N,N-bis(2-hydroxyethyl)dodecanamide

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:

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

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

KL2 due to RA

Justification for type of information:

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

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

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 weeks
- 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

Duration of treatment / exposure:

28 d

Frequency of treatment:

Daily once, 5 times/wk

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Days 1-28

Dose / conc.:

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

Remarks:

Days 1-28

Dose / conc.:

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

Remarks:

Days 1-14

Dose / conc.:

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

Remarks:

Days 15-28

No. of animals per sex per dose:

10/sex/dose for main study; 5/sex/dose for 4 month recovery period

Control animals:

yes, concurrent no treatment

Details on study design:

According to standard procedure

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

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)

Based on:

test mat.

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:

no

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 read across substance, C12-18 and C18-unsatd. DEA, according to design based on OECD Guideline 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:

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 March 10, 2021 to February 4, 2022

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 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

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.

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

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

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.

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.

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

Key result

Critical effects observed:

no

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 read across substance C16-18 and C18-unsatd. DEA in accordance with OECD 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, 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).

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From June 1965 to September 1965

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Rats were administered dietary levels of the test substance at 0, 0.1, 0.5, 1 and 2% for 90 d. The animals were observed daily for clinical signs, body weights were recorded weekly and haematological, clinical chemistry and urine examinations were carried out at termination. Gross and histopathological examinations were also performed.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Carworth Farm E

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Age at study initiation: Weanlings
- Housing: Five animals per cage
- Diet: Powdered Spillers Small Laboratory Animal Diet, ad libitum
- Water: Water, ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 d

Frequency of treatment:

Daily

Remarks:

0, 0.1, 0.5, 1 and 2% in diet (nominal)

No. of animals per sex per dose:

15

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 and weekly once thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg bw/day: Yes
- Time schedule for examinations: Day 0 and weekly once thereafter

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study
- Parameters checked in table [No.2]: Total erythrocyte count, hematocrit, hemoglobin, reticulocyte count, total and differential leucocyte counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study
- Parameters examined: Liver and kidney function tests and levels of serum glutamic-oxaloncetic and glutamic-pyruvic transaminases and of blood urea checked

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the study
- Parameters checked in table [No.3] were examined: Urine was examined for colour, pH, microscopic constituents, protein, reducing substances, bile salts and blood and activity of glutamic-oxaloacetic transaminase. Volume and the specific gravity of the urine excreted were measured during a 6 h period of water deprivation and during 4 h period commencing 16 h after a water load of 25 mL/kg.

OTHER: At autopsy, the absolute and relative organ weight of the brain, heart, liver, kidneys, adrenals and gonads were recorded.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (At autopsy, the gross appearance of the brain, heart, liver, kidneys, adrenals and gonads were recorded)
HISTOPATHOLOGY: Yes (Paraffin wax sections of brain, heart, liver, kidneys, adrenals and gonads together with a wide range of other organs were stained with haematoxylin and eosin for histological examination, smears of femoral marrow were stained by the May Grunwald-Giemsa method)

Other examinations:

Palatability test: Pairs of male rats were allowed access to stock diet and to diet containing either one of the four dietary test levels of test material. The consumption of both diets was recorded for a period of 8 d.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In general rats remained in good health apart from 2 males on 1% test material which were killed on Days 23 and 58 because of weight loss and respiratory distress.

Mortality:

mortality observed, treatment-related

Description (incidence):

In general rats remained in good health apart from 2 males on 1% test material which were killed on Days 23 and 58 because of weight loss and respiratory distress.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight gain was observed from 0.5% onwards.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intake was reduced at all dietary levels except the lowest level of 0.1%.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Reductions in hemoglobin levels, hematocrit values and red cell counts in females at 1 and 2% levels but these effects were much less pronounced in males.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Serum levels of glutamic-oxaloacetic transaminases were significantly elevated at 0.5% and above in females, but only at 0.5% level in males.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Proteinurea was comparable in test and control groups. Tests for blood, bile salts and reducing substances were negative in all groups. Level of Glutamic-oxaloacetic transaminase were significantly elevated at dietary levels of 0.5% and above in females but only at the 0.5% level in males. No significant effect was seen in Glutamic-pyruvic transaminase level at all dietary levels.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The principal organ weight changes were increases in the relative kidney weight in all test groups except at 0.1% in females and at 0.1 and 0.5% in males and increases in relative liver weight in female on the two highest levels.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No significant findings were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Examination of the femoral marrow smears showed no deviation from normality. There were no adverse histopathological findings in any organs.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Description (incidence and severity):

OTHER FINDINGS: In the palatability test, exclusive preference was shown to the control diet, virtually no test diet being consumed at any of the dietary levels incorporated. This observation suggests that toxic anorexia was not the cause of reduced food intake.

Key result

Dose descriptor:

NOEL

Effect level:

0.1 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: equivalent to 50 mg/kg bw/day.

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 in rats was considered to be 0.1% in the diet, equivalent to 50 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the repeated dose oral toxicity of the test substance, C12 DEA. The rats were administered test substance concentrations of 0, 0.1, 0.5, 1 and 2% in diet for 90 d. The animals were observed daily for clinical signs. Body weight was recorded weekly and haematological, clinical chemistry and urine examinations were carried out at termination. Gross and histopathological examinations were also performed at termination. No adverse effect on the appearance or condition of the animals was observed. Growth retardation was associated with diminished food intake from the dose level of 0.5%. Food refusal was demonstrably due to an effect of the test material on palatability of the diet. Terminal haematological examination revealed a reduction in the haemoglobin level, haematocrit and red cell count at the 1 and 2% dose levels in females but less pronounced effects were seen in males. Serum levels of glutamic-oxaloacetic transaminase were elevated at 0.5% and above in females but only at 0.5 % in males. No untoward effect was observed in the renal function tests. The principal organ weight changes were: increases in the relative kidney weight in all test groups except at 0.1% in females and at 0.1 and 0.5% in males, and increases in the relative liver weight in females on the two highest levels. The types and incidence of histological lesions were comparable in control and test groups. Under the study conditions, the 90 d NOEL was considered to be 0.1% in diet, equivalent to 50 mg/kg bw/day (Gaunt, 1965).

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study planned

Study period:

As of 2025 at the earliest. Depending on the results of the ongoing OECD 421 study and testing proposal acceptance.

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Amides, C8-18 (even numbered) and C18-unsatd., N,N-bis(hydroxyethyl (C8-18 and C18-unsatd. DEA), EC No. 931-329-6

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies
: none
- Available non-GLP studies
: none
- Historical human data
: none
- (Q)SAR
: not applicable
- In vitro methods
: not applicable
- Weight of evidence
: not sufficient
- Grouping and read-across
: not sufficient
- Substance-tailored exposure driven testing [if applicable]
: none
- Approaches in addition to above [if applicable] : not applicable
- Other reasons [if applicable]
: none

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Proposed adaptations were not considered sufficient to address this endpoint.
This was discussed with ECHA in the frame of a Dossier Improvement Action Plan (DIAP).

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:

- Proposed study design: according to OECD Guideline 408

Planned study period: As of 2025 at the earliest. Depending on the results of the ongoing OECD 421 study and testing proposal acceptance.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

rat

Route of administration:

oral: gavage

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

700 mg/kg bw/day

Study duration:

subacute

Species:

rat

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

other justification

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

Reference 1

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 29, 1991 to April 16, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Deviations:

not specified

Principles of method if other than guideline:

A 14-week toxicity study in mice was conducted to evaluate the cumulative toxic effects of repeated dermal exposure to the test substance.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: Approximately 7 wk
- Housing: Housed individually in Polycarbonate cages
- Bedding: Sani-Chip® heat-treated hardwood chips (PJ Murphy Forest Products Corp., Montville, NJ), changed weekly
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (and changed weekly)
- Water: Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum (cleaned every 2 wks)
- Acclimatization period: 14 d
- Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly and rotated every 2 weeks
- Animal number per cage: 1 Bedding: Sani-Chip® heat-treated hardwood chips (PJ Murphy Forest Products Corp., Montville, NJ), changed weekly
- Cage Filters: Spun-bonded polyester Du Pont 2024 (Snow Filtration, Co., Cincinnati, OH), changed every 2 weeks
- Racks: Stainless steel drawer-type (Lab Products, Inc., Maywood, NJ), changed and rotated every 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature : 21.1-23.9°C
- Relative humidity : 42-57%
- Air changes : 10/hr
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: 1992-01-13 To: 1992-04-14

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared every 3 weeks by liquefying and stirring the test substance at approximately 70°C. A weighed amount of the test substance was mixed with approximately half the required 95% ethanol, and the mixture was sonicated until it appeared to be in solution. The solution was allowed to cool and was then diluted to volume with 95% ethanol to give the required concentrations.The test substance formulations were applied on shaved skin of the test animals.
-Stability studies of the 10 mg/mL dose formulation were performed by the study laboratory using HPLC. When stored in sealed glass containers and protected from light, the dose formulations were stable for at least 28 days between -20°C and room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of the test substance from the beginning, middle, and end of the studies were analyzed at the study laboratory using HPLC. All the samples from the formulations were within 10% of the target concentration.
-Stability studies of the 10 mg/mL dose formulation were performed by the study laboratory using HPLC. When stored in sealed glass containers and protected from light, the dose formulations were stable for at least 28 days between -20°C and room temperature.

Duration of treatment / exposure:

14 wks

Frequency of treatment:

5 exposures/wk

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Method of animal grouping: Distributed randomly into groups of approximately equal initial mean body weights.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly, and at the end of the studies

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

OTHER: Organs weighed were heart, right kidney, liver, lung, right testis, and thymus

Sacrifice and pathology:

SACRIFICE: At the end of the 14 wk study, blood was collected from the retroorbital sinus of all core study mice for hematology and clinical chemistry analyses. Thereafter the test animals were anesthetised with a carbon dioxide/oxygen mixture.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes. Complete histopathology was performed on 0 and 400 mg/kg bw core study mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition, the skin was examined in all mice.

Other examinations:

Sperm motility and vaginal cytology: At the end of the studies, samples were collected for sperm motility or vaginal cytology from all mice in the 0, 100, 200, and 400 mg/kg bw groups for evaluations.
- The following sperm motility parameters was evaluated: spermatid heads per gram of testis, spermatid heads per testis, spermatid count, motility, and concentration in cauda epididymis. The left cauda, epididymis, and testis were weighed.
- Vaginal samples were collected for 12 consecutive days prior to the end of the studies for vaginal cytology evaluations. The length of the estrous cycle and the length of time spent in each stage of the cycle were evaluated.

Statistics:

- Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends.
- Analysis of neoplasm and non-neoplastic lesion incidences: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pair wise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided. Values of P greater than 0.5 are presented as 1-P with the letter N added to indicate a lower incidence or negative trend in neoplasm occurrence relative to the control group (e.g., P=0.99 is presented as P=0.01N).
- Analysis of Continuous Variables: Organ and body weight data were analyzed using the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across dose levels.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Irritation of the skin at the site of application was noted in all males and females administered 400 or 800 mg/kg bw/day.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased absolute and/or relative kidney weights of in males at ≥100 mg/kg bw/day and in females at 800 mg/kg bw/day; increased liver weights in females at ≥200 mg/kg bw/day and reduced thymus weights in males at 400 and 800 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Including epidermal and sebaceous gland hyperplasia, chronic inflammation, parakeratosis and ulcer in males and females at ≥200 mg/kg bw/day.

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were no significant differences in reproductive tissue evaluations or in estrous cycle characterization between dosed and vehicle control groups.

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Remarks:

(local effects)

Effect level:

ca. 100 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

no

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

Conclusions:

Under the study conditions, the 14-weeks NOAELs for systemic and local effects in mice were 50 and 100 mg/kg bw/day, respectively.

Executive summary:

A study was conducted to determine the repeated dose dermal toxicity of the test substance, C12 DEA, according to design based on OECD Guidance 411, in compliance with GLP. Groups of 10 male and 10 female mice were dermally exposed to 0, 50, 100, 200, 400 or 800 mg/kg bw/day in ethanol for 5 d/week during a period of 14 weeks. Mortality, clinical findings, body weight and histopathology were evaluated at specific time intervals. All animals survived until the end of the study and final mean body weights and body weight gains of dosed mice were generally similar to those of the vehicle control groups. Skin irritation at the site of application was noted in all males and females administered 400 or 800 mg/kg bw/day. The kidney weights of males receiving 100, 400, or 800 mg/kg bw/day and females receiving 800 mg/kg bw/day were significantly greater than those of the vehicle controls. Liver weights of females at 200 mg/kg bw/day or greater were significantly higher than those of vehicle controls. Increased incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, chronic inflammation parakeratosis and ulcer, were observed in animals receiving 200 mg/kg bw/day or greater. Under the study conditions, the 14-week NOAELs for systemic and local effects in mice were 50 and 100 mg/kg bw/day, respectively (NTP, 1999).

Reference 2

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 31, 1991 to April 16, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Deviations:

not specified

Principles of method if other than guideline:

A 14-week toxicity study in rats was conducted to evaluate the cumulative toxic effects of repeated dermal exposure to the test substance.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: Approximately 7 wk
- Housing: Housed individually in Polycarbonate cages
- Bedding: Sani-Chip® heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (and changed weekly)
- Water: Tap water (Columbus municipal supply) via automatic watering system (Edstrom Industries, Inc., Waterford, WI), available ad libitum (cleaned every 2 wks)
- Acclimatization period: 14 d
- Cages: Polycarbonate (Lab Products, Inc., Maywood, NJ), changed weekly and rotated every 2 weeks
- Animal number per cage: 1
- Bedding: Sani-Chip® heat-treated hardwood chips (P.J. Murphy Forest Products Corp., Montville, NJ), changed weekly
- Cage Filters: Spun-bonded polyester Du Pont 2024 (Snow Filtration, Co., Cincinnati, OH), changed every 2 weeks
- Racks: Stainless steel drawer-type (Lab Products, Inc., Maywood, NJ), changed and rotated every 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21.1-23.9°C
- Relative humidity: 42-57%
- Air changes: 10/hr
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From: 1992-01-13 To: 1992-04-14

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared every 3 wks by liquefying and stirring the test substance at approximately 70°C. A weighed amount of the test substance was mixed with approximately half the required 95% ethanol, and the mixture was sonicated until itappeared to be in solution. The solution was allowed to cool and was then diluted to volume with 95% ethanol to give the required concentrations.The test substance formulations were applied on shaved skin of the test animals.
- Stability and homogeneity in the vehicle: Stable up to 28 d when stored between -20˚C and room temperature; Stable up to 3 h when exposed to air and light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Periodic analyses of the dose formulations of the test substance from the beginning, middle, and end of the studies were analyzed at the study laboratory using HPLC. All the samples from the formulations were within 10% of the target concentration.

Duration of treatment / exposure:

14 wks

Frequency of treatment:

5 exposures/wk

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Method of animal grouping: Distributed randomly into groups of approximately equal initial mean body weights.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially, weekly, and at the end of the studies

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On days 4 and 24 (blood was collected from the retro-orbital sinus of clinical pathology study male and female rats from each dose group) of dosing
- Anaesthetic used for blood collection: Yes, carbon dioxide/oxygen mixture
- How many animals: All animals
- Parameters examined: Hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; leukocyte count and differential

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day 4 and 24 (blood was collected from the retroorbital sinus of clinical pathology study male and female rats from each dose group) of dosing
- How many animals: All animals
- Parameters examined: Blood urea nitrogen; creatinine; total protein; albumin; alanine aminotransferase; alkaline phosphatase; sorbitol dehydrogenase; and total bile acids

OTHER: Organs weighed were heart, right kidney, liver, lung, right testis, and thymus

Sacrifice and pathology:

SACRIFICE: At the end of the 14 wk study, blood was collected from the retroorbital sinus of all core study rats for hematology and clinical chemistry analyses. Thereafter the test animals were anesthetised with a carbon dioxide/oxygen mixture.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes. Complete histopathology was performed on 0 and 400 mg/kg core study rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus. In addition, the skin was examined in all rats.

Other examinations:

Sperm motility and vaginal cytology: At the end of the studies, samples were collected for sperm motility or vaginal cytology from all rats in the 0, 100, 200, and 400 mg/kg bw/day groups for evaluations.
- The following sperm motility parameters was evaluated: spermatid heads per gram of testis, spermatid heads per testis, spermatid count, motility, and concentration in cauda epididymis. The left cauda, epididymis, and testis were weighed.
- Vaginal samples were collected for 12 consecutive days prior to the end of the studies for vaginal cytology evaluations. The length of the estrous cycle and the length of time spent in each stage of the cycle were evaluated.

Statistics:

- Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends.
- Analysis of neoplasm and non-neoplastic lesion incidences: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and non-neoplastic lesion prevalence. Tests of significance included pair wise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided. Values of P greater than 0.5 are presented as 1-P with the letter N added to indicate a lower incidence or negative trend in neoplasm occurrence relative to the control group (e.g., P=0.99 is presented as P=0.01N).
- Analysis of Continuous Variables: Organ and body weight data were analyzed using the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
- Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data were analyzed using the nonparametric multiple comparison methods of Shirley (1977) and Dunn (1964). Prior to statistical analysis, extreme values identified by the outlier test of Dixon and Massey. Average severity values were analyzed for significance with the Mann-Whitney U test (Hollander and Wolfe, 1973). Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976) to the transformed data to test for simultaneous equality of measurements across dose levels.

Clinical signs:

no effects observed

Description (incidence and severity):

The primary clinical finding was irritation of the skin at the site of application in males administered 100 mg/kg bw or greater and in females administered 200 or 400 mg/kg bw.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Irritation of the skin at the site of application in males administered 100 mg/kg bw or greater and in females administered 200 or 400 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

All animals survived to the end of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Final mean body weights and body weight gains of males administered 200 or 400 mg/kg bw were significantly less than those of the vehicle control group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Segmented neutrophil counts were significantly increased on days 4 and 24 and at wk 14 in males and only at wk 14 in females administered 400 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alkaline phosphatase activity was minimally increased in 400 mg/kg bw/day males.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The kidney weights of females administered 200 or 400 mg/kg bw were significantly greater than those of the vehicle control group. In males administered 400 mg/kg bw, the absolute liver weight was significantly less than that of the vehicle control group; this difference may be related to significantly lower mean body weights in this group of males.

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Dose dependent increase in the incidences of non-neoplastic lesions of the skin at the site of application (including hyperplasia of the epidermis and sebaceous gland, chronic inflammation, parakeratosis and ulceration).

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

There were no significant differences in reproductive tissue evaluations or in estrous cycle characterization between dosed and vehicle control groups.

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

ca. 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

ca. 25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

dermal irritation

histopathology: non-neoplastic

Critical effects observed:

no

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

Conclusions:

Under the study conditions, the 14-weeks NOAELs for systemic and local effects in rats were 100 and 25 mg/kg bw/day, respectively.

Executive summary:

A study was conducted to determine the repeated dose dermal toxicity in Fischer 344 rats of the test substance, C12 DEA, according to design based on OECD Guidance 411, in compliance with GLP. Groups of 10 male and 10 female rats were dermally exposed to 0, 25, 50, 100, 200 or 400 mg /kg bw/day in ethanol for 5 d/week during a period of 14 weeks. Survival, clinical findings, body weight, haematology, clinical chemistry and histopathology were evaluated at specific time intervals. All animals survived until study end. Final mean bodyweights and body weight gains of males receiving 200 or 400 mg/kg bw/day were significantly lower than those of the vehicle control group. Irritation of the skin at the site of application was observed in males receiving 100 mg/kg bw/day or greater and in females receiving 200 or 400 mg/kg bw/day. Kidney weights of females administered 200 or 400 mg/kg bw/day were significantly greater than those of the vehicle control group. There was a dose-dependent increase in the incidence of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, chronic inflammation, parakeratosis and ulcer. The effects at 25 and 50 mg/kg bw/day were similar and included only minimal hyperplasia. Under the study conditions, the 14-weeks NOAELs for systemic and local effects in rats were 100 and 25 mg/kg bw/day, respectively (NTP, 1999).

Reference 3

Endpoint:

chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

A two-year dermal study was conducted to evaluate the chronic toxicity of the test substance in mice at dose levels of 0, 100, or 200 mg/kg/d bw. 5 exposures per week were given for 105-106 weeks. The animals were observed twice daily, body weights and clinical findings were recorded periodically. Necropsy was performed on all animals and complete histopathology was performed.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate , changed weeklyand rotated every 2 wk
- Diet : NIH-07 open formula pelleted diet, ad libitum
- Water : Tap water via automatic watering system available cleaned every 2 wk, ad libitum
- Acclimation period: 13 d (males); 14 d (females)

ENVIRONMENTAL CONDITIONS
- Temperature : 20.5-26.1°C
- Humidity : 33-64%
- Air changes : 10 / hr
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: Dec. 30, 1992 To: Jan. 06, 1995

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

VEHICLE
Purity: ranged from 97 to 103% relative to the reference standard

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose preparations were analysed approximately every 2 m using HPLC. All dose formulations analysed during the 2-yr studies were within 10% of the target concentration. In addition to dose formulation analysis prior to dosing, samples collected after dosing (animal room samples) were analysed periodically.

Duration of treatment / exposure:

104-105 weeks

Frequency of treatment:

Five exposures per week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Dose selection was based primarily on the increased incidences of a spectrum of skin lesions at the site of application. Doses of 400 or 800 mg/kg bw were associated with high incidences of chronic inflammation and ulceration and were thus considered to be inappropriate for a 2-yr study. A marked reduction in toxic response occurred at 200 mg/kg bw, and 100 mg/kg bw was a no-effect level in male mice. Therefore, 200 mg/kg bw was selected as the high dose for the 2-yr study and 100 mg/kg bw as the low dose.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At study initiation, at 4-wk intervals during the study, and at necropsy

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially at study initiation, weekly during weeks 1-13, at 4-wk intervals thereafter, and at the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE: Carbon dioxide asphyxiation

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, Complete histopathology was performed on all animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

Survival analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Possible dose-related effects on survival were analysed by Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses were two sided.

Analysis of Neoplasm and Nonneoplastic Lesion Incidences: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided.

Clinical signs:

no effects observed

Description (incidence and severity):

No significant difference clinical findings was observed between the dosed groups and the vehicle control groups.

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Description (incidence):

No significant difference in survival was observed between the dosed groups and the vehicle control groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant difference was observed in the mean body weights between the dosed groups at 100 mg/kg bw and the vehicle control groups. The mean body weight of females that received 200 mg/kg bw was 94% that of the vehicle controls by week 33 and remained lower throughout the remainder of the study.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Skin, site of application: Incidences of nonneoplastic lesions of the skin at the site of application were significantly increased in dosed males and females. The lesions were of minimal to moderate severity and consisted mostly of thickening of the epidermis (epidermal hyperplasia) and sebaceous gland hyperplasia. Compared to the vehicle controls, the incidences of chronic inflammation and hyperkeratosis were significantly greater in all groups of dosed males and females, and the incidences of parakeratosis were significantly greater in males and females administered 200 mg/kg bw. Ulcers occurred in a few mice at 200 mg/kg bw and were indicative of more severe local irritation.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver: The incidences of hepatocellular adenoma or carcinoma (combined) were significantly increased in dosed female groups compared to that in the vehicle control group, as was the incidence of hepatocellular adenoma in 100 mg/kg bw females. These incidences exceeded the historical control ranges for these neoplasms. There were also increases in the incidences of eosinophilic foci in dosed female mice, and the increase was significant in females at 200 mg/kg bw. The incidences of hepatocellular adenoma, carcinoma, and adenoma or carcinoma (combined) were not significantly increased in dosed male mice. These increases were associated with free diethanolamine, which was present as a contaminant of lauric acid diethanolamine condensate.

Thyroid Gland: Incidences of focal hyperplasia of thyroid gland follicular cells were increased in dosed male mice; the incidence in the 200 mg/kg bw group was significantly greater than that in the vehicle control group. There were no corresponding increases in the incidences of follicular cell neoplasms.

Key result

Dose descriptor:

LOAEL

Remarks:

(systemic effects)

Effect level:

ca. 100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

ca. 100 mg/kg bw/day

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

no

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

Conclusions:

Under the study conditions, the 2-yr LOAELs for local and systemic effects in mice were both considered to be 100 mg/kg bw/day.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal toxicity of the test substance, C12 DEA, in compliance with GLP. Groups of 50 male and 50 female mice were exposed to dermal doses equivalent to 0, 100 or 200 mg/kg/day, 5 d/week, for 105-106 weeks. The animals were observed twice daily. Body weights and clinical findings were recorded periodically. Necropsy and complete histopathology was performed on all animals at test end. Mean body weights of females that received 200 mg/kg bw/day were lower than those of the vehicle controls beginning at week 33. The incidence of hepatocellular adenoma or carcinoma (combined) was significantly increased in dosed females compared to the vehicle controls, as was the incidence of hepatocellular adenoma in the 100 mg/kg bw/day female group. These tumour findings were considered rat-specific and not relevant for human risk assessment (for more details see section on Carcinogenicity). There were dose-related increases in the incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, hyperkeratosis, chronic inflammation and parakeratosis. Dosed males had greater incidences of thyroid gland follicular cell focal hyperplasia than did the vehicle controls; the incidence in the 200 mg/kg bw/day group was significantly greater than that in the vehicle control group. Under the study conditions, the 2-yr LOAELs for local and systemic effects in mice were both 100 mg/kg bw/day (NTP, 1999).

Reference 4

Endpoint:

chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not available

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

A two-year dermal study was conducted to evaluate the chronic toxicity of the test substance in rats at dose levels of 0, 100, or 200 mg/kg/d bw. 5 exposures per week were given for 105-106 weeks. The animals were observed twice daily, body weights and clinical findings were recorded periodically. Necropsy was performed on all animals and complete histopathology was performed.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, NY
- Age at study initiation: 6 wk
- Housing: Housed individually in Polycarbonate , changed weekly and rotated every 2 wk
- Diet : NIH-07 open formula pelleted diet, ad libitum
- Water : Tap water via automatic watering system, ad libitum
- Acclimation period: 11 d (males); 12 d (females)

ENVIRONMENTAL CONDITIONS
- Temperature : 21.1-26.7°C
- Humidity : 36-59%
- Air changes : 10/h
- Photoperiod : 12 h dark/12 h light

IN-LIFE DATES: From: Dec. 14, 1992 To: Dec. 14, 1994

Type of coverage:

open

Vehicle:

ethanol

Details on exposure:

VEHICLE
- Purity: ranged from 97% to 103% relative to the reference standard

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose preparations were analysed approximately every 2 m using HPLC. All dose formulations analysed during the 2-yr studies were within 10% of the target concentration. In addition to dose formulation analysis prior to dosing, samples collected after dosing (animal room samples) were analysed periodically.

Duration of treatment / exposure:

104-105 wk

Frequency of treatment:

Five exposures per week

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Dose selection was based primarily on the increased incidences of a spectrum of skin lesions at the site of application. Doses of 200 and 400 mg/kg bw caused high incidences of chronic inflammation and ulceration in males and females. Final mean body weights and body weight gains of male rats treated with 200 or 400 mg/kg bw were also less than those of the vehicle controls. Therefore, 200 and 400 mg/kg bw were considered inappropriate for the study. There was a very obvious reduction in toxic response of the skin at 100 mg/kg bw, with chronic inflammation and ulceration being absent in females and present in only one male; thus, 100 mg/kg bw was selected as the high dose for the 2-yr rat study. The responses observed at 25 and 50 mg/kg bw were very similar and consisted of only minimal hyperplasia; therefore, 50 mg/kg bw was selected as the low dose for the study.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At study initiation, at 4-wk intervals during the study, and at necropsy

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed initially at the study initiation, weekly during weeks 1-13, at 4-wk intervals thereafter, and at the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE: Carbon dioxide asphyxiation

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes, Complete histopathology was performed on all animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone and marrow, brain, clitoral gland, esophagus, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (and epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.

Statistics:

Survival analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Possible dose-related effects on survival were analysed by Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses were two sided.

Analysis of Neoplasm and Nonneoplastic Lesion Incidences: The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. Tests of significance included pairwise comparisons of each dosed group with controls and a test for an overall dose-related trend. Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided.

Clinical signs:

no effects observed

Description (incidence and severity):

No significant difference in clinical findings was observed between the dosed groups and the vehicle control groups.

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Description (incidence):

No significant difference in survival was observed between the dosed groups and the vehicle control groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant difference was observed in the mean body weights between the dosed groups and the vehicle control groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes of minimal to moderate severity occurred in the skin at the site of application in treated male and female rats. The major alterations from normal skin were thickening of the epidermis (epidermal hyperplasia) and sebaceous gland hyperplasia (which usually occurred along with epidermal hyperplasia). Incidences of chronic inflammation, hyperkeratosis, and parakeratosis in all dosed groups were significantly greater than those in the vehicle controls, as were the incidences of ulceration in 100 mg/kg bw males and females. The nonneoplastic skin lesions at the site of application were considered to be indicative of local irritation with no neoplastic or preneoplastic changes.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No significant differences was reported between the vehicle control groups and dosed groups in the incidences of neoplasms.

Other effects:

not examined

Details on results:

OTHER FINDINGS: The incidence of forestomach ulcer was significantly lower in males that received 100 mg/kg bw/d than in the vehicle controls. The incidences of inflammation of the nasal mucosa were significantly lower in dosed males than in the vehicle controls. The incidence of chronic inflammation of the liver was significantly lower in females administered 100 mg/kg bw than in the vehicle controls.

Key result

Dose descriptor:

NOAEL

Remarks:

(systemic effects)

Effect level:

ca. 100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed

Key result

Dose descriptor:

LOAEL

Remarks:

(local effects)

Effect level:

ca. 50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

no

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

Conclusions:

Under the study conditions, the 2-yr NOAEL for systemic effects in rats was considered to be the highest tested dose of 100 mg/kg bw/day and the 2-yr LOAEL for local effects was 50 mg/kg/bw/day.

Executive summary:

A study was conducted to evaluate the long-term repeated dose dermal toxicity in Fischer 344 rats of the test substance, C12 DEA, in compliance with GLP. Groups of 50 male and 50 female rats were exposed to dermal doses equivalent to 0, 50 or 100 mg/kg/day, 5 d/week, for 104-105 weeks. The animals were observed twice daily. Body weights and clinical findings were recorded periodically. Necropsy and complete histopathology was performed on all animals at test end. There were no significant differences between vehicle control and dosed males or females in survival or mean body weights. There were no treatment-related differences in incidences of neoplasm. Dose-related increases occurred in the incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, hyperkeratosis, chronic inflammation, parakeratosis, and ulcer. Under the study conditions, the 2-yr NOAEL for systemic effects was considered to be the highest tested dose of 100 mg/kg bw/day and the 2-yr LOAEL for local effects in rats was 50 mg/kg/bw/day (NTP, 1999).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

chronic

Species:

rat

Additional information

Oral (28 day) 

A study was conducted to evaluate the repeated dose oral toxicity of the read across substance, C12-18 and C18-unsatd. DEA, according to a design based on OECD Guideline 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 and reproductive/developmental screen)

A combined repeated dose toxicity study with the reproduction / developmental toxicity screening was conducted with the read across substance C16-18 and C18-unsatd. DEA in accordance with OECD 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, 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).

Oral (90 day)

A study was conducted to evaluate the repeated dose oral toxicity of the test substance, C12 DEA. The rats were administered read across substance concentrations of 0, 0.1, 0.5, 1 and 2% in diet for 90 d. The animals were observed daily for clinical signs. Body weight were recorded weekly and haematological, clinical chemistry and urine examinations were carried out at termination. Gross and histopathological examinations were also performed at termination. No adverse effect on the appearance or condition of the animals was observed. Growth retardation was associated with diminished food intake from the dose level of 0.5%. Food refusal was demonstrably due to an effect of the test material on palatability of the diet. Terminal haematological examination revealed a reduction in the haemoglobin level, haematocrit and red cell count at the 1 and 2% dose levels in females but less pronounced effects were seen in males. Serum levels of glutamic-oxaloacetic transaminase were elevated at 0.5% and above in females but only at 0.5% in males. No untoward effect was observed in the renal function tests. The principal organ weight changes were: increases in the relative kidney weight in all test groups except at 0.1% in females and at 0.1 and 0.5% in males, and increases in the relative liver weight in females on the two highest levels. The types and incidence of histological lesions were comparable in control and test groups. Under the study conditions, the 90 d NOEL in rats was considered to be 0.1% in the diet, equivalent to 50 mg/kg bw/day (Gaunt, 1965).

Furthermore, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), a testing proposal is submitted for the conduct of repeated dose toxicity study according to OECD Guideline 408 with the read across substance, C8-18 and C18-unsatd. DEA.

 Dermal (14 week)

A study was conducted to determine the repeated dose dermal toxicity of the test substance, C12 DEA, according to design based on OECD Guidance 411, in compliance with GLP. Groups of 10 male and 10 female mice were dermally exposed to 0, 50, 100, 200, 400 or 800 mg/kg bw/day in ethanol for 5 d/week during a period of 14 weeks. Mortality, clinical findings, body weight and histopathology were evaluated at specific time intervals. All animals survived until the end of the study and final mean body weights and body weight gains of dosed mice were generally similar to those of the vehicle control groups. Skin irritation at the site of application was noted in all males and females administered 400 or 800 mg/kg bw/day. The kidney weights of males receiving 100, 400, or 800 mg/kg bw/day and females receiving 800 mg/kg bw/day were significantly greater than those of the vehicle controls. Liver weights of females at 200 mg/kg bw/day or greater were significantly higher than those of vehicle controls. Increased incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, chronic inflammation parakeratosis and ulcer, were observed in animals receiving 200 mg/kg bw/day or greater. Under the study conditions, the 14-week NOAELs for systemic and local effects in mice were 50 and 100 mg/kg bw/day, respectively (NTP, 1999).

A study was conducted to determine the repeated dose dermal toxicity in Fischer 344 rats of the test substance, C12 DEA, according to design based on OECD Guidance 411, in compliance with GLP. Groups of 10 male and 10 female rats were dermally exposed to 0, 25, 50, 100, 200 or 400 mg /kg bw/day in ethanol for 5 d/week during a period of 14 weeks. Survival, clinical findings, body weight, haematology, clinical chemistry and histopathology were evaluated at specific time intervals. All animals survived until study end. Final mean bodyweights and body weight gains of males receiving 200 or 400 mg/kg bw/day were significantly lower than those of the vehicle control group. Irritation of the skin at the site of application was observed in males receiving 100 mg/kg bw/day or greater and in females receiving 200 or 400 mg/kg bw/day. Kidney weights of females administered 200 or 400 mg/kg bw/day were significantly greater than those of the vehicle control group. There was a dose-dependent increase in the incidence of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, chronic inflammation, parakeratosis and ulcer. The effects at 25 and 50 mg/kg bw/day were similar and included only minimal hyperplasia. Under the study conditions, the 14-weeks NOAELs for systemic and local effects in rats were 100 and 25 mg/kg bw/day, respectively (NTP, 1999).

Dermal (2 year)

A study was conducted to evaluate the long-term repeated dose dermal toxicity of the test substance, C12 DEA, in compliance with GLP. Groups of 50 male and 50 female mice were exposed to dermal doses equivalent to 0, 100 or 200 mg/kg/day, 5 d/week, for 105-106 weeks. The animals were observed twice daily. Body weights and clinical findings were recorded periodically. Necropsy and complete histopathology was performed on all animals at test end. Mean body weights of females that received 200 mg/kg bw/day were lower than those of the vehicle controls beginning at week 33. The incidence of hepatocellular adenoma or carcinoma (combined) was significantly increased in dosed females compared to the vehicle controls, as was the incidence of hepatocellular adenoma in the 100 mg/kg bw/day female group. These tumour findings were considered rat-specific and not relevant for human risk assessment (for more details see section on Carcinogenicity). There were dose-related increases in the incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, hyperkeratosis, chronic inflammation and parakeratosis. Dosed males had greater incidences of thyroid gland follicular cell focal hyperplasia than did the vehicle controls; the incidence in the 200 mg/kg bw/day group was significantly greater than that in the vehicle control group. Under the study conditions, the 2-yr LOAELs for local and systemic effects in mice were both 100 mg/kg bw/day (NTP, 1999).

A study was conducted to evaluate the long-term repeated dose dermal toxicity in Fischer 344 rats of the test substance, C12 DEA, in compliance with GLP. Groups of 50 male and 50 female rats were exposed to dermal doses equivalent to 0, 50 or 100 mg/kg/day, 5 d/week, for 104-105 weeks. The animals were observed twice daily. Body weights and clinical findings were recorded periodically. Necropsy and complete histopathology was performed on all animals at test end. There were no significant differences between vehicle control and dosed males or females in survival or mean body weights. There were no treatment-related differences in incidences of neoplasm. Dose-related increases occurred in the incidences of non-neoplastic lesions of the skin at the site of application, including epidermal and sebaceous gland hyperplasia, hyperkeratosis, chronic inflammation, parakeratosis, and ulcer. Under the study conditions, the 2-yr NOAEL for systemic effects was considered to be the highest tested dose of 100 mg/kg bw/day and the 2-yr LOAEL for local effects in rats was 50 mg/kg/bw/day (NTP, 1999). 

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 and dermal repeated dose toxicity studies in rodents with the substance itself or the read across substances C12-18 and C18-unsatd. DEA and C16-18 and C18-unsatd. DEA, the test substance does not require classification for this endpoint according to CLP (EC 1272/2008) criteria.