N-(2-hydroxyethyl)dodecanamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422 (2018): No Observed Adverse Effect Level (NOAEL) of >= 1000 mg/kg bw/day for reproductive toxicity (fertility)

Link to relevant study records

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:

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

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:

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:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

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 (20 ± 5 ºC) in the dark and in a dessicator.
- Stability of formulation under test conditions: Homogeneity (Accuracy), For continuous data, a parametric analysis was performed if Bartlett's test for variance homogeneity is not significant at the 1% level. Treated groups were compared to control using Williams' test, unless there is evidence against a monotonic dose-response when Dunnett's test was performed instead.
- Solubility and stability of the test substance in the formulation: yes
.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

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

OTHER SPECIFICS: n/a

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Hannover Wistar rats

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: 275-340 g & Females: 196-238 g.
- Fasting period before study: no
- Housing: Cages with standard, granulated, S8-15 sawdust bedding, Premating period (maximum 5 animals/cage) Makrolon type-IV cages, Mating period (one male and one female/cage) Makrolon type-III cages & Postmating, gestation and lactation periods (individual) Makrolon type-III.
- Diet Pelleted standard Teklad 2014C / Teklad 2018C rat/mouse maintenance diet ad libitum (supplied by Envigo RMS, S.L.).
- Water (e.g. ad libitum): Tap water in bottles ad libitum
- Acclimation period: 5 days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: See above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 ºC
- Humidity (%): 35 - 55 %
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 07 November 2017 To 05 March 2018

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

1. The necessary amount of the test item was weighed in a single-use container.
2. The test item was transferred to a sufficiently large mortar and it was pulverized with a pestle.
3. Small amounts of vehicle were added and mixed with the pestle. Any lumps were broken up at this point, resulting in a suspension.
4. The suspension was transferred to a volumetric flask or graduated measuring cylinder that was previously moistened with the vehicle. The mortar was rinsed and the single-use container completely with the vehicle to ensure that there were no remnants of the test item. This vehicle was added to the volumetric container.
5. The suspension was transferred to a suitable container and mixed with a shear mixer until the suspension was homogenized.
6. After the formulations were mixed for 5 to 30 minutes, the samples were taken when necessary.
NOTE: The density was taken the first day each concentration was prepared.
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.

Details on mating procedure:

Paired for mating: After minimum of 2 weeks of treatment.
Male/female ratio: 1:1
Duration of pairing: 1-4 days
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 70 at 100 mg/kg/day and female 75 at 350 mg/kg/day) were sacrificed 26 days after the last day of the mating period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to treatment start, an analytical method (M0345_HPLC_DFA_N-(2-hydroxyethyl) dodecanamide_Formulation_Vehicle_ISV) was validated in the present study. Formulations at two concentration levels (concentrations: 10 and 250 mg/mL) were prepared. The validation parameters and acceptance criteria were met.

The formulations prepared at three different concentrations were analyzed twice over the course of the study to verify its correct preparation.

Control (vehicle) formulations were analyzed to confirm the absence of test item or other substances at the retention time of the test item.

The formulation analysis results (including validation and DFA) were calculated using the Empower (version 2.0, Waters, USA). Other parameters such as accuracy, mean, SD and CV were calculated using an Excel spread sheet.

Duration of treatment / exposure:

5-8 weeks

Frequency of treatment:

Once daily

Details on study schedule:

F0 males: Two weeks prior to mating start until the day before sacrifice (after 5 weeks of treatment). They were then killed.
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 / 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 vehicles only

Details on study design:

Dose selection rationale: It was considered a suitable dose level range, based on the preliminary results obtained in the previous non-GLP study FV29DL 14-day Oral (Gavage) Dose-Range Toxicity Study for OECD 422 conducted at in Envigo CRS, S.A.U.:
- The high dose was selected as no toxicity was observed in the preliminary study at 1000 mg/kg/day and considering it as a limit dose to be tested.
- Intermediate and low dose levels were selected considering approximately a 3-fold interval between doses. 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.

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.

Frequency of administration: Once daily
-F0 males: Two weeks prior to mating start until the day before sacrifice (after 5 weeks of treatment). They were then killed.
-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: 5 mL/kg

Duration of treatment period: 5-8 weeks

Storage of formulation in animal housing (for administration): At room temperature and in agitation.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation was recorded at 1000 mg/kg/day in males from day 7 of treatment and in females from day 9 until the end of the study. Salivation was also recorded occasionally in males at 100 and 350 mg/kg/day and in females at 350 mg/kg/day. It tended to occur approximately within ½ hour after dosing. Incidence was higher in males than in females.

No other signs considered related to the administration of N-(2-hydroxyethyl) dodecanamide were observed during the study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female no. 54, administered at 0 mg/kg/day, was found dead on lactation day 6. No clinical signs were observed in this female during the treatment period and no findings were observed in the macroscopic examination during necropsy.

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:

effects observed, non-treatment-related

Description (incidence and severity):

In males, after 5 weeks of treatment at 1000 mg/kg/day, statistically significant differences from Controls were observed in hematocrit, hemoglobin and lymphocytes (lower values with respect to Control) and Red Cell Distribution Width (higher values with respect to Control). The significant differences observed in mean Activated partial thromboplastin time (SAPT) values in males at 1000 mg/kg/day with respect to Control were considered non-adverse, given the magnitude of the differences between means.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry after 5 weeks of treatment in males revealed statistically lower than Control bile acids and higher than Control glucose at 350 and 1000 mg/kg/day. CK mean values at 1000 mg/kg/day were statistically lower with respect to Control. These differences were considered fortuitous in nature and were devoid of any toxicological significance (due to the individual values). They were attributed to the normal biological variability taking into account the common ranges observed in the historical control data in animals following the same study conditions.

Statistically significant differences in mean potassium, chloride and calcium values were recorded in males at 1000 mg/kg/day with respect to Control; however, these differences could be considered not toxicological relevant. In the absence of an effect in the total proteins or albumin, the significant differences observed in protein electrophoresis were considered not to have any relevancy.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Grip strength recordings revealed significantly lower mean forelimb and mean hindlimb values among males in all test-item administered groups with respect to Control. However based on the high variability observed in the individual males at 0 mg/kg/day (increasing mean values), there was no dose-effect relationship or evidence of this effect in females and there were no clinical signs observed that can corroborate this finding in males and could be related to the high deviation recorded in Control males.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs observed among the F1 litters that could be clearly attributable to parental treatment with N-(2-hydroxyethyl) dodecanamide.
Although little or no milk was observed in the stomach in some pups on lactation day 1, the majority of the offspring survived the first days of the phase, with no cannibalization or loss of offspring from day 2 onwards, with the exception of a pup (94-7F) at 350 mg/kg/day, which was missing on day 6 of lactation.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not specified

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:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There was no effect observed in male nipple observations.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

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

Tables containing raw data are attached in background material.

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 (NOAEL) 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, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.

Executive summary:

In a study conducted in accordance with OECD 422, three groups of 10 male and 10 female rats received N-(2-hydroxyethyl) dodecanamide 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 32 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, arachis oil.

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:

No test-item-related mortality was observed during the study, and administration of N-(2-hydroxyethyl) dodecanamide at 100, 350 or 1000 mg/kg/day was considered not to have any relevant effects on clinical condition, body weight, food consumption, grip strength, sensory reactivity and motor activity, pre-coital interval, mating performance and fertility, or gestation length. Salivation was recorded in all test-item-administered groups; the incidence was dose-related in both sexes and is most likely due to taste aversion (from gavage dosing).

Estrous cycles before and during treatment were considered not to be altered by the test item. At termination, all reproductive phase females showed diestrus.

In males administered at 1000 mg/kg/day, hematocrit, hemoglobin and lymphocytes were affected with respect to Control. Although it cannot be considered an adverse effect as the males’ physiology was not affected, and it was not possible to positively attribute the finding to the test item. There were no differences in coagulation, clinical biochemistry or organ weights considered treatment-related. T4 analyses of samples in F0 males and F1 offspring on day 13 did not reveal any differences that could be attributable to treatment.

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

Histopathology revealed that there were no treatment-related effects in any of the examined organs or in the reproductive organs or mammary glands.

There were no adverse effects observed in the offspring. So, offspring survival, litter size, clinical signs, body weights, ano-genital distances or macropathology were not affected by N‑(2-hydroxyethyl) dodecanamide administration.

In conclusion, the effects of oral (gavage) administration of N-(2-hydroxyethyl) dodecanamide 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, taking into account that findings observed in clinical pathology did not affect the general well-being, growth or development of males.

The No Observed Effect Level (NOEL) for systemic toxicity was considered to be 1000 mg/kg/day for females, taking into account that there was no effect on body weight, food consumption, clinical signs, clinical pathology, organ weights or histopathology.

Reproductive / developmental toxicity:

The No Adverse Effect Level (NOAEL) 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, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.

Effect on fertility: via oral route

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 Klimish rating of 1. The available data meet the data requirements for the REACH tonnage band.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a study conducted in accordance with OECD 422, three groups of 10 male and 10 female rats received N-(2-hydroxyethyl) dodecanamide 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 32 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, arachis oil.

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:

No test-item-related mortality was observed during the study, and administration of N-(2-hydroxyethyl) dodecanamide at 100, 350 or 1000 mg/kg/day was considered not to have any relevant effects on clinical condition, body weight, food consumption, grip strength, sensory reactivity and motor activity, pre-coital interval, mating performance and fertility, or gestation length. Salivation was recorded in all test-item-administered groups; the incidence was dose-related in both sexes and is most likely due to taste aversion (from gavage dosing).

Estrous cycles before and during treatment were considered not to be altered by the test item. At termination, all reproductive phase females showed diestrus.

In males administered at 1000 mg/kg/day, hematocrit, hemoglobin and lymphocytes were affected with respect to Control. Although it cannot be considered an adverse effect as the males’ physiology was not affected, and it was not possible to positively attribute the finding to the test item. There were no differences in coagulation, clinical biochemistry or organ weights considered treatment-related. T4 analyses of samples in F0 males and F1 offspring on day 13 did not reveal any differences that could be attributable to treatment.

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

Histopathology revealed that there were no treatment-related effects in any of the examined organs or in the reproductive organs or mammary glands.

There were no adverse effects observed in the offspring. So, offspring survival, litter size, clinical signs, body weights, ano-genital distances or macropathology were not affected by N‑(2-hydroxyethyl) dodecanamide administration.

In conclusion, the effects of oral (gavage) administration of N-(2-hydroxyethyl) dodecanamide 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, taking into account that findings observed in clinical pathology did not affect the general well-being, growth or development of males.

The No Observed Effect Level (NOEL) for systemic toxicity was considered to be 1000 mg/kg/day for females, taking into account that there was no effect on body weight, food consumption, clinical signs, clinical pathology, organ weights or histopathology.

Reproductive / developmental toxicity:

The No Adverse Effect Level (NOAEL) 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, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.

Effects on developmental toxicity

Description of key information

OECD 422 (2018): No Observed Adverse Effect Level (NOAEL) of >= 1000 mg/kg bw/day for developmental toxicity

Link to relevant study records

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.

Qualifier:

according to guideline

Guideline:

other: OECD 422 Guideline for testing of chemicals adopted 29 July 2016: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

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

Principles of method if other than guideline:

OECD 422 Guideline for testing of chemicals adopted 29.07.16: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

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 (20 ± 5 ºC) in the dark and in a dessicator.
- Stability of formulation under test conditions: Homogeneity (Accuracy), For continuous data, a parametric analysis was performed if Bartlett's test for variance homogeneity is not significant at the 1% level. Treated groups were compared to control using Williams' test, unless there is evidence against a monotonic dose-response when Dunnett's test was performed instead.
- Solubility and stability of the test substance in the formulation: yes
.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

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

OTHER SPECIFICS: n/a

Species:

rat

Strain:

Wistar

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: 275-340 g & Females: 196-238 g.
- Fasting period before study: no
- Housing: Cages with standard, granulated, S8-15 sawdust bedding, Premating period (maximum 5 animals/cage) Makrolon type-IV cages, Mating period (one male and one female/cage) Makrolon type-III cages & Postmating, gestation and lactation periods (individual) Makrolon type-III.
- Diet Pelleted standard Teklad 2014C / Teklad 2018C rat/mouse maintenance diet ad libitum (supplied by Envigo RMS, S.L.).
- Water (e.g. ad libitum): Tap water in bottles ad libitum
- Acclimation period: 5 days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: See above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 ºC
- Humidity (%): 35 - 55 %
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 07 November 2017 To 05 March 2018

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

1. The necessary amount of the test item was weighed in a single-use container.
2. The test item was transferred to a sufficiently large mortar and it was pulverized with a pestle.
3. Small amounts of vehicle were added and mixed with the pestle. Any lumps were broken up at this point, resulting in a suspension.
4. The suspension was transferred to a volumetric flask or graduated measuring cylinder that was previously moistened with the vehicle. The mortar was rinsed and the single-use container completely with the vehicle to ensure that there were no remnants of the test item. This vehicle was added to the volumetric container.
5. The suspension was transferred to a suitable container and mixed with a shear mixer until the suspension was homogenized.
6. After the formulations were mixed for 5 to 30 minutes, the samples were taken when necessary.
NOTE: The density was taken the first day each concentration was prepared.
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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to treatment start, an analytical method (M0345_HPLC_DFA_N-(2-hydroxyethyl) dodecanamide_Formulation_Vehicle_ISV) was validated in the present study. Formulations at two concentration levels (concentrations: 10 and 250 mg/mL) were prepared. The validation parameters and acceptance criteria were met.
The formulations prepared at three different concentrations were analyzed twice over the course of the study to verify its correct preparation
Control (vehicle) formulations were analyzed to confirm the absence of test item or other substances at the retention time of the test item
The formulation analysis results (including validation and DFA) were calculated using the Empower (version 2.0, Waters, USA). Other parameters such as accuracy, mean, SD and CV were calculated using an Excel spread sheet.

Details on mating procedure:

Paired for mating: After minimum of 2 weeks of treatment.
Male/female ratio: 1:1
Duration of pairing: 1-4 days
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 70 at 100 mg/kg/day and female 75 at 350 mg/kg/day) were sacrificed 26 days after the last day of the mating period.

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:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: It was considered a suitable dose level range, based on the preliminary results obtained in the previous non-GLP study FV29DL 14-day Oral (Gavage) Dose-Range Toxicity Study for OECD 422 conducted at in Envigo CRS, S.A.U.:
- The high dose was selected as no toxicity was observed in the preliminary study at 1000 mg/kg/day and considering it as a limit dose to be tested.
- Intermediate and low dose levels were selected considering approximately a 3-fold interval between doses. 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.

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.

Frequency of administration: Once daily
- F0 males: Two weeks prior to mating start until the day before sacrifice (after 5 weeks of treatment). They were then killed.
- 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: 5 mL/kg

Duration of treatment period: 5-8 weeks

Storage of formulation in animal housing (for administration): At room temperature and in agitation.

Maternal 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
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.

Ovaries and uterine content:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Fetal examinations:

Individual litter values tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 and 13 of age.
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

Indices:

litter size, sex ratio or offspring survival

Historical control data:

Yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Salivation was recorded at 1000 mg/kg/day in males from day 7 of treatment and in females from day 9 until the end of the study. Salivation was also recorded occasionally in males at 100 and 350 mg/kg/day and in females at 350 mg/kg/day. It tended to occur approximately within ½ hour after dosing. Incidence was higher in males than in females.

No other signs considered related to the administration of N-(2-hydroxyethyl) dodecanamide were observed during the study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female no. 54, administered at 0 mg/kg/day, was found dead on lactation day 6.No clinical signs were observed in this female during the treatment period and no findings were observed in the macroscopic examination during necropsy.

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:

effects observed, non-treatment-related

Description (incidence and severity):

In males, after 5 weeks of treatment at 1000 mg/kg/day, statistically significant differences from Controls were observed in hematocrit, hemoglobin and lymphocytes (lower values with respect to Control) and Red Cell Distribution Width (higher values with respect to Control). The significant differences observed in mean Activated partial thromboplastin time (SAPT) values in males at 1000 mg/kg/day with respect to Control were considered non-adverse, given the magnitude of the differences between means.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry after 5 weeks of treatment in males revealed statistically lower than Control bile acids and higher than Control glucose at 350 and 1000 mg/kg/day. CK mean values at 1000 mg/kg/day were statistically lower with respect to Control. These differences were considered fortuitous in nature and were devoid of any toxicological significance (due to the individual values). They were attributed to the normal biological variability taking into account the common ranges observed in the historical control data in animals following the same study conditions.

Statistically significant differences in mean potassium, chloride and calcium values were recorded in males at 1000 mg/kg/day with respect to Control; however, these differences could be considered not toxicological relevant. In the absence of an effect in the total proteins or albumin, the significant differences observed in protein electrophoresis were considered not to have any relevancy.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Grip strength recordings revealed significantly lower mean forelimb and mean hindlimb values among males in all test-item administered groups with respect to Control. However based on the high variability observed in the individual males at 0 mg/kg/day (increasing mean values), there was no dose-effect relationship or evidence of this effect in females and there were no clinical signs observed that can corroborate this finding in males and could be related to the high deviation recorded in Control males.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

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.

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

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:

NOAEL

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

Tables containing raw data are attached in background material.

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 (NOAEL) 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, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.

Executive summary:

In a study conducted in accordance with OECD 422, three groups of 10 male and 10 female rats received N-(2-hydroxyethyl) dodecanamide 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 32 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, arachis oil.

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:

No test-item-related mortality was observed during the study, and administration of N-(2-hydroxyethyl) dodecanamide at 100, 350 or 1000 mg/kg/day was considered not to have any relevant effects on clinical condition, body weight, food consumption, grip strength, sensory reactivity and motor activity, pre-coital interval, mating performance and fertility, or gestation length. Salivation was recorded in all test-item-administered groups; the incidence was dose-related in both sexes and is most likely due to taste aversion (from gavage dosing).

Estrous cycles before and during treatment were considered not to be altered by the test item. At termination, all reproductive phase females showed diestrus.

In males administered at 1000 mg/kg/day, hematocrit, hemoglobin and lymphocytes were affected with respect to Control. Although it cannot be considered an adverse effect as the males’ physiology was not affected, and it was not possible to positively attribute the finding to the test item. There were no differences in coagulation, clinical biochemistry or organ weights considered treatment-related. T4 analyses of samples in F0 males and F1 offspring on day 13 did not reveal any differences that could be attributable to treatment.

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

Histopathology revealed that there were no treatment-related effects in any of the examined organs or in the reproductive organs or mammary glands.

There were no adverse effects observed in the offspring. So, offspring survival, litter size, clinical signs, body weights, ano-genital distances or macropathology were not affected by N‑(2-hydroxyethyl) dodecanamide administration.

In conclusion, the effects of oral (gavage) administration of N-(2-hydroxyethyl) dodecanamide 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, taking into account that findings observed in clinical pathology did not affect the general well-being, growth or development of males.

The No Observed Effect Level (NOEL) for systemic toxicity was considered to be 1000 mg/kg/day for females, taking into account that there was no effect on body weight, food consumption, clinical signs, clinical pathology, organ weights or histopathology.

Reproductive / developmental toxicity:

The No Adverse Effect Level (NOAEL) 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, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.

Effect on developmental toxicity: via oral route

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 Klimish rating of 1. The available data meet the data requirements for the REACH tonnage band.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Refer to "Effects on Fertility - Additional Information"

Mode of Action Analysis / Human Relevance Framework

The test item did not induced any effects on the exposured animals and as such, no mode of action could be deduced.

Justification for classification or non-classification

The substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP).