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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Well conducted and comparable to guideline study, no information on GLP status

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Mus-Rattus, Brunntal, Germany
- Strain: Wistar rat, MuRa Han 67 SPF
- Age at study initiation: between 6-7 wk
- Weight at study initiation: 109 (f) - 114 (m) g
- Housing: plastic cages, 3 males and 5 females/cage
- Diet (e.g. ad libitum): ad libitum (Altromin Ratdiet No. 1424 DK, Altromin GmbH, Lage, Germany)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 60-80
- Air changes (per hr): 11
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

Doses were adapted weekly to the body weight; application volume - 5 mL/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

no information

Duration of treatment / exposure:

28 d

Frequency of treatment:

daily once, 5 times/wk

Dose / conc.:

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

Remarks:

(Days 1-28)
Basis:
actual ingested

Dose / conc.:

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

Remarks:

(Days 1-28)
Basis:
actual ingested

Dose / conc.:

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

Remarks:

(Days 1-14)
Basis:
actual ingested

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Remarks:

(Days 15-28)
Basis:
actual ingested

No. of animals per sex per dose:

10/sex/dose for main; 5/sex/dose for 4 months recovery period

Control animals:

yes, concurrent vehicle

Details on study design:

according to standard procedure

Positive control:

not necessary

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: end of study

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study
- Anaesthetic used for blood collection: Yes (ether)
- How many animals: 10 per dose and sex
- Parameters checked: Hematocrit, erythrocytes, leukocytes, hemoglobin, thrombocytes, mean corpuscular volume, white blood cell differential

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study
- How many animals: 10 per sex and dose
- Parameters checked: GPT, GOT, AP, glucose, urea, total protein, calcium, phosphate, cholesterol

URINALYSIS: Yes
- Time schedule for collection of urine: end of study
- Metabolism cages used for collection of urine: No
- Parameters checked: urea, creatinine, sodium, potassium, glucose, calcium, ap

NEUROBEHAVIOURAL EXAMINATION: No

Other: Groups of 5 male and 5 female rats kept for an additional 4 month recovery period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
the following tissues/organs were examined:
adrenal gland
aorta thoracica
brain (cornu ammonis)
coagulating gland with seminal vesicle
epididymis
eye with optic nerve
heart
intestine, large
intestine, small
kidney
liver
lungs
lymph node (cervical)
lymph node (mesenteric)
mucles
oesophagus
ovary
pancreas
prostate
salivary glands (mandibular, parotid and sublingual gland)
skin
spleen
stomach
testicle
thymus
thyroid (incl. parathyroids)
tongue
trachea (incl. larynx)
urinary bladder
uterus (incl. cervix and oviducts)

HISTOPATHOLOGY: Yes
see gross pathology

Other examinations:

None

Statistics:

t-test used for statistical analysis of all parameters except organ weight (U-test)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

The doses up to 1500 mg/kg body weight/day were tolerated by all animals without lethality.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight gain and total increase of body weights did not differ from the control group.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical parameters calcium, blood sugar, urea, creatinine, cholesterine, GGT, GOT, GPT and LDH did not show any critical signs. Only slight shifts which were not dose-dependent could be observed. These signs were considered as not substance depending.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative organ weights showed no significant changes in the substance groups compared to the control group, except the organ weight of the liver which is slightly increased for the males of group 4 (750/1500 mg/kg bw) and increased adrenal glands weight in high dose females. This result is considered of no relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

The pathological and histological evaluation did not show significant compound related gross or microscocopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose dependent reversible local findings were restricted to the fore stomach mucosa (important hyperplasia and ulcerations, in some cases also hyper- and parakeratosis of the forestomach of males and females at the high dose. Similar effects but less intense at the medium and low doses).

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The pathological and histological evaluation did not show significant compound related gross or microscocopic organ injury of liver, kidneys, adrenals, heart, lung, spleen and gonads; dose dependent reversible local findings were restricted to the fore stomach mucosa (important hyperplasia and ulcerations, in some cases also hyper- and parakeratosis of the forestomach of males and females at the high dose. Similar effects but less intense at the medium and low doses).

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

> 750 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: No biologically relevant treatment-related effects observed on any of the parameters recorded at any dose, also test animals treated with 1500 mg/kg bw (Days 15-28) showed no adverse effect

Key result

Critical effects observed:

not specified

Conclusions:

Under the study conditions, the 28 d NOAEL to rats was considered to be >750 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the test substance, C12-18 and C18-unsatd. MEA, according to a design based on OECD 407. Groups of 10 male and 10 female Wistar rats were orally gavaged with the substance diluted in olive oil, 5 d/week for 28 d at doses of 0, 70, 250, 750 (Days 1-14) and 1500 (Days 15-28) mg/kg bw/d. Clinical signs, bodyweight, haematology, clinical chemistry, urinalysis, gross and microscopic pathology were recorded. Additional groups of 5 male and 5 female rats were kept for a 4 month recovery period. No treatment-related adverse effects were observed at any of the doses. Changes in the forestomach at some doses including controls were attributed to the use of olive oil and found to be reversible after end of exposure. Under the study conditions, the 28 d NOAEL to rats was considered to be >750 mg/kg bw/day (Potokar, 1983).

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

Not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sprague-Dawley rats (Vivo Bio Tech Ltd), age: 6–7 weeks

Route of administration:

oral: gavage

Vehicle:

other: water with Tween 80 (0.5% w/v) and carboxymethyl cellulose (1% w/v)

Details on oral exposure:

Preliminary tests found that the test substance was suitable for oral gavage by suspension in water with Tween 80 (0.5% w/v) as a surfactant and carboxymethyl cellulose (1% w/v) as a suspending agent.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses performed to verify the concentrations of the test substance in dosing formulations showed that they were within an acceptable range compared to their respective nominal concentrations.

Duration of treatment / exposure:

90 d

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 (10 males and 10 females)

Control animals:

yes

Details on study design:

Rats were divided into six groups. Four groups of 20 (10 males and 10 females) were treated for 90 d by oral gavage with the vehicle control, the low dose of 250 mg/kg bw/day, the mid dose of 500 mg/kg bw/day, or the high dose of 1000 mg/kg bw/day. An additional 10 animals (five males and five females) in the control and in the high-dose groups were allowed to recover for an additional 28 days.

Observations and examinations performed and frequency:

Observations:

Mortality (daily), clinical signs (weekly),
Ophthalmological examinations: before study initiation and at study termination,
Neurological parameters: qualitative and quantitative assessment of sensory reactivity, grip strength, motor activity, frequency of urination, defecation, rearing, and landing foot splay,
Body weight and food consumption: weekly,
Samples for hematology and clinical chemistry analysis: taken just prior to sacrifice.

Hematological parameters include hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, and blood clotting time.

Clinical chemistry determinations include sodium, potassium, glucose, total cholesterol, urea, blood urea nitrogen, creatinine, total protein and albumin, and two or more enzymes that indicate hepatocellular effects.

Urine samples taken during the last week before sacrifice are examined for appearance, volume, osmolality or specific gravity, pH, protein, glucose, and presence of blood or blood cells.

Sacrifice and pathology:

At study termination, gross necropsy is performed including complete external body examination and organ weights recorded for liver, kidneys, adrenals, testes, epididymides, uterus, ovaries thymus, spleen, brain, and heart. Histopathological examination is performed on tissue samples from: all gross lesions, representative brain regions, spinal cord at three levels, pituitary, thyroid, parathyroid, thymus, esophagus, salivary glands, stomach, small and large intestines (including Peyer’s patches), liver, pancreas, kidneys, adrenals, spleen, heart, trachea, lungs, aorta, gonads, uterus, accessory sex organs, female mammary gland, prostate, urinary bladder, lymph nodes, peripheral nerve, bone marrow section, skin, and eyes if changes were observed upon examination. Tissue samples are fixed in 10% neutral buffered formalin before embedding in paraffin wax. Sections of 5 μm thickness are stained with hematoxylin and eosin for microscopic examination.

Statistics:

The results were analyzed statistically with IBM SPSS Statistical Software (version 23). Following assessment of homogeneity, using Levene’s test, of body weight, food consumption, hematology, clinical chemistry, organ weight, and neurological examination data, different groups were subjected to one-way analysis of variance (ANOVA). Comparisons between treated and control groups were analyzed by t tests with variance evaluated at the 5% level of significance.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs were found in any group other than respiratory rales in one control male (day 84 until termination at day 91) and one treated, mid-dose (500 mg/kg bw/d) male from day 80 until day 91; these observations were incidental, not dose-related, and of no toxicological significance.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average weight of male animals in the high treatment recovery group, was less than the average of males in the control recovery group, a difference that was statistically significant (P < 0.05). Based on the combined data from all dose groups and food consumption data, it is concluded that consumption of the test substance did not have an adverse effect on body weight or body weight gain.

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:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The results are normal with no differences between control and treated groups except for elevated total WBC counts for the high-dose group at the end of the recovery period. Although statistically higher than concurrent controls, the value 10.08+E3/cm2 is well within the historical range for control rats in 90-day studies at the test facility.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few isolated measurements were statistically different from controls, but are considered to be incidental and without biological significance due to lack of any dose dependency as well as their values falling within ranges of the laboratory’s historical controls.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The only statistically significant difference in mean organ weights noted at end of the 90 d treatment was that of the relative, but not absolute, heart weight in females being lesser than the control, which in absence of dose dependence, was considered to be incidental. At the end of the 28 d recovery period, absolute and relative adrenal weights only in male rats were statistically higher in the high dose group as compared to controls, and absolute liver weights were significantly lower in the highest dose group females only than in control rats. These results appear to be incidental due to the lack of any changes in other correlated parameters, such as necropsy findings, histopathology findings, clinical hematology, and clinical chemistry.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross necropsy revealed an absence of any remarkable gross abnormalities in all but two male animals at the end of the 90 d treatment period and two males at the end of the 28 d recovery phase. One mid-dose rat had multiple abscesses in the lungs and another mid dose rat animal had underweight testes and epididymides. These incidental findings were found only in this group and are concluded to be unrelated to dosing with the test substance. Moderate splenic enlargement was found in two high dose males at the end of the recovery period, but subsequent histopathologic examination showed this to be due to splenic congestion, a common condition considered to be incidental in this case.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The evaluation of tissues and organs from control and high-dose groups showed no incidence of any remarkable findings that could be related to treatment due to lack of any dose dependency as well as their values falling within ranges of the laboratory’s historical controls. Single animals in different groups showed isolated (1 in 20 animals) findings.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

Key result

Critical effects observed:

no

For detailed results tables kindly refer to the attached background materials section of the IUCLID.

Conclusions:

Under the study conditions, the 90 d NOEL was considered to be 1000 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the read-across substance, C16 MEA, according to a design based on OECD Guideline 408, in compliance with GLP. Preliminary tests found that the test substance was suitable for oral gavage by suspension in water with Tween 80 (0.5% w/v) as a surfactant and carboxymethyl cellulose (1% w/v) as a suspending agent. Sprague-Dawley rats were divided into six groups. Four groups of 20 (10 males and 10 females) were treated for 90 d by oral gavage with the vehicle control, the low dose of 250 mg/kg bw/day, the mid dose of 500 mg/kg bw/day, or the high dose of 1000 mg/kg bw/day. An additional 10 animals (five males and five females) in the control and in the high-dose groups were allowed to recover for an additional 28 d. The following observations and examinations were performed: mortality (daily), clinical signs (weekly), functional observations and locomotor activity (end of treatment), body weight and food consumption (weekly), clinical pathology (end of treatment), ophthalmological examinations (before study initiation and at study termination), macroscopy at termination, organ weights and histopathology on a selection of tissues. There were some incidental findings that are concluded to be unrelated to treatment and of no toxicological significance. No treatment-related adverse effects were found up to the highest tested dose level of 1000 mg/kg bw/day. Under the study conditions, the 90 d NOEL was considered to be 1000 mg/kg bw/day (Nestmann, 2016).

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

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

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

From December 23, 2020 to March 4, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The formulation analysis at the end of treatment was done in Week 4, even if males were sacrificed on Week 5. The weight of females at arrival ranged between 198-213 grams. These deviations had no impact on the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

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

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, on 26 November 2020, the weight range for each sex was determined (200-218 g for males, 198-213 g for females) 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 34 days 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. Nesting material was 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 and one female showing damaged eye (animal pretest no. 21) 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. No replacements occurred after the first dose was administered.

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 item 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, 30 and 100 mg/mL), according to stability data from ERBC study No. A4105. 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. A4105 in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in ERBC validation protocol (r > 0.99; accuracy 80-120%; precision CV < 10%). In ERBC Study no. A4105, 28-hour stability at room temperature and 8-day stability at 2-8°C were verified in the range from 10 to 100 mg/mL. According to ERBC SOPs, suspensions are considered to be stable if concentration and homogeneity, after the defined period of storage, are still acceptable (80%-120% for concentration and CV < 10% for homogeneity). The proposed preparation procedure for the test substance was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) in ERBC Study no. A4105 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 on Weeks 1 and 4 (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, for a total of 33/35 days. 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, for a total of 50 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/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, concurrent vehicle

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. From Day 1 to Day 3, clinical signs were done as follows: 0.5-1 hour post-dose, 2-2.5 hours post-dose, 4-4.5 hours post dose. From Day 4, observations were done 0.5-1 hour post-dose. From Day 9, due to the presence of post-dose reaction (salivation) the observations were done within 15 minutes from treatment. 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), all animals were 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), all animals were selected from each group and the motor activity 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: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on gestation 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 during the pre-mating period starting from Day 1 of dosing. Individual food consumption for the 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 (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), coagulation (prothrombin time) and clinical chemistry (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) were collected by random selection from 5 males and 5 females (females with viable litters) of each group, under condition of food deprivation.

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 using a computer-generated random order). 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.

Blood collection and thyroid hormone determination (T4 and TSH) (delegated phase): Blood collection for hormone determination was performed from all animals at termination.

Immunoanalysis: Thyroid hormone determination (T4 and TSH) (delegated phase) Immunoanalysis was carried out by the Test Site, BioVetim (Study Phase number: BIOV X1620), according to the following immunoanalytical methods validated at the Test Site: - RV-T4/R/S-BKM/RIA-002 for T4 - RV-TSH/R/S-IZO/RIA-002 for TSH.

Sacrifice and pathology:

- Parental animals that completed the scheduled test period were killed by exsanguinations under isofluorane anaesthesia.
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.

- 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: 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 examination: 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.

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 non-parametric 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was the only treatment-related clinical signs recorded in animals treated at 300 and 1000 mg/kg/day during the study, affecting both genders. Animals of the control group and those treated at 100 mg/kg/day did not show any sign during the whole treatment period. The number of animals affected by salivation as well as the duration of the sign increased with increasing dose, affecting all animals treated at 1000 mg/kg/day.
This clinical sign, however, was considered not adverse since it was recorded only after each administration and not during the afternoon observations, at the end of each day. Furthermore, salivation was never recorded during the weekly detailed clinical signs, thus confirming its transitory nature.
The subcutaneous mass in mammary area noted in one female (no. X1620061) receiving 1000 mg/kg/day was confirmed at macroscopic observations.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Group mean body weight or body weight gain was comparable between control and all tested dose levels, both on males and females.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No changes of toxicological significance were observed in food consumption during the study in either males or females. The slight but statistically significant decrease observed in females dosed at 1000 mg/kg/day at Day 20 post coitum, was considered as sporadic and of no toxicological significance, since it was recorded only on a single occasion.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were recorded. Similarly, no treatment-related changes were recorded for coagulation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes were recorded. Some females dosed at 1000 mg/kg/day showed increases of alkaline phosphatase (78%), alanine aminotransferase (42%) and aspartate aminotransferase (62%) and a decrease of triglycerides (41%). Males of the same group showed an increase of alanine aminotransferase (29%). The above changes were considered to be within the range of expected biological variation and therefore considered to be incidental.

Endocrine findings:

no effects observed

Description (incidence and severity):

No changes of toxicological relevance were observed in parental males.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed. The statistically significant increase of diuresis recorded in males dosed at 1000 mg/kg/day (54%) was considered to be incidental due to the absence of other related changes.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in terminal body weight and organ weights when compared to the controls. Any organ weight variations, including the decrease in absolute and relative mean epididymides and spleen weights in high dose treated males (-11% for relative epididymides weight and -15% for relative spleen weight compared to controls) were not correlated to any histopathological changes, in the range of expected spontaneous changes in rats of the same age and considered unrelated to treatment.

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, including the subcutaneous mass observed during necropsy of a high dose female (no. X1620061, mammary gland adenocarcinoma), 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.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No treatment-related alterations in motor activity, grip strength 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, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopic observations at the end of the treatment period. Any microscopic observations 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.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 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 signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

neuropathology

sperm measures

urinalysis

water consumption and compound intake

other endocrine activity endpoints

Critical effects observed:

no

Conclusions:

Under the study conditions, the NOAEL for general toxicity was considered to be 1000 mg/kg/day for males and females.

Executive summary:

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted in rats with the read across substance C8-18 and C18-unsatd. MEA according to OECD Guideline 422, 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, 300 and 1000 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 to 35 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 50 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. Salivation was the only treatment-related clinical sign recorded in males and females treated at 300 and 1000 mg/kg/day, during the study. However, this clinical sign was considered not treatment-related since it was only recorded soon after each administration and not in the afternoon observations. This was also supported by the absence of salivation during the weekly detailed clinical observations, thus confirming its transitory nature. 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 item. 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 item. Under the study conditions, the e NOAEL for general toxicity was considered to be 1000 mg/kg/day for males and females (De Marzi, 2021).

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

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

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From February 10, 2021 to November 15, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 29 July 2016

Deviations:

yes

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

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 (210-221 g for males, 179-199 g for females) 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. At allocation to the study, the body weight variation of animals did not exceed 20% of the mean weight of each sex. 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.

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 item 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, 30 and 100 mg/mL), according to stability data from ERBC study No. A4124. 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. A4124 in the range from 10 to 100mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 80-120%; precision CV < 10%). In ERBC Study no. A4124, a 28 hour stability at room temperature and a 10 day stability at 2-8°C were verified in the range from 10 to 100mg/mL. According to ERBC SOPs, suspensions are considered to be stable if concentration, after the defined period of storage, is still acceptable (80-120%). 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. A4124 to confirm that the method was suitable. Final results for all levels were within the acceptability limits stated in the validation protocol for concentration (80-120%) and homogeneity (CV < 10%). Samples of the preparations prepared on Week 1 and Last week 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, for a total of 33/34 days. 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 (up to at least 51 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/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

- Mortality:
Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day. Severely debilitated animals were observed carefully. One animal showing difficulty in parturition was sacrificed for humane reasons. A complete necropsy was performed in all cases.

- 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, 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. 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, according to internal 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 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).

-Blood collection for metabolomics analysis:
At the end of treatment period, 1 mL was taken from all surviving males and females, under conditions of food deprivation. Blood samples were collected (from the retro-orbital sinus in the males, from the abdominal vena cava in the females) and immediately transferred with a 1 mL pipette tip into an Eppendorf tube containing 10 µL of 10% Titriplex III solution. The tube was immediately closed and gently mixed 5-6 times by inverting it. Blood was kept cool in ice water (approximately 4°C) during the collection period of up to 24 minutes. To separate plasma, the blood was centrifuged at 4°C, 20000 g for 2 minutes. Blood cells and plasma were separated; 0.5 mL of the supernatant plasma layer were pipetted with a 1 mL pipette tip into the second labelled Eppendorf tube and covered with a N2 atmosphere. Tubes were closed with the lid which was wrapped with Parafilm-M, and frozen at -80°C within at maximum 30 minutes after centrifugation, then stored at -80°C until despatch for additional investigations (metabolomics analysis) at an external laboratory. Results of the metabolomic analysis are reported in Section 13.

Sacrifice and pathology:

-Euthanasia: Parental animals and those that had completed the scheduled test period were killed by exsanguinations under isofluorane anaesthesia. Animals sacrificed for humane reasons were killed with carbon dioxide. The males were killed after the mating of all females, up to a total of 33/34 days. The females with live pups were killed on Day 14 post partum. The female which did not gave birth 25 days after positive identification of mating was killed shortly after.

- 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.
Females: All females were examined also for the following: 1) number of visible implantation sites (pregnant animals) and 2) number of corpora lutea (pregnant animals). Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

- Organ weights:
Parental animals: From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed: abnormalities, adrenal glands, bone marrow, aecum, clitoral gland, colon, duodenum, epididymides, eyes, femur, heart, ileum, jejunum, kidneys, liver, lung, lymph nodes, mammary gland, nasal cavity, oessophagus, ovaries, parathyroid gland, pituitary gland, penis, prostate gland, rectum, sciatic nerve, seminal vesicles, spinal column, spinal cord, skeletal muscle, splee, stomatch, testes, thymus, thyroid gland, trachea, urinary bladder, uterus, vagina. 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 examination:
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 tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out. The examination was restricted to 1) tissues from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term 2) all abnormalities in all groups.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables 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. 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 theWilliams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation noted occasionally in high dose males (2 out of 10) and high dose females during post coitum (10 out of 10) and post partum (8 out of 10) periods was considered to be a treatment-related sign. This treatment-related clinical sign is not considered to be adverse, due to its occasional occurrence and in the absence of effects on the health status of the affected animals. Other findings recorded were considered as incidental or related to in-life procedures and were deemed as minor clinical signs, not related to treatment with the test substance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Animal no. X1660064 (high dose male) was found dead on Day 17 of mating phase. Macroscopically, dark red colour of lungs, white foam in trachea and red staining of muzzle were observed. At microscopic observation, diffuse congestion and haemorrhage of the lungs, mild alveolar macrophages aggregations laden with brown pigment (test item like) were observed. Death was considered to be related to misgavage. Animal no. X1660015 (control female) was sacrificed for humane reasons on Day 22 of the gestation phase. On the basis of the in vivo signs such as prolonged time without compleating the parturition, impaired parturition was considered to be the reason for sacrifice. At macroscopic observation, enlarged liver and dark left lobe of thymuswere noted. No abnormalities were detected at microscopic observation. The gross and microscopic evaluation did not allow to establish the cause of impaired parturition.

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 comparable between treated and control groups through the study. The statistically significant decrease in body weight gain recorded in mid-dose females prior to dosing was considered incidental since it occurred before the start of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by treatment 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. The statistically significant difference of large unstained cells recorded between control and females dosed at 300 mg/kg/day (2 fold increase) was not dose-related and values were within the range of historical control data, therefore it was considered to be incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were recorded. Some statistically significant differences between control and treated animals were recorded, such as: increase of alanine aminotransferase in males dosed at 1000 mg/kg/day (29%) increase of protein and albumin in females receiving 300 and 1000 mg/kg/day (approximately 10%) and increase of bile acids in females dosed at 100 mg/kg/day (95%). Changeswere not dose-related and/or within the range of historical data, therefore they were considered to be unrelated to treatment. In addition, two females dosed at 300 mg/kg/day (nos. X1660043 and X1660049) and one female
receiving 1000 mg/kg/day (X1660061) showed a marked increase of bile acids. Changes were 11- to 15-fold. Due to the low incidence (2/10), the absence of dose-relation and other related changes (i.e. liver parameters), the bile acids increments were considered to be unrelated to treatment.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were recorded. In the absence of other correlated changes, the statistically significant increase of TSH observed in parental males, was considered to be incidental.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes were observed in treated animals in comparison to controls.

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:

effects observed, non-treatment-related

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. In particular, the female no. X1660045 (mid-dose female) found not pregnant at Day 27 post coitum showed at macroscopic observations distended uterus with pale creamy contents; microscopically this correlated with chronic inflammation of endometrium and myometrium in the uterus and was considered incidental.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related alterations in motor activity, grip strength and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment. The statistically significant decrease in motor activity in females of Group 3 may have been due to the low value of one animal out of 5 (female no. X1660043) and, in the absence of a dose-relation, it was not considered to be treatment-related.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopic observations at the end of the treatment period. Any microscopic observations 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 substance-related microscopic observations in the testis (stage aware evaluation on PAS-stained slides).

Histopathological findings: neoplastic:

not examined

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

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

neuropathology

organ weights and organ / body weight ratios

urinalysis

other: serum/plasma biochemistry (migrated information); serum/plasma hormone analyses (migrated information)

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, the NOAEL for general toxicity was considered to be 1000 mg/kg/day for males and females.

Executive summary:

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted in rat with the read across substance C16-18 MEA according to OECD Guideline 422, 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, 300 and 1000 mg/kg/day). All doses were administered by gavage 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 51 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 treatment related mortality occurred throughout the study. Salivation was the only treatment-related clinical sign recorded in males and females treated at 1000 mg/kg/day, during the study. However, this clinical sign did not reveal any treatment related changes. 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. Under the study conditions, the NOAEL for general toxicity was considered to be 1000 mg/kg/day for males and females (Liberati, 2022).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Sufficient data is available to evaluate this endpoint.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral (28 days)

A study was conducted to evaluate the repeated dose oral toxicity of the test substance, C12-18 and C18-unsatd. MEA, according to a design based on OECD 407. Groups of 10 male and 10 female Wistar rats were orally gavaged with the substance diluted in olive oil, 5 d/week for 28 d at doses of 0, 70, 250, 750 (Days 1-14) and 1500 (Days 15-28) mg/kg bw/d. Clinical signs, bodyweight, haematology, clinical chemistry, urinalysis, gross and microscopic pathology were recorded. Additional groups of 5 male and 5 female rats were kept for a 4 month recovery period. No treatment-related adverse effects were observed at any of the doses. Changes in the forestomach at some doses including controls were attributed to the use of olive oil and found to be reversible after end of exposure. Under the study conditions, the 28 d NOAEL to rats was considered to be >750 mg/kg bw/day (Potokar, 1983).

Oral (combined repeated dose toxicity and reproductive-developmental toxicity screening)

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted in rats with the read across substance C8-18 and C18-unsatd. MEA according to OECD Guideline 422, 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, 300 and 1000 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 to 35 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 50 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. Salivation was the only treatment-related clinical sign recorded in males and females treated at 300 and 1000 mg/kg/day, during the study. However, this clinical sign was considered not treatment-related since it was only recorded soon after each administration and not in the afternoon observations. This was also supported by the absence of salivation during the weekly detailed clinical observations, thus confirming its transitory nature. 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 item. 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 item. Under the study conditions, the e NOAEL for general toxicity was considered to be 1000 mg/kg/day for males and females (De Marzi, 2021).

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted in rat with the read across substance C16-18 MEA according to OECD Guideline 422, 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, 300 and 1000 mg/kg/day). All doses were administered by gavage 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 51 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 treatment related mortality occurred throughout the study. Salivation was the only treatment-related clinical sign recorded in males and females treated at 1000 mg/kg/day, during the study. However, this clinical sign did not reveal any treatment related changes. 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. Under the study conditions, the NOAEL for general toxicity was considered to be 1000 mg/kg/day for males and females (Liberati, 2022).

Oral (90 days)

A study was conducted to evaluate the repeated dose oral toxicity of the read-across substance, C16 MEA, according to a design based on OECD Guideline 408, in compliance with GLP. Preliminary tests found that the test substance was suitable for oral gavage by suspension in water with Tween 80 (0.5% w/v) as a surfactant and carboxymethyl cellulose (1% w/v) as a suspending agent. Sprague-Dawley rats were divided into six groups. Four groups of 20 (10 males and 10 females) were treated for 90 d by oral gavage with the vehicle control, the low dose of 250 mg/kg bw/day, the mid dose of 500 mg/kg bw/day, or the high dose of 1000 mg/kg bw/day. An additional 10 animals (five males and five females) in the control and in the high-dose groups were allowed to recover for an additional 28 d. The following observations and examinations were performed: mortality (daily), clinical signs (weekly), functional observations and locomotor activity (end of treatment), body weight and food consumption (weekly), clinical pathology (end of treatment), ophthalmological examinations (before study initiation and at study termination), macroscopy at termination, organ weights and histopathology on a selection of tissues. There were some incidental findings that are concluded to be unrelated to treatment and of no toxicological significance. No treatment-related adverse effects were found up to the highest tested dose level of 1000 mg/kg bw/day. Under the study conditions, the 90 d NOEL was considered to be 1000 mg/kg bw/day (Nestmann, 2016).

Additional considerations 

Based on an in-depth analysis of the available information (see read-across justification in Section 13 of the IUCLID dataset), it is the hypothesis that a read-across within and across MEA, DEA and MIPA derived alkanolamides is scientifically plausible and justified. While there is at present no evidence for putting the read-across hypothesis in question, some limitations, predominantly related to the quality of individual endpoint studies (including quality of the test substance characterisations) and existing higher tier endpoint data gaps, are recognized. Accordingly, additional physico-chemical and toxicology data were generated in Tier 1 of a tiered testing programme to strengthen the toxicokinetic and toxicological link within and across the members of the DEA, MEA, and MIPA subcategories.

In Tier 1, a series of bridging studies according to OECD TG 421 and 422 were conducted with a representative short- and a long-chain substance of each subcategory (i.e., DEA, MEA, and MIPA). Additionally, taking advantage of the bridging studies samples, metabolomics analyses were conducted to enhance the quality and quantity of data from a biological perspective.

Overall, the results of the Tier 1 testing confirmed and supported the hypothesis of a similar toxicological profile within and across the different sub-categories. All investigated substances displayed in line with existing data a similar systemic toxicity profile with no observed repeated dose toxicity at the highest tested dose (i.e., NOAELs ≥ 700 mg/kg/day) and absence of reproductive or developmental toxicity. The absence of significant metabolome changes is in line with the Tier 1 in vivofindings, and thereby further confirming the read-across hypothesis that there is no significant difference in terms of type and strength of effects within and across the FAA subcategories.

In the present dossier update, proposed Tier 2 studies have been included with the aim to generate a complete set of higher toxicology data for a selected >1000 tpa substance (i.e., OECD TG 408/443/414 (rats)/414 (2nd species). The testing in Tier 2 will be conducted with C8-18 and C18-unsatd. DEA, the substance that is generally perceived to be the most critical from a toxicological point of view due to the proposed hazard classification of DEA. Additionally, a few minor non-significant metabolomic changes were noted for the investigated DEA-FAA substance (i.e., C16-18 and C18-unsatd. DEA), suggesting some type of biological activity, possibly explaining some findings in the 1000 mg/kg bw/day dose group in the dose-range finding study. These observations support the selection and recommendation to investigate a DEA-FAA substance as a 'worst case' for the FAA category in Tier 2.

The strategy and status overview are detailed in the document entitled‘ECHA-DIAP - FAA testing strategy summary status overview – Oct 22’, attached in Section 13 of the IUCLID dataset.

Justification for classification or non-classification

Based on the results of oral repeated dose toxicity studies in rodents with the test substance and various read-across substances, the test substance does not require classification for this endpoint according to CLP (EC 1272/2008) criteria.