Isotridecanol, ethoxylated

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Aug 2019 - 27 Jan 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Jun - 18 Jul 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

- Principle of test: Preliminary toxicity study in rats using daily oral administration for 2 weeks (dose range finding study). The data generated was used to decide on dose levels for a subsequent subchronic (90-day) repeated dose toxicity study (RTC study no. A3596).
- Parameters analysed / observed: Mortality, clinical signs, body weight, food consumption, terminal body weight, organ weights and macroscopic observations.

GLP compliance:

no

Remarks:

2 weeks preliminary toxicity (dose range finding) study

Limit test:

no

Species:

rat

Strain:

other: Hsd: Sprague Dawley SD

Details on species / strain selection:

The Sprague Dawley SD rat was selected because it is accepted by many regulatory authorities and there are ample experience and background data available.

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy and supplied by Envigo Netherlands.
- Females: nulliparous and non-pregnant
- Age at delivery: 27 - 29 days
- Weight at delivery: males 90.3 - 91.4 g, females 94.3 - 96.6 g
- Housing: animals were housed up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages; nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet: rodent diet 4 RF 21 (Mucedola S.r.l., SettimoMilanese (MI), Italy), ad libitum
- Water: ad libitum, via water bottles
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY
No known contaminants in the feed or water at levels that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 25 Jun 2019 To: 18 Jul 2019

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is a possible route of exposure of the test item in man.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The required amount of test item was suspended in the vehicle. The preparations were made daily.
- Concentration of test substance in vehicle: 50, 100 and 200 mg/mL
- Dose volume: 5 mL/kg

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

Daily, 7 days / week

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day for mortality, once daily for clinical signs

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule: on the day of allocation to treatment group, on the day that treatment commenced, twice weekly thereafter and just prior to necropsy

FOOD CONSUMPTION:
- Time schedule: weight of food consumed by each cage of rats was recorded weekly; group mean daily intake per rat was calculated.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 1 under 'Any other information on materials and methods incl. tables')

HISTOPATHOLOGY: No

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: No relevant clinical signs in males. In females the following clinical signs were observed: slight to moderate decreased activity and hunched posture (4/4, Days 2 - 5), slight to moderate piloerection (4/4, Days 1 - 9), slight ataxia (2/4, Days 7 - 8 or 9) and swollen abdomen (2/4, Days 11 - 15). No relevant clinical signs were observed in any other animal at any dose level.

Mortality:

mortality observed, treatment-related

Description (incidence):

1000 mg/kg bw/day: One female was found dead on Day 9 immediately after dosing. A slight red staining of the muzzle was observed. At necropsy, the gross findings were red or dark colour of lungs, thyroid gland and thymus, red staining of muzzle (head) and red area (glandular region) and content of the stomach. The cause of the death was unknown. All other animals survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

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):

500 and 1000 mg/kg bw/day: A statistically significant increase in relative and/or absolute mean liver weight in males (approx. 17% and 31% in the mid- and high-dose group, respectively, for relative organ weight) and females (approx. 16% and 40% in the mid- and high-dose group, respectively, for relative organ weight) was observed. Moreover, a significant decrease in relative mean spleen weight in female rats (approx. 13% and 11% in the mid- and high-dose group, respectively, for relative organ weight) was noted. No other relevant changes were observed in treated animals of both sexes,

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day: Multiple red areas were noted in 1/4 male rat.
1000 mg/kg bw/day: Distended stomach and swollen abdominal cavity were observed in 1/4 females. No other relevant changes were found in animals of both sexes in all groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

dose level: for main study

Effect level:

500 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

clinical signs

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

A dose level of 500 mg/kg bw/day was concluded to be applied in the main study.

Critical effects observed:

no

Data source

Reference

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isotridecanol, ethoxylated

EC Number:

500-241-6

EC Name:

Isotridecanol, ethoxylated

Cas Number:

69011-36-5

Molecular formula:

not available, isomeric structures

IUPAC Name:

Isotridecanol, ethoxylated

Test animals

Species:

rat

Strain:

other: Hsd: Sprague Dawley SD

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS srl, San Pietro al Natisone (UD), Italy and supplied by Envigo Netherlands.
- Females: nulliparous and non-pregnant
- Age at delivery: 41 - 43 days
- Weight at delivery: Males 99.1 - 120.3 g, females 93.2 - 101.7 g
- Housing: The animals were housed up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages as indicated in the relevant SOP. Nesting material was provided inside suitable bedding bags and changed at least twice a week.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 18 days

DETAILS OF FOOD AND WATER QUALITY
No known contaminants in the feed or water at levels that would interfere with the objectives of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19 Aug 2019 To: 26 Nov 2019

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is a possible route of exposure of the test item in man.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Trial preparations were performed to select the vehicle (corn oil) and to establish a suitable formulation procedure.
- The required amount of test item was suspended in the vehicle. The preparations were made weekly.
- Concentration of test substance in vehicle: 10, 30 and 100 mg/mL

- Dose volume: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate the analytical method and the preparation procedure and to verify the stability of the preparations (RTC Study no. A3597). The preparations, in the concentrations range of 10 - 200 mg/mL were stable for 28 h at room temperature and for 8 days at +4 °C. Samples of the preparations prepared in Weeks 1 and 13 of the current study were analysed to check the concentration. Results of the analyses were within the acceptability limits stated in the test facility's SOPs for concentration of solutions (85 - 115%).

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily, 7 days / week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected based on information from a preliminary, non-GLP compliant 2 weeks toxicity study (ERBC study no. E0407).
During the last week of treatment, individual overnight urine samples were collected from all male and female animals from each main phase group under conditions of food and water deprivation. Prior to necropsy, blood samples were collected under isoflurane anaesthesia from the retro-orbital sinus and/or from the vena cava for thyroid hormone determination, haematology, coagulation and clinical chemistry of the same animals, under conditions of food deprivation.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once per week during the study from the start of treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Time schedule: The weight of food consumed by each cage of rats was recorded at weekly intervals starting from treatment.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule and dose groups for examinations: Both eyes of all animals were examined prior to the commencement of treatment. The eyes of all animals from high dose and control groups were re-examined during Week 13 of treatment.

HAEMATOLOGY, CLINICAL CHEMISTRY AND PLASMA/SERUM HORMONES: Yes
- Time schedule for collection of blood: During the last week of treatment from the retro-orbital sinus of the same animals for thyroid hormone determination and haematology and clinical chemistry investigations. During the necropsy procedure, blood samples were collected under isoflurane anaesthesia from the abdominal vena cava for coagulation determination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals.
- Haematology parameters: 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 (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets and Prothrombin time.
- Clinical chemistry parameters: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Bile acids, Glucose, Triglycerides, Inorganic phosphorus, Total bilirubin, Total cholesterol, HDL, LDL, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium and Chloride.
- Thyroid hormone investigated: Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH).

URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of treatment, individual overnight urine samples were collected from all male and female animals from each group under conditions of food and water deprivation.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters checked: Urine samples were stored at -20 °C, but not analysed, since no treatment-related effects were observed.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during Week 12 of treatment.
- Dose groups that were examined: All animals in all dose groups.
- Battery of functions tested: sensory activity (auditory, visual and proprioceptive stimuli), grip strength and motor activity.

IMMUNOLOGY: No

OTHER:
- Oestrous cycle: At the end of the treatment period, just prior to necropsy, vaginal smears were taken from all surviving females and the oestrous cycle phase was recorded.

Sacrifice and pathology:

GROSS PATHOLOGY AND ORGAN WEIGHTS: All animals were subjected to a full post mortem examination and the following organs weighed: Brain, epididymides, adrenal glands, parathyroid, prostate incl. seminal vesicles, thyroid, heart, kidney, liver, ovaries, spleen, testes, thymus, uterus and cervix.

HISTOPATHOLOGY: Histopathology was conducted for all animals in the control and high-dose groups for the following tissues: Adrenal glands, Aorta, Bone marrow (from sternum), Brain (cerebrum, cerebellum, medulla/pons), Caecum, Coagulating glands, Colon, Duodenum, Epididymides, Eyes (2), Femur with joint (2), Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Liver, Lungs (including mainstem bronchi), Lymph nodes (cervical and mesenteric), Mammary gland (Males and Females), Oesophagus, Ovaries, Oviducts, Pancreas, Parathyroid glands, Pituitary gland, Prostate gland, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach, Testes, Thymus (where present), Thyroid gland, Trachea, Urinary bladder, Uterus incl. cervix and Vagina.

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Statistical analysis of histopathological finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during the study. Salivation was recorded in animals of all treatment groups, starting from approximately 2 - 3 weeks after the start of treatment in animals of low and mid-dose groups and approximately 7 - 10 days after the start of treatment in those of the high-dose group. Since this sign was occasionally recorded also in animals of the control group, receiving the vehicle alone, it could be considered not adverse and probably related to the high density of the preparations and vehicle used for the formulation.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No difference in body weight and body weight gain was noted in treated animals, when compared to controls during the study.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption was comparable between treated and control animals. A slight and statistically significant increase in food consumption was occasionally recorded during the study (on Day 71 in males of Group 4; on Day 15 in females of Groups 2 and 4), and, therefore, considered of no toxicological relevance.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ocular findings were noted before the start of treatment and during Week 13 of treatment, at the ophthalmoscopic examination performed on animals of the control and high dose group.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological changes recorded in animals of the treatment groups when compared to controls, are summarised in Table 1. Due to the lack of severity and the absence of other changes, the findings were considered to be not adverse. The statistically significant decrease of haemoglobin recorded in males dosed at 150 mg/kg bw/day was not dose-related. Therefore, it was considered to be incidental.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in some biochemical parameters recorded in animals of the treatment groups when compared to controls, are summarised in Table 2. Although changes in bilirubin values showed some dose-relation, this finding was considered not to be adverse, since the decrease of bilirubin usually has no pathological significance and there were no other related changes. Due to their minimal severity and/or the lack of a dose-relation, the other changes were considered to be non-adverse.

Endocrine findings:

no effects observed

Description (incidence and severity):

Thyroid stimulating hormone (TSH) was increased in males dosed at 500 mg/kg bw/day. Compared with mean control data, the increment was 2.0 fold. Due to the absence of other related findings, as changes of the other hormones or changes in absolute and relative thyroid weights, this change was not considered to be of toxicological significance.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant changes were observed. The increase in diuresis recorded in males dosed at 150 mg/kg bw/day (42% above controls) was not dose-related, therefore it was considered to be incidental.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Slight, but statistically significant decreases in rearing were recorded in females dosed at 150 and 500 mg/kg bw/day during the study. Since these findings were occasionally observed during the study with a high individual variability, this sign is not considered to be indicative of neurotoxicity. The decrease in rearing occasionally observed in male animals, although statistically significant, was considered incidental and not treatment related.

No signs of neurotoxicity were observed at functional tests (sensory reactivity, landing footsplay, grip strength) performed at the end of the treatment period. A statistically significant increase in mean grip strength (+32%) was recorded in male animals of the high dose group (Group 4). This change is not considered to be adverse, since it is not associate with any other signs of neurotoxicity. Motor activity measurements performed at the end of the treatment period did not show any toxicologically significant differences between treated animals and controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in absolute and relative mean liver weights was recorded in males and females treated at 150 mg/kg bw/day (increase in relative weight of approximately 7% for males and 10% for females) and at 500 mg/kg bw/day (increase in relative weight of approximately 17% for males and 21% for females). This variation was considered treatment-related. A statistically significant increase in absolute and relative mean kidneys weight was observed in animals of mid- and high dose groups (increase in relative weight of approximately 9% for males in mid- and high dose groups, approximately 12% for females of the high-dose group), when compared to the controls. However, since the weight variation of kidneys was not accompanied by corroborating histopathological changes, this change was considered not toxicologically relevant. All other organ weight variations between control and treated animals were considered incidental since individual values were within the physiological range of Sprague Dawley SD rats of this age.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The only relevant change observed following gross pathology examination was thickening of the stomach (non-glandular region) in 1/10 males in the high-dose group. All other observed changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were observed in the liver and stomach (non-glandular region) of high-dose animals of both sexes.
– Liver: Minimal centrilobular hepatocellular hypertrophy was seen in 8/10 females and 3/10 males treated at the high-dose. Hepatocyte hypertrophy could be associated with microsomal enzyme induction secondary to the exposure to the test item and was distributed mainly in centrilobular areas; hepatocellular cytoplasm showed a pale, ground glass appearance and could be considered an adaptative change and not adverse.
– Stomach: Treatment-related changes were seen in the non-glandular region of the stomach (forestomach) in 9/10 males and 9/10 females of the high-dose group and consisted of minimal to moderate squamous epithelial hyperplasia, characterised by uniform thickening of all layers of the epithelium, with convolutions of the epithelium with downward projections of rete peg-like structures. Frequently, this finding was associated with oedema in the submucosa of minimal degree. The changes observed in the forestomach were judged to be a local irritant effect. As humans lack a non-glandular stomach, the finding is considered relevant to the rat but not to human.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted in all control and treated males. Normal physiology of the oestrous cycle (oestrous, metestrous, diestrous and proestrous) was noted in control and treated females. No morphological changes were detected when compared to each “oestrous phase” in the ovaries, uterus/cervix and vagina.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic and local toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically effects relevant for humans observed

Remarks on result:

other: Changes observed in the stomach were evaluated to be a local irritant effect, were not sufficently severe to become adverse and not relevant for humans due to their location.

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1. Haematological changes recorded in animals of the treatment groups when compared to controls

Dose level (mg/kg bw/day)	Sex	WBC	MCHC	PLT	HGB
150	M	-	-	-	-5%
500	F	+37%	-2%	-14%	-

Table 2. Changes in some biochemical parameters recorded in animals of the treatment groups when compared to controls

Dose level (mg/kg bw/day)	Sex	TBIL	HDL	LDL	Cl	K	IP	Na
50	M	-	-17%	-19%	-	-	-	-
	F	-21%	-	-	-	+7%	-	-
150	M	-43%	-16%	-	-	-5%	-	-
	F	-32%	-	-	-	-	-	-
500	M	-58%	-	-	-1%	-	-	-
	F	-49%	-	-	-	+10%	+17%	-1%

Additional tables are provided under 'Attached background material' as pdf documents.