Amides, C12-18 and C18-unsatd., N,N-bis(hydroxyethyl)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined repeated dose toxicity study with the reproduction / developmental toxicity screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 10, 2021 to February 4, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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:

adopted on 29 July 2016

Deviations:

yes

Remarks:

Females and males were supplied in the weight range of 178-235 and 201-222 g and not 175-200 and 200-225 g. Motor activity measurements were carried out Week 5 and not Week 4, as indicated in the Study Protocol. These deviations had no impact on the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C16-18 and C18-unsatd., N,N-bis(hydroxyethyl)

EC Number:

271-653-9

EC Name:

Amides, C16-18 and C18-unsatd., N,N-bis(hydroxyethyl)

IUPAC Name:

Amides, C16-18 (even numbered) and C18-unsatd., N, N-bis(hydroxyethyl)

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Hsd: Sprague Dawley SD rats

Details on species / strain selection:

The Sprague Dawley rat is the species and strain of choice because it is accepted by many regulatory authorities and there is ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Animal supply and acclimatization:
A total of 102 Hsd: Sprague Dawley SD rats (45 males and 57 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy. After arrival, the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian. An acclimatisation period of approximately 4 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

- Animal husbandry:
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C±2°C and 55%±15%, respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. From arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week. Drinking water was supplied ad libitum to each cage via water bottles, except in the case of urinalysis investigations. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum throughout the study. There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC. Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

- Allocation to groups:
On the day of allocation all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. Furthermore, female animals that exhibited anomalies in the oestrous cycle were not allocated to the main groups. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed five per sex per cage. The cages were identified by a label and recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each group were distributed to minimise possible environmental effects and or contamination. Any animal showing signs of ill health during the period between allocation and the start of treatment was subjected to pathological examination as considered appropriate and replaced with a surplus animal selected from the same batch.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item will be administered orally, by gavage. The oral route has been selected as it is a possible route of exposure of the test substance in man.

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC

Details on oral exposure:

The required amount of test substance was suspended in the vehicle. The formulations were prepared weekly or daily (concentrations of 10, 25 and 70 mg/mL), according to stability data from ERBC study No. A4125. Concentrations were calculated and expressed in terms of test substance as supplied. The test substance was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical method was validated in ERBC Study no. A4125 in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation study (r > 0.99; accuracy 80-120%; precision CV < 10%). In ERBC Study no. A4125, a 48 hour stability at room temperature and a 9 day stabiliy at 2-8°C were verified in the range from 10 to 100mg/mL. According to ERBC SOPs, suspensions were considered to be stable if concentration and homogeneity, after the defined period of storage, were still acceptable (80%-120% for concentration and CV < 10% for homogeneity). The proposed preparation procedure for the test item was checked in the range from 10 to 100mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study no. A4125 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in ERBC SOPs for concentration (80-120%) and homogeneity (CV < 10%). Samples of the preparations prepared onWeek 1 and 5 (last week with males and females) were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (80-120% for concentration and CV < 10% for homogeneity). Chemical analysis was carried out by the Analytical Chemistry Department. The software used for this activity was Empower® 2 Build No. 2154.

Duration of treatment / exposure:

Males: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing, through the pairing period and thereafter until the day before necropsy or the day before sacrifice, up to a total of 33/34 days for surviving animals. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females: Animals were dosed once a day, 7 days a week, for 14 days prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice (for a total of 43 to 63 days). Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation and lactation periods, dose volumes were calculated according to the last recorded body weight.

Frequency of treatment:

Once daily, 7 days/weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

700 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

- Mortality:
Throughout the study, all animals were checked early in the morning and in the afternoon each working day. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

- Clinical signs:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs was recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions. Observations were performed at the same time interval each day (approximately 5 - 10 minutes and 1.30 - 2 hours post-dose), the interval was selected taking into consideration the presence of post-dose reactions. All observations were recorded for individual animals.

- Clinical Observations (Functional Observation Battery Tests):
Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination (ERBC SOP no. ANI/344). Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. All observations were recorded for individual animals.

- Grip strength and sensory reactivity to stimuli:
Once during the study, towards the end of treatment (during Week 5 for males and Day 12 post partum for females with viable litters, where possible), 5 out of 10 males and 5 out of 10 females were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength (ERBC SOP no. ANI/344). Measurements were performed using a computer-generated random order.

- Motor activity assessment (MA):
Once during the study, towards the end of treatment (during Week 5 for males and on Day 12 post partum for females with viable litters where possible), 5 males and 5 females were randomly selected from each group and the motor activity (MA) were measured (for approximately 5 minutes) by an automated activity recording device (ERBC SOP no. ANI/346). Measurements were performed using a computer-generated random order.

- Body weight - Parental animals:
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.
Dams were weighed on Days 1, 4, 7, 13 post partum and just before to necropsy.

- Food consumption:
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting from Day 1 of dosing up to mating. Individual food consumption for mated females was measured on gestation Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.

- Clinical pathology investigations
Blood collection was performed for hormone determination (0.8 mL) from all parental animals at termination under condition of food deprivation. Blood samples for haematology, clinical chemistry and coagulation were collected by random selection from 5 males and 5 females (females with viable litters) of each group, under condition of food deprivation. Following haematology and coagulation paramaters were assessed: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count, platelets and prothrobin time. Clinical chemistry parameters assessed corresponds to : alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, triglycerides, bile acids, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G Ratio, sodium, potassium, calcium, chloride, inorganic phosphorus.

-Urinalysis (Only males randomly selected)
During the last week of treatment, individual overnight urine samples were also collected from the same animals selected for clinical pathology investigations (5 males/group, randomly selected). Before starting urine collection water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis. The measurements performed on urine samples are as follows: appearance, volume (manually recorded), specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood.
These parameters were analysed by Menarini Aution Max AX 4280/Aution Eleven AE 4020, according to in- ternal procedures. The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for: epithelial cells, leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal components.

- Blood collection and thyroid hormone determination (T4 and TSH)
Blood collection for hormone determination was performed from all animals at termination.
Males
Blood samples (approximately 0.8 mL) for hormone determination were collected under isoflurane anaesthesia from the retro-orbital sinus. The order of collection was equalised between groups.
Females
As a part of the necropsy procedure, blood samples (approximately 0.8 mL) for hormone determination was withdrawn from the abdominal vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.

- Immunoanalysis - Thyroid hormone determination (T4 and TSH)
Samples were assayed to determine the serum levels of Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA).

Sacrifice and pathology:

-Necropsy
The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

-Organ weights
From all animals completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

-Tissues fixed and preserved
Samples of all the tissues (all parental animals) were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

-Histopathological analyses
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

-Sacrifice/Euthanasia: Parental animals and those that had completed the scheduled test period were killed by exsanguinations under isofluorane anaesthesia. The animal sacrificed for humane reasons was killed with carbon dioxide. Pups: Pups killed for humane reasons or those that had completed the scheduled test period (Day 4 post partum or Day 14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal. Pups selected for blood collection for hormone determination were killed on the day of blood sampling. Parental males: The males were killed after the mating of all females (up to Day 35 of the study). Parental females: The females with live pups were killed on Day 14 post partum. One female with total litter loss was killed shortly after the occurrence of total litter loss. The females showing no evidence of copulation were killed 25 days after the last day of the mating session. The females which did not gave birth 25 days after positive identification of mating was killed shortly after

Statistics:

Standard deviations were calculated as appropriate. For variables such as body weight, food consumption, clinical pathology parameters and organ weight, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was observed in animals of both sexes dosed at 250 (10 out of 10 males and 5 out of 10 females) and 700 mg/kg/day (all males and females) with a dose-related frequency and incidence, from the first few days of the pre-mating phase up to termination. This treatment-related clinical sign is not considered to be adverse, since no effects on the health status of the affected animals were evident. No clinical signs were observed in animals dosed at 100 mg/kg/day.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight and body weight gain for male and female animals were generally comparable between the treated and control groups, before and during mating, during gestation and post partum periods.
In a single occasion (Day 0 post coitum), females receiving 700 mg/kg/day showed a very slight but statistically significant decrease in body weight (-5%) compared to the control group. This isolated change was followed by a regular growth of the animals. Due to its occasional occurrence and to its low magnitude, this change was not considered treatment related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment in both gender during the study.

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):

No treatment-related changes were recorded.
A statistically significant decrease of monocytes was recorded between control and males dosed at 700mg/kg/day (49%). Values were within the range of historical control data and no other related finding was recorded, therefore this change was considered to be unrelated to treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No effects were obseved.

Endocrine findings:

no effects observed

Description (incidence and severity):

No effects were obseved.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes were observed.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in the open arena (neurotoxicity assessment) did not reveal changes attributable to the test substance, compared to controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in terminal body weight and organ weights when compared to the controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No treatment-related alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic observations associated with the oral administration of test substance were present in the non glandular region of the stomach of high dose animals of both sexes. Forestomach: treatment-related changes were present in the non-glandular region of the stomach (forestomach) in 4/5 males and 4/5 females of the high dose group and consisted of mild to moderate epithelial hyperplasia (1). Epithelial hyperplasia was associated with hyperkeratosis (thickening of the stratum corneum). Chronic inflammation and oedema were observed in the submucosa in association with the hyperplasia, and, in two instances (animals nos. X1670071 and X1670077), there was also mucosal erosion/ulceration. No treatment-related changes were observed in the non glandular region of the stomach of the selected animals of both sexes from the mid-dose group. Any other microscopic observations other than that listed above had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment. There were no test item-related microscopic observations in the testis (stage aware evaluation on PAS-stained slides).

(1) Toxicologic Pathology, 2016; 29 (1 Suppl): 1S–124S - Thomas Nolte.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Observation of animals at removal from the cage and in the open arena (neurotoxicity assessment) did not reveal changes attributable to the test item, compared to controls.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

neuropathology

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the NOAEL for general toxicity was considered to be 700 mg/kg/day for males and females.

Executive summary:

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted with the test substance C16-18 and C18-unsatd. DEA in accordance with OECD 422 and in compliance with GLP. Four groups of ten Sprague Dawley rats (males and females) were exposed by oral gavage to increasing concentrations of the test substance (0, 100, 250 and 700 mg/kg/day). All doses were administered at a constant volume of 10 mL/kg body weight. 0.5% aqueous solution of carboxymethylcellulose was used as a vehicle. According to the study design, males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, for a total of 33/34 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post-partum, for a total of 43 to 63 days. The following investigations were performed: body weight, clinical signs (including neurotoxicity assessment, motor activity and sensory reactivity to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry in five randomly selected animals/sex/group), macroscopic observations, organ weights. Routine histopathological examination was performed in control and high dose groups (five randomly selected animals/sex/group). No mortality occurred throughout the study due to the administration of the test substance. Salivation was the only treatment-related clinical sign recorded in males and females treated at 250 and 700 mg/kg/day, during the study. These clinical signs were not considered to be adverse since no effects on the health status were evident in the affected animals. Neurotoxicity assessment (removal from the home cage and observations in an open arena), motor activity, grip strength and sensory reactivity to stimuli did not reveal changes attributable to the test substance. Body weight and body weight gain of treated animals did not show differences throughout the study when compared to the control group. The food consumption was comparable in all groups. No adverse findings were recorded in clinical pathology investigations (haematology including coagulation parameters and clinical chemistry). Hormone analysis did not show any relation to treatment. Urinalysis in male animals did not reveal changes attributable to the test substance. Changes were observed in the stomach of the majority of males and females dosed at 700mg/kg/day at microscopic examination, due to a local irritant effect of the test substance but these signs were not considered to be severe. Under the study conditions, the NOAEL for general toxicity was considered to be 700 mg/kg/day for males and females (Longobardi, 2022).