5 lactating females with litter per group)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (5 males/group) and (5 lactating females with litter per group).
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 5 and lactation day 7 – 9.
- Dose groups that were examined: All groups – five surviving male and five lactating females (length of separation of dam from litter must be minimized)
- Battery of functions tested: sensory activity / grip strength / motor activity.
IMMUNOLOGY: No
- Time schedule for examinations: n/a
- How many animals: n/a
- Dose groups that were examined: n/a - Oestrous cyclicity (parental animals):
- 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. - Sperm parameters (parental animals):
- Testes: detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings was noted
- Litter observations:
- Individual litter values tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 and 13 of age.
- Postmortem examinations (parental animals):
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - Postmortem examinations (offspring):
- Offspring examinations:
Individual values tabulated.
Ano-genital distance presented as distance in mm – was normalized to a measure of pup size using body weight as a covariate for statistical analysis. Nipple/areolae counts presented as mean and individual numbers for males per litter and an overall group mean calculated.
Absolute and adjusted for terminal body weight: Group mean values and SD calculated.
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). - Statistics:
- In-house statistical analysis
- Reproductive indices:
- litter size, sex ratio or offspring survival
- Offspring viability indices:
- litter size, sex ratio or offspring survival
- Clinical signs:
- no effects observed
- Dermal irritation (if dermal study):
- not examined
- 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
- 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
- Reproductive function: oestrous cycle:
- no effects observed
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- 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 absence of adverse toxic effects
- Key result
- Dose descriptor:
- NOEL
- Remarks:
- Reproductive toxicity
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- 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.
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There was no relevant effect of treatment on mean body weights of male or female offspring on day 1 of age or on subsequent body weight measurements until day 13 of age. The statistically significant high mean values observed at 1000 mg/kg/day were not considered to be adverse. Although there was no obvious explanation for the larger mean pup weights at 1000 mg/kg/day, it was considered that this increase may have reflected the marginally smaller litter size and the slightly larger number of females with a 22 day gestation period.
- Food consumption and compound intake (if feeding study):
- not examined
- 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
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
- Other effects:
- no effects observed
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Generation:
- F1
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- Exposure to the test item up to 1000 mg/kg bw/day caused no evident toxicity related to clinical signs, body weights, food consumption or effects on sensory reactivity. 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.No cell or stage-specific (testes and seminiferous tubules) abnormalities were noted in treated males.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.
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:
OECD 422 (2018): A study was conducted to assess the general systemic toxic potential of the test item, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, when administered to Wistar rats by oral gavage administration for 5-8 weeks.
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 the test item 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.
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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
Materials and methods
Test guideline
- 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
Test material
- Reference substance name:
- Fatty Acids, C14 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Molecular formula:
- C34H68N2O3
- IUPAC Name:
- Fatty Acids, C14 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Reference substance name:
- Fatty Acids, C15 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Molecular formula:
- C35H70N2O3
- IUPAC Name:
- Fatty Acids, C15 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Reference substance name:
- Fatty Acids, C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Molecular formula:
- C36H72N2O3
- IUPAC Name:
- Fatty Acids, C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Reference substance name:
- Fatty Acids, C15 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Molecular formula:
- C37H74N2O3
- IUPAC Name:
- Fatty Acids, C15 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Reference substance name:
- Fatty Acids, C16 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Molecular formula:
- C38H76N2O3
- IUPAC Name:
- Fatty Acids, C16 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Reference substance name:
- Fatty Acids, C17 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Molecular formula:
- C39H78N2O3
- IUPAC Name:
- Fatty Acids, C17 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Reference substance name:
- Fatty Acids, C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Molecular formula:
- C40H80N2O3
- IUPAC Name:
- Fatty Acids, C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol
- Reference substance name:
- Stearic acid
- EC Number:
- 200-313-4
- EC Name:
- Stearic acid
- Cas Number:
- 57-11-4
- Molecular formula:
- C18H36O2
- IUPAC Name:
- stearic acid
- Reference substance name:
- Palmitic acid
- EC Number:
- 200-312-9
- EC Name:
- Palmitic acid
- Cas Number:
- 57-10-3
- Molecular formula:
- C16H32O2
- IUPAC Name:
- palmitic acid
- Reference substance name:
- Myristic acid
- EC Number:
- 208-875-2
- EC Name:
- Myristic acid
- Cas Number:
- 544-63-8
- Molecular formula:
- C14H28O2
- IUPAC Name:
- myristic acid
- Reference substance name:
- 2-(2-aminoethylamino)ethanol
- EC Number:
- 203-867-5
- EC Name:
- 2-(2-aminoethylamino)ethanol
- Cas Number:
- 111-41-1
- Molecular formula:
- C4H12N2O
- IUPAC Name:
- 2-(2-aminoethylamino)ethanol
- Test material form:
- solid
- Details on test material:
- Identification: N-(2-{[C16-18 (EVEN NUMBERED) ALKANOYL]AMINO}ETHYL)-N-(2-HYDROXYETHYL)[C16-18 (EVEN NUMBERED) ALKYLAMIDE
Storage Conditions: Room temperature
Constituent 1
Constituent 2
Constituent 3
Constituent 4
Constituent 5
Constituent 6
Constituent 7
Constituent 8
Constituent 9
Constituent 10
Constituent 11
- 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
Test animals
- 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
Administration / exposure
- 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
Doses / concentrationsopen allclose all
- 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
Examinations
- 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
Results and discussion
Results of examinations
- 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.
Effect levels
- 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
Target system / organ toxicity
- Key result
- Critical effects observed:
- no
Any other information on results incl. tables
Tables containing raw data are attached in background material.
Applicant's summary and conclusion
- 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.
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