Alcohols, C12-15, ethoxylated

Endpoint summary

Description of key information

Oral (subacute, rat, m/f, OECD 422): NOAEL (systemic toxicity) = 1000 mg/kg bw/day

Oral (subacute, rat, m/f, OECD 422): NOAEL (local toxicity) = 1000 mg/kg bw/day

 

Oral (subchronic, rat, m/f, OECD 408): NOAEL (systemic toxicity) = 1000 mg/kg bw/day

Oral (subchronic, rat, m/f, OECD 408): NOAEL (local toxicity) = 300 mg/kg bw/day

 

Conclusion based on data obtained with alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5) and considering all the available data on repeated dose toxicity in the Alcohol Ethoxylates (AE) category, in a Weight-of-Evidence approach.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18 MAR 2020 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females: nulliparous and non-pregnant
- Age at study initiation: males 10-11 weeks, females 13-14 weeks
- Weight at study initiation: males 266-317 g, females 201-271 g
- Housing: On arrival and during the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages.
During the post-mating phase, males were housed in their home cage (plastic cages) with a maximum of 5 males/cage. Females were individually housed in plastic cages.
During the lactation phase, females were housed in plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 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): 20-21
- Humidity (%): 46-56
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES
From: 18 MAR 2020 to 20 MAY 2020

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
- Test item dosing formulations were kept at room temperature until dosing.
- Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select corn oil as the suitable vehicle and to establish a suitable formulation procedure (Test Facility Study No. 20219687).
- Concentration in vehicle: 4 mL/kg
- Supplier: Sigma-Aldrich, Steinheim, Germany
- Specific gravity: 0.92

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analyses were performed using a validated analytical procedure (Test Facility Study No. 20219687).
- Concentration analysis was conducted. Results were considered acceptable if mean sample concentration results were within or equal to ± 10% for suspensions of target concentration.
- Homogeneity analysis was conducted. Results were considered acceptable if the coefficient of variation (CV) of concentrations was less than or equal to 10%.
- Stability analyses were performed previously in conjunction with the method development and validation study.

Duration of treatment / exposure:

Males were treated for 29 days, up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-64 days.
Females which failed to deliver or had a total litter loss were treated for 40-43 days.

Frequency of treatment:

Daily, 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 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 selection rationale: The dose levels were selected based on the results of a 10-day Dose Range Finding Study with oral gavage administration of the test material in rats (Test Facility Reference No. 20219688), and in an attempt to produce graded responses to the test item.
- Fasting period before blood sampling for clinical biochemistry: F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Clinical observations were conducted twice daily.

BODY WEIGHT: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION: Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

CLINICAL CHEMISTRY AND HAEMATOLOGY: Blood of all F0-animals was collected on the day of scheduled necropsy. Samples were collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthasia (isoflurane). F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.

SERUM HORMONES: Measurement of total T4 was conducted for F0-males.

NEUROBEHAVIOURAL EXAMINATION: 5 males during Week 4 of treatment and 5 females during the last week of lactation (i.e. PND 6-13) were assessed. Tests were performed after dosing, after completion of clinical observations. All dose groups were assessed. The battery of functions tested were: sensory activity / grip strength / motor activity.

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.

Sacrifice and pathology:

SACRIFICE
- Male animals: All surviving animals were sacrificed as soon as possible after the last litters in each generation were produced.
- Maternal animals: All surviving animals were sacrificed after the last litter of each generation was weaned.

GROSS PATHOLOGY AND ORGAN WEIGHTS: All animals were subjected to a full post mortem examination and the following organs weighed; Brain, cervix, epididymis, adrenal, coagulation gland, parathyroid, prostate, seminal vesicle, thyroid, heart, kidney, liver, ovaries, spleen, testes, thymus, uterus

HISTOPATHOLOGY: Histopathology was conducted for the following tissues: Aorta, nasopharynx, bone marrow, femur, sternum, brain, cervix, epididymides, esophagus, eye, adrenal, coagulation gland, harderian, lacrimal, mammary, parathyroidc, pituitary, prostate, salivary, seminal vesicle, thyroid, gut-associated lymphoid tissue, heart, kidney, large intestine, cecum, colon, rectum, larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, trachea urinary bladder uterus, vagina.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six different covariance structures, were used in order to select the best fitting statistical model.
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. Pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted during daily detailed clinical observations and no findings were noted during the weekly arena observations in this study.
Salivation seen after dosing among all animals of the 300 and 1000 mg/kg/day dose groups and some animals of the 100 mg/kg/day dose group was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Incidental findings that were noted included rales, alopecia and scabs. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered not to be signs of toxicological relevance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item up to 1000 mg/kg/day.
One female of the control group and one female at 300 mg/kg/day were euthanized on Lactation Days 7 and 3, respectively, as they had a total litter loss.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was considered not affected by treatment with the test item up to 1000 mg/kg/day.
At 1000 mg/kg/day, mean relative food consumption was increased (1.27x of control; statistically significant) in females over lactation Days 1-4. No toxicologically relevance was attached to this finding, as food intake had returned to control levels in the subsequent intervals.
Any statistically significant changes in food consumption before or after correction for body weight observed in Groups 2 and 3 were considered to be unrelated to treatment since no trend was apparent regarding dose.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological parameters of treated rats were considered not to have been affected by treatment with the test item up to 1000 mg/kg/day.
Coagulation parameters of treated rats were considered not to have been affected by treatment with the test item up to 1000 mg/kg/day.
The statistically significant increase in mean activated partial thromboplastin time (APTT) in males at 100 and 300 mg/kg/day was considered unrelated to administration of the test item due to absence of a dose response.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered affected by treatment with the test item at 300 and 1000 mg/kg/day.
The following statistically significant changes distinguished treated from control animals:
• Mean albumin concentration was slightly increased (both 1.06x; above historical control range) in males at 300 and 1000 mg/kg/day.
• Mean urea concentration was increased (1.37x; within historical control range) in males at 1000 mg/kg/day.
• Mean cholesterol concentration was decreased (0.74 and 0.65x, respectively; both below historical control range) in males at 300 and 1000 mg/kg/day.
• Mean bile acid concentration was increased (2.90x; within historical control range) in females at 1000 mg/kg/day.
These changes were considered as non-adverse since they were not associated with any adverse pathological alterations.

Any other statistically significant changes in clinical chemistry parameters achieving a level of statistical significance when compared to controls, occurred in the absence of a dose-related response. As such, these slight differences were considered not related to treatment.

Endocrine findings:

no effects observed

Description (incidence and severity):

Serum levels of total T4 in F0-males were considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional tests were inadvertently not performed in females.
Functional observation parameters were considered not affected by treatment with the test item up to 1000 mg/kg/day in males.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined males up to 1000 mg/kg/day. Grip strength was similar between control and high dose males.
Motor activity was similar between treated and control male groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight changes were noted in the liver of males and females, in the thyroid glands of males and in the adrenal glands of females. These changes (which were considered to be non-adverse) are summarized in Table 1.
Any other differences, including those that reached statistical significance (i.e. absolute and relative to body weight uterus weight at 100 mg/kg/day, and heart- and kidney weight relative to body weight in females at 1000 mg/kg/day) were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
There were no other test item-related organ weight changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, test item-related macroscopic enlarged liver was observed in a single male and female and enlarged adrenal glands were observed in a single female. These changes were considered to be non-adverse.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the liver, jejunum and forestomach of males and females at 1000 mg/kg/day, in the thyroid gland of males starting at 100 mg/kg/day and in the adrenal gland of females at 1000 mg/kg/day. These changes are summarized in Table 2, 3 and 4.
Liver: Centrilobular hepatocellular hypertrophy was noted at 1000 mg/kg/day in all males up to slight degree and in 2/6 females at minimal degree.
Jejunum: Vacuolation in the lamina propria was noted at 1000 mg/kg/day in 4/5 males up to slight degree and in 3/5 females at minimal degree.
Forestomach: Minimal squamous cell hypertrophy was noted at 1000 mg/kg/day in 4/5 males and in a single female.
Thyroid glands: An increased incidence and/or severity of follicular cell hypertrophy was noted starting at 100 mg/kg/day in males up to slight degree.
Adrenal glands: Diffuse cortical hypertrophy was noted at 1000 mg/kg/day in 3/5 females up to slight degree.
All of these changes were considered non-adverse.
There were no other test item-related histologic changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically relevant effects observed

Key result

Critical effects observed:

no

Table 1. Mean Percent Organ Weight Differences from Control Group

		Males			Females
Dose level (mg/kg/day):	100	300	1000	100	300	1000
LIVER
Absolute	14*	18**	33**	0	27**	39**
Relative to body weight	8*	14**	36**	3	20**	36**
THYROID GLANDS
Absolute	5	2	15	na	na	na
Relative to body weight	2	0	18*	na	na	na
ADRENAL GLANDS
Absolute	na	na	na	4	16	35**
Relative to body weight	na	na	na	13	17	39**

na = not applicable

*: P<0.05, **: P<0.01

Table 2. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Males				Females
Dose level (mg/kg/day):	0	100	300	1000	0	100	300	1000
LIVER ^a	5	5	5	6	6	5	6	6
Centrilobular hypertrophy
Minimal	-	-	-	1	-	-	-	2
Slight	-	-	-	5	-	-	-	-
JEJUNUM ^a	5	5	5	5	5	5	5	5
Vacuolation
Minimal	-	-	-	3	-	-	-	3
Slight	-	-	-	1	-	-	-	-
FORESTOMACH ^a	5	5	5	5	5	5	5	5
Squamous cell hyperplasia
Minimal	-	-	-	4	-	-	-	1

^a = Number of tissues examined from each group.

Table 3. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Males
Dose level (mg/kg/day):	0	100	300	1000
THYROID GLANDS ^a	5	5	5	5
Follicular cell hypertrophy
Minimal	-	3	3	4
Slight	-	-	1	1

^a = Number of tissues examined from each group.

Table 4. Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

		Females
Dose level (mg/kg/day):	0	100	300	1000
ADRENAL GLANDS ^a	5	5	6	5
Diffuse cortical hypertrophy
Minimal	-	-	-	1
Slight	-	-	-	2

^a = Number of tissues examined from each group.

 

 

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Jun - 21 Dec 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

- Temperature (min. 17 °C) outside the targeted range for 2 days - Clinical biochemistry did not include blood urea nitrogen parameter

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl: WI(Han)

Details on species / strain selection:

outbred, SPF-quality

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 157 - 207 g (males) and 112 - 154 g (females)
- Fasting period before study: no
- Housing: Up to 5 animals of the same sex and same dosing group housed together in polycarbonate cages (Makrolon type IV, height 18 cm or Makrolon type 2000P, height 21.5 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, DE) and equipped with water bottles. During locomotor activity monitoring, animals are housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: pelleted diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany); ad libitum (except during motor activity measurements)
- Water: municipal tap water; ad libitum (except during motor activity measurements)
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY:
Results of analysis for nutritional components, environmental contaminants and water analysis are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 22
- Humidity (%): 47 - 71
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 29 Jun 2021 To: 26 Oct 2021

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared at least weekly and stored at 4 °C. Formulations (w/w) were homogenized to visually acceptable levels by stirring continuously during dosing. An adjustment was made for he specific gravity of the test item and the vehicle. No correction was made for purity / composition of the test item. The dosing volume was 5 mL/kg bw. The dose volume for each animal was calculated and adjusted based on the most recent body weight measurement.

VEHICLE
- Justification for use and choice of vehicle: test item stable in vehicle, as demonstrated by stability analysis; trial preparations were performed and showed suitability of the vehicle
- Concentration in vehicle: 20, 60 and 200 mg/mL for the low-, mid- and high-dose group, respectively.

Analytical verification of doses or concentrations:

yes

Remarks:

in Weeks 1, 6 and 12

Details on analytical verification of doses or concentrations:

Accuracy: The concentrations analyzed in the formulations of Group 2 - 4 prepared for use in Weeks 1, 6 and 12 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 90 - 110% of target concentration). No test item was detected in the Group 1 formulations (controls).
Homogeneity: The formulations of Groups 2 and 4 prepared for use in Weeks 1, 6 and 12 were homogeneous (i.e., coefficient of variation = 10%).

Duration of treatment / exposure:

Main study: 13 weeks
Recovery: 28 days

Frequency of treatment:

once daily, 7 days / week

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

Main study: 10 (Group 1 - 4)
Recovery groups: 5 (Group 1 and 4)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily; from Day 1 at 0 to 1 h postdose; twice daily checked for mortality/moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly; from Week 1 and throughout the study, and on the day of necropsy
- Arena observations: observed for clinical signs outside the home cage in a standard arena once before the first test item administration, and weekly during treatment and recovery periods

BODY WEIGHT: Yes
- Time schedule for examinations: weekly; from at least Day 1 and throughout the study

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No (measured per cage)
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: Yes (qualitatively)
- Time schedule for examinations: on a regular basis throughout the study

OPHTHALMOSCOPIC EXAMINATION: Yes
Pretreatment Period: all main study and recovery animals once
Dosing Period: all Group 1 and 4 main study animals during Week 13.
Recovery Period: examination of the recovery animals at the end of the recovery period only upon occurrence of treatment-related effects at the end of treatment period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the final day of treatment or recovery, prior to sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: White Blood Cell (WBC), Neutrophils (absolute), Lymphocytes (absolute), Monocytes (absolute), Eosinophils (absolute), Basophils (absolute), Large unstained cells (LUC) (absolute), Red Blood Cell (RBC), Reticulocytes (absolute), Red Blood Cell Distribution Width (RDW), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelets, Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the final day of treatment or recovery, prior to sacrifice
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline Phosphatase (ALP), total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, triglycerides, HDL and LDL cholesterol, sodium, potassium, chloride, calcium, and inorganic phosphate (Inorg. Phos).

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: on the final day of treatment or recovery, prior to sacrifice
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: Triiodothyronine (T3), Thyroxine (T4), and Thyroid-Stimulating Hormone (TSH).

URINALYSIS: Yes
- Time schedule for collection of urine: on the final day of treatment or recovery, prior to sacrifice
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, overnight
- Parameters checked: pH, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the dosing period (Week 12 - 13)
- Dose groups that were examined: first 5 animals per sex per group
- Battery of functions tested: sensory activity, grip strength and motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; a complete necropsy examination was performed, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

HISTOPATHOLOGY: Yes; the full list of tissues was collected from main study and recovery animals of all groups and weighed (see Attachment 2, Table 16 under section "Attached background material"). The full list of tissues from Group 1 and 4 were subjected to histopathological examination, while only gross lesions and target tissues were evaluated for Group 2 and 3, and recovery groups. For histopathology, representative samples of tissues were collected and preserved in 10% neutral buffered formalin or modified Davidson's solution.

For examined parameters, see Attachment 2, Table 16 under section "Attached background material".

Other examinations:

-Estrous Stage Determination: On the day of sacrifice, a vaginal smear was taken from all females to determine the stage of estrous.
- Sperm analysis: from all surviving males; evaluated for sperm motility, progressive motility, morphology and sperm numbers

Statistics:

Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers, and/or incidences are reported as appropriate by dataset. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted. The following statistical tests were used: Levene’s test, ANOVA F-test, Kruskal-Wallis Dunnett’s and Dunn’s test, ANCOVA, Fisher's exact test, and Steel-test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Abnormal breathing sounds were noted in 4/10 males and 1/10 females at 300 mg/kg bw/day and 2/15 males and 3/15 females at 1000 mg/kg bw/day on a few days during the dosing period. As these signs were observed incidentally and animals fully recovered, this was considered not toxicologically relevant.
Erected fur was observed in 2/15 males at 1000 mg/kg bw/day once or on two consecutive days. Based on the low occurrence and short duration, this observation was considered not toxicologically relevant.
Salivation was noted in males and females starting at 100 mg/kg bw/day (3/10 males and 1/10 females) during the dosing period, as well as in a 1/15 control females. 8/10 males and 5/10 females were affected at 300 mg/kg bw/day, and all males and females at 1000 mg/kg bw/day. Salivation was not considered toxicologically relevant, taking into account the nature of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
No test item-related clinical signs were noted during weekly arena observations and during the recovery period. Any other clinical signs (e.g. fur loss, skin lesion, skin scabs and/or skin laceration) noted during the dosing period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
For details on clinical signs, please refer to Attachment 1 - Tables 1 - 2 under section "Attached background material".

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight and body weight gain of males and females up to 300 mg/kg bw/day remained in the same range as controls over the study period.
For males at 1000 mg/kg bw/day, body weight and body weight gain were lower than controls from Day 57 onwards, resulting in a 7% lower mean body weight at the end of the dosing period (Day 91; not statistically significant). Slight body weight loss (up to -5%) was noted incidentally in a few males. During the recovery period, a trend towards higher body weight gain was noted for males at 1000 mg/kg bw/day, resulting in a 5% lower mean body weight than controls (not statistically significant). The apparent higher body weights in females at 1000 mg/kg bw/day were attributed to the higher mean body weight at the start of the dosing period. The difference in mean body weight compared to concurrent controls remained similar throughout the dosing period and was therefore considered not to be related to treatment with the test item.
For details on body weight and body weight changes, please refer to Attachment 1 - Figure 1 and Tables 3 - 4 under section "Attached background material".

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes in food consumption were recorded during the dosing and recovery period.
Females of the main study at 1000 mg/kg bw/day showed a slightly increased food consumption from Day 8 onwards, with an overall food consumption of 8% higher compared to control animals over the whole duration of the dosing period (Days 1 - 91, not statistically significant). This was in line with the slightly higher mean body weight observed from the start of the dosing period onwards and is therefore considered not to be related to treatment with the test item.
For details on food consumption, please refer to Attachment 1 - Figure 2 and Table 5 under section "Attached background material".

Food efficiency:

not examined

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ophthalmology findings in Week 13.
The nature and incidence of ophthalmology findings noted during the pre-treatment period was similar among the groups and occurred within the range considered normal for rats of this age and strain. As these findings occurred at pre-treatment only, they were unrelated to treatment with the test item.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology parameters at the end of the dosing period of males up to 300 mg/kg bw/day and females up to 300 mg/kg bw/day were considered not to have been affected by the test item.
At the end of the dosing period, increased hematocrit (HCT) levels were noted in males at 1000 mg/kg bw/day (0.4911 L/L; 1.04 x of control, statistically significant). At the end of the recovery period the HCT values were comparable to control, indicating recovery.
Remaining differences in hematology parameters, including changes at the end of the recovery period, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
For details on haematological findings, please refer to Attachment 1 - Table 8 under section "Attached background material".
Coagulation:
Coagulation parameters at the end of the dosing period of males up to 1000 mg/kg bw/day and females up to 300 mg/kg bw/day were considered not to have been affected by the test item. Activated partial thromboplastin time (APTT) at the end of the dosing period was shortened in females at 1000 mg/kg bw/day (20.45 sec; 0.92 x of control, statistically significant). At the end of the recovery period, no difference between animals dosed at 1000 mg/kg bw/day and control animals was observed suggesting full recovery.
For details on coagulation, please refer to Attachment 1 - Table 9 under section "Attached background material".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical chemistry parameters at the end of the dosing period for males and females at 100 mg/kg bw/day were considered not to have been affected by the test item.
Changes in clinical chemistry parameters at the end of the dosing period comprised an increase in aspartate aminotransferase (AST) activity in males at 300 and 1000 mg/kg bw/day (1.31 and 1.27 x of control, respectively, statistically significant), and an increase in alkaline phosphatase (ALP) activity in males and females at 1000 mg/kg bw/day (1.26 and 1.30 x, respectively; not statistically significant for females).
Total bilirubin (TBIL) concentration at the end of the dosing period was statistically significantly decreased in males at 1000 mg/kg bw/day (0.81 x). Furthermore, urea concentration in females at 300 and 1000 mg/kg bw/day (1.37 and 1.42 x, respectively, statistically significant) and creatinine (CREAT) concentration in females at 1000 mg/kg bw/day (1.25 x; not statistically significant) was increased at the end of the dosing period. Glucose (GLUC) concentration was significantly increased in males at 1000 mg/kg bw/day (1.16 x), while HDL cholesterol (HDL) was significantly decreased in males at 300 and 1000 mg/kg bw/day (0.69 and 0.68 x, respectively) at the end of the dosing period.
At the end of the recovery period, TBIL, UREA, CREAT and HDL were partially recovered, and ALP and GLUC were comparable to control suggesting full recovery.
Remaining differences in clinical chemistry parameters, including changes at the end of the recovery period, regardless of statistical significance, were considered not test item-related based on the absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
For details on clinical biochemistry findings, please refer to Attachment 1 - Table 10 under section "Attached background material".

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Thyroxine (T4) levels were statistically significantly decreased in males at 1000 mg/kg bw/day (0.78 x) but increased in females at 1000 mg/kg bw/day (1.21 x, not statistically significant). All mean values were within the historical control range (Period 2020-2021: Thyroxine (T4; ng/mL) Male: mean = 43.41; P5 - P95: 28.50–60.34 (n=139) Thyroxine (T4; ng/mL) Female: mean = 27.14; P5 - P95: 16.30–43.46 (n=139), and T4 values were comparable to controls at the end of the recovery period for both sexes. This effect was considered not toxicologically relevant.
In addition, thyroid stimulating hormone (TSH) levels were increased in females at 1000 mg/kg bw/day at the end of the dosing period (3.63 x, statistically significant) and at the end of the recovery period (4.27 x, not statistically significant). As the control mean was low when compared to the historical control range, whereas the mean at 1000 mg/kg/day was within the historical control range (Period 2020-2021: Thyroid stimulating hormone (TSH; mU/L) Female: mean = 0.0852; P5 - P95: 0.0020 - 0.3819 (n=184)), this difference in TSH was considered to be unrelated to treatment with the test item.
Finally, TSH was decreased in males at 1000 mg/kg bw/day at the end of the recovery period (0.72 x; not statistically significant). As this change was not observed at the end of the dosing period and lacked correlating morphological findings in the thyroid gland, this was considered to be unrelated to treatment with the test item.
For details on thyroid hormones, please refer to Attachment 1 - Table 10 under section "Attached background material".

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis parameters of males up to 300 mg/kg bw/day and females up to 1000 mg/kg bw/day were considered not to have been affected by treatment with the test item. In males at 1000 mg/kg bw/day, the mean urinary volume was increased (2.09 x of control, not statistically significant) at the end of the dosing period. At the end of the recovery period, urinary volume was comparable between animals dosed with the test item and controls suggesting full recovery.
No effect was observed on the pH of the urine.
For details on urinalysis, please refer to Attachment 1 - Table 11 under section "Attached background material."

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were similar in all examined animals. Grip strength and motor activity was similar between control and test item-dosed animals. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. For details on behavioural findings, please refer to Attachment 1 - Tables 6 - 7 under section "Attached background material".

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test item-related higher organ weight was noted in the liver (starting at 300 mg/kg bw/day) and kidney (1000 mg/kg bw/day) of males and females and the adrenal gland of females only (1000 mg/kg bw/day):
Liver: Higher liver weights were noted in males and females starting at 300 mg/kg bw/day and were statistically significant as relative to body weight only at 300 mg/kg bw/day (difference to controls: 14% (males) and 19% (females), and both as absolute (difference to controls: 26% (males) and 54% (females)) and relative values (difference to controls: 38% (males) and 48% (females)) at 1000 mg/kg bw/day. This correlated to centrilobular hepatocellular hypertrophy microscopically. Following the recovery period, at 1000 mg/kg bw/day, liver weight in females was higher only when expressed relative to body weight (7% difference to controls), and of smaller magnitude than at the end of dosing (partial recovery); and considered to be recovered in males. In males at 100 mg/kg bw/day at the end of dosing and 1000 mg/kg bw/day at the end of recovery, the difference in liver weight in males was interpreted as not test item-related due to a combination of factors including the low magnitude and statistically significance relative to body weight only.
Adrenals: higher adrenal gland weight was noted at the end of the dosing period in females at 1000 mg/kg bw/day, significant as absolute value (26% difference to controls). Following the recovery period, higher adrenal gland weights were noted in females at a slightly higher magnitude than at the end of dosing (32% difference to controls), but statistically significant only when expressed relative to body weight (11% difference to controls). There was no histologic correlate for this finding.
Kidney: Higher kidney weight was noted in males and females at 1000 mg/kg bw/day. In males, this was statistically significant only relative to body weight (18% difference to controls); in females, it was statistically significant as absolute value (22% difference to controls) and relative to body weight (17% difference to controls). There was no histologic correlate. Following the recovery period there was no difference in kidney weight of either sex.
Other findings: In males at the end of dosing, a few organs demonstrated apparent higher weights at 1000 mg/kg bw/day when expressed relative to body weight only. This included the heart (8%) and testis (12%) and was interpreted to reflect the lower body weight of this group (-9%). The few other statistically significant changes in at the end of the dosing (higher epididymis, seminal vesicle gland weight and testis weight at 100 mg/kg bw/day) were considered to be unrelated to treatment with the test item, as they occurred in the absence of a dose-related trend. As regards liver, adrenals and kidney weights, the effects seen were considered treatment-related, but not toxicologically relevant.
For details on organ weights, please refer to Attachment 1, Tables 13 - 14 under section "Attached background material".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic observations were noted at 1000 mg/kg bw/day the end of dosing only in the liver (males and females) and stomach (males only):
Liver: At 1000 mg/kg bw/day, prominent lobular architecture noted in 6/10 males and 1/10 females, was considered to be test item-related and may correlate to centrilobular hypertrophy. The low incidence of this finding at 100 and 300 mg/kg bw/day in males (2/10 and 1/10, respectively) was interpreted as unlikely related to the test item due to the low and non dose-related incidence, and lack of correlation to organ weight or microscopic findings.
Stomach: At 1000 mg/kg bw/day (males), an increased incidence (compared to control males) of dark red foci in the glandular mucosa (4/10, correlating to acute mucosal hemorrhage), and irregular surface of the non-glandular stomach (1/10, correlated to squamous hyperplasia) were noted. The low incidence of dark red foci at 100 mg/kg bw/day was comparable to the control group.
There were no macroscopic findings in the liver and stomach at the end of the recovery period.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
For details on macroscopic findings, please refer to Attachment 1 - Table 15 under section "Attached background material".

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the thyroid gland, liver, jejunum, and stomach of males and females, and in the kidney of males:
Thyroid: Colloid alteration (6/10 males and 4/10 females) and/or follicular cell hypertrophy (4/10 males and 3/10 females) were observed in males and females at 1000 mg/kg bw/day. The incidence and severity of follicular cell hypertrophy and colloid alteration noted at 100 and 300 mg/kg bw/day in males was generally comparable to the control males, i.e. 3/10 minimal incidences of follicular cell hypertrophy in either group, compared to 2/10 minimal incidences in the control group, and 3/10 or 2/10 incidences of colloid alteration for the 100 and 300 mg/kg bw/day groups, respectively, as compared to and 2/10 minimal colloid alteration incidences in the control group. Following the recovery period, minimal colloid alteration was noted in 1/5 females and 3/10 males (increased incidence as compared to 0 and 1 incidences in the controls, respectively) while incidences of hypertrophy were the same as for controls (males; 1/5, minimal severity) or not present (females), consistent with partial recovery of the microscopic thyroid gland observations.
Liver: centrilobular hepatocellular hypertrophy was noted in 6/10 males (minimal) and all females (3/10 minimal, 7/10 mild) at 1000 mg/kg bw/day and correlated to higher liver weights, and likely also correlated to prominent lobular architecture noted macroscopically. This effect was absent in any other dose group, including the control group. Centrilobular hypertrophy was not observed in males or females after the recovery period.
Stomach: a combination of findings was noted in the non-glandular stomach (forestomach) of males and females at 1000 mg/kg bw/day, including squamous mucosal hyperplasia in 8/10 males and all females (up to moderate; correlated to irregular surface in one male) with hyperkeratosis in 6/10 males and 8/10 females (minimal), edema in 6/10 males and 3/10 females (up to mild), and ulceration in 3/10 males and 1/10 females (up to mild; most often at the limiting ridge). Non-glandular ulceration and vacuolation of minimal degree also occurred in 1/10 males dosed at 300 mg/kg bw/day, while 1/10 females at 100 and 300 mg/kg bw/day each also had squamous mucosal hyperplasia of minimal degree. In the control group, only 2/10 minimal and 1/10 mild incidences of squamous mucosa edema occurred in males and 1/10 control females had glandular mucosa edema of mild severity. At 100 mg/kg bw/day, 1/10 males and females each had squamous mucosa edema of minimal degree. At 300 mg/kg bw/day, 2/10 males (minimal degree) and 1/10 females (mild degree) were observed with the same lesion. In the glandular mucosa in males, a slight increase in the incidence and severity of focal acute mucosal hemorrhage was also noted (3/10 males and 1/10 females affected at the high dose, and 1/10 case of minimal severity in 300 mg/kg bw/day dosed males) and correlated to dark red foci macroscopically. The low incidence (single occasion) of a few of these observations noted in both sexes at 100 and 300 mg/kg bw/day were not considered to be strong evidence of a test item-related effect. Following the recovery period, minimal squamous mucosal hyperplasia and hyperkeratosis was noted in 2/5 males and 3/5 females of the 1000 mg/kg bw/day dose group only, consistent with a partial recovery.
Jejunum: Vacuolation of the villous lamina propria was observed in all males and females at 1000 mg/kg bw/day. This was also noted at minimal degree in 1/10 males at 300 mg/kg bw/day, but at this low incidence and severity it is no strong evidence of a test item-related effect. This finding was characterized by discrete small to large clear vacuoles in the lamina propria of the villi, and it is most likely that these vacuoles represent dilated lacteals. In some vacuoles a lining of attenuated endothelial-like cells were observed. This finding was not observed in control animals. At the end of the recovery period, the incidence and severity in males and females was comparable to the end of dosing, suggesting no recovery.
Kidney: In males, an increased severity (compared to the concurrent control group) of hyaline droplet accumulation was noted starting at 300 mg/kg bw/day (8/10 affected males, of which 5 were minimal and 3 were mild, compared to 6/10 minimal incidences in the controls and 100 mg/kg bw/day group each). At the high dose group, 7/10 animals presented with this finding, of which 3 were of minimal, one of mild, and 2 of moderate severity. Following the recovery period at 1000 mg/kg bw/day, the incidence and severity of this observation was comparable to the control males (1000 mg/kg bw/day: 3/5 affected, minimal severity versus control: 4/5 affected of which 3 were of minimal and one was of mild severity), suggesting recovery.
Remaining histologic changes were considered to be incidental findings or were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. Only the findings in the stomach are considered toxicologically relevant.
For details on histopathological findings, please refer to Attachment 1 - Table 16 under section "Attached background material".

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Estrous stage determination: no effects observed
- Sperm analysis: No test item-related effects on sperm were observed. A decreased percentage of progressive sperm was observed in males at 100, 300 and 1000 mg/kg bw/day (24, 22 and 23%, respectively, versus 31% for control). In addition, an increased number of cells with other tails was noted at 1000 mg/kg bw/day only (4 cells versus 1 in control). As all values remained well within the historical control range (Period 2017-2021: Progressive sperm (%): mean = 27; P5 - P95: 7.0 – 44.0 (n=353); other tails (No. of cells): mean = 2; P5 - P95: 0.0 – 6.0 (n=349)) and lacked a clear dose response, these slight differences were considered unrelated to treatment with the test item. Any other (statistically significant) changes in sperm analysis parameters were considered to be unrelated to the test item, as these occurred in the absence of a dose-related trend.
For details on sperm analysis, please refer to Attachment 1 - Table 12 under section "Attached background material".

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to and including this dose level

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level.

Key result

Dose descriptor:

LOAEL

Remarks:

local

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

no

Conclusions:

The present study was conducted under GLP and according to OECD test guideline 408 (2018). Under the conditions of the study, administration of the test substance once daily by oral gavage for at least 90 days resulted in adverse effects in the stomach of males and female Han Wistar rats treated at 1000 mg/kg bw/day, consisting of ulceration of the non-glandular mucosa. No adverse systemic effects were observed. Based on this result, a No Observed Adverse Effect Level (NOAEL) systemic = 1000 mg/kg bw/day in males and females was determined, and a NOAEL local at 300 mg/kg bw/day in males and females was established.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 1) studies performed with the registered substance. The selected studies are thus sufficient to fulfil the standard information requirements set out in Annexes VIII - X, Section 8.6, of the REACH Regulation (EC) No. 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data on repeated dose toxicity are available for alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5) as well as several member substances of the Alcohol Ethoxylates (AE) category.

Studies with alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5)

Alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5) was tested in Wistar Han rats in a combined repeated dose toxicity study with the reproductive / developmental toxicity screening test according to OECD guideline 422 under GLP conditions (Shell, 2021). Groups of 10 animals per sex received doses of 100, 300 and 1000 mg/kg bw/day by daily oral gavage, 7 days a week for a minimum of 28 days. A similarly constituted control group was dosed with the vehicle (corn oil) only. Males were treated for 28-29 days whereas females that delivered were treated for 50-63 days (14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery). Females which failed to deliver or had a total litter loss were treated for 42-54 days. The following parameters and endpoints were evaluated: mortality/moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0 males), gross necropsy findings, organ weights and histopathologic examinations. In addition, a number of reproduction / developmental parameters were investigated. The details of the reproductive/developmental screening test are summarised in IUCLID section 7.8.1.

A slightly increased plasma albumin concentrations in males at 300 and 1000 mg/kg bw/day, increased plasma urea concentrations in males at 1000 mg/kg bw/day, decreased plasma cholesterol concentrations in males at 300 and 1000 mg/kg bw/day and increased bile acid concentrations in females at 1000 mg/kg bw/day were considered as non-adverse since these changes were not associated with any adverse pathological alterations. Non-adverse test item-related morphologic alterations were present in males and females at 1000 mg/kg bw/day in the liver (macroscopically enlarged liver, centrilobular hypertrophy, increased weights starting at 100 mg/kg bw/day in males and 300 mg/kg bw/day in females), forestomach (squamous cell hyperplasia) and jejunum (vacuolation in the lamina propria), in males starting at 100 mg/kg bw/day in the thyroid gland (follicular cell hypertrophy and increased weights at 1000 mg/kg bw/day) and in females at 1000 mg/kg/day in the adrenal gland (macroscopically enlarged adrenal gland, diffuse cortical hypertrophy, and increased weights at 1000 mg/kg bw/day). No toxicologically significant changes were noted in any of the remaining parameters investigated in this study, i.e. mortality, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), body weight, food consumption, hematology and clotting parameters, male T4 thyroid hormone levels.

Based on the findings of this study, a No-Observed-Adverse-Effect-Level (NOAEL) of = 1000 mg/kg bw/day for parental systemic toxicity was determined.

 

Alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5) was administered to male and female Wistar (Crl: WI(Han) rats in a Repeated dose 90-day oral toxicity study according to OECD guideline 408 under GLP conditions (Shell, 2022). Groups of 10 animals/sex were administered doses of 100, 300 and 1000 mg/kg bw/day by oral gavage, 7 days a week for a minimum of 90 days. The control group was treated according to the same protocol and received the vehicle (corn oil) only. A satellite group of 5 animals/sex was included in the control and 1000 mg/kg bw/day group to assess the recovery from any treatment-related effects. Following the treatment period, the recovery period for the satellite animals was 28 days.

The following parameters were recorded: mortality/moribundity, clinical signs, detailed clinical observations, body weight and food consumption, water consumption, opthalmoscopic examination, estrous cycle determination, sperm analysis, haematological parameters, clinical chemistry parameters, measurement of thyroid hormones (T3, T4 and TSH), urinalysis, neurobehavioural examination, gross necropsy, organ weights and histopathologic examination.

Salivation was observed on several occasions shortly after dosing in 3/10 males and 1/10 females in the low-dose group, as well as in 1/15 control females. In total 8/10 males and 5/10 females were affected in the mid-dose group, and all males and females in the high-dose group. No salivation was noted during the recovery period. The salivation was not considered toxicologically relevant and was considered a physiological response rather than a sign of systemic toxicity. Abnormal breathing sounds were noted in 4/10 males and 1/10 females in the mid-dose group and 2/15 males and 3/15 females in the high-dose group on a few days during the dosing period. Erected fur was observed in 2/15 males in the high-dose group once or on two consecutive days. Based on the low occurrence and short duration, these observations were not considered to be toxicologically relevant. No toxicologically relevant clinical signs were noted during weekly arena observations and during the recovery period.

For males in the high-dose group, the body weight gain was lower than controls from Day 57 onwards, resulting in a 7% lower mean body weight compared with the control at the end of the dosing period (Day 91; not statistically significant). During the recovery period, a trend towards higher body weight gain was noted for high-dose males, resulting in a 5% lower mean body weight than for the controls (not statistically significant) at the end of the recovery period. The apparent higher body weights in high-dose females were attributed to the higher mean body weight at the start of the dosing period. The difference in mean body weight compared to concurrent controls remained similar throughout the dosing period and was therefore not considered related to treatment with the test substance in females. In males, the body weight gain changes were not considered adverse.

At the end of the dosing period, increased hematocrit levels were noted in males in the high-dose main group (0.4911 L/L; 1.04 x of control, statistically significant). At the end of the recovery period, the hematocrit values were comparable to control. The activated partial thromboplastin time (APTT) at the end of the dosing period was shortened in high-dose females only (20.45 sec; 0.92 x of control, statistically significant). At the end of the recovery period, no difference between high-dose animals and control animals was observed. These results were therefore not considered to be toxicologically relevant.

Changes in clinical chemistry parameters at the end of the dosing period showed an increase in aspartate aminotransferase (AST) activity in mid- and high-dose males (1.31 and 1.27 x of control, respectively, statistically significant), and an increase in alkaline phosphatase (ALP) activity in high-dose males and females (1.26 and 1.30 x, respectively; not statistically significant for females). At the end of the recovery period, ALP levels in males were still significantly increased (1.51x) and ALP levels for males and females were comparable to control. The mean values of these results remained within the historical control data range and are not considered to be toxicologically relevant.

Thyroxine (T4) levels were statistically significantly decreased in high-dose males of the main group (0.78 x) but increased in high-dose females of the main group (1.21 x, not statistically significant). As all mean values were within the historical control range and the results in the treatment groups were comparable to controls at the end of the recovery period for both sexes, this effect was not considered toxicologically relevant. In addition, thyroid stimulating hormone (TSH) levels were increased in high-dose females at the end of the dosing period (3.63 x, statistically significant) and at the end of the recovery period (4.27 x, not statistically significant). As the control mean was low when compared to the historical control range, whereas the mean in the high-dose group was within the historical control range, this difference in TSH was not considered to be treatment-related.

A decreased percentage of progressive sperm was observed in males in the low- mid- and high-dose group: 24, 22 and 23%, respectively, versus 31% for control. In addition, an increased number of cells with other tails was noted in the high-dose group only (4 cells versus 1 in control). As all values remained well within the historical control range and lacked a clear dose response, these slight differences were considered unrelated to treatment with the test substance.

In the main group, increased liver weights were noted in males and females in the mid- and high-dose group, compared with the controls. The increases were statistically significant relative to body weight at the mid-dose (males: 13%, females: 19%), and both as absolute (males: 25%, females: 54%) and relative values (males: 38%, females: 48%) in the high-dose group, compared with control. This correlated to centrilobular hepatocellular hypertrophy observed microscopically. Following the recovery period the liver weight in the satellite group was comparable to the control. The increase in liver weight is considered to be a treatment-related adaptation. A higher adrenal gland weight was noted at the end of the dosing period in high-dose females compared with control; the 25% increase in absolute weight was statistically significant. Following the recovery period, higher absolute adrenal gland weights were noted in females at a slightly higher magnitude than at the end of dosing (32%, not statistically significant), and relative to body weight (10%, statistically significant). There was no histologic correlate for this finding and it is not considered to be toxicologically relevant. An increase in kidney weight was noted in high-dose males and females of the main group, compared with the control. In males, this was statistically significant only relative to body weight (18%), and in females, it was statistically significant as an absolute value (22%) and relative to body weight (17%). There was no histologic correlate. Following the recovery period there was no difference in kidney weight in either sex.

Treatment-related macroscopic findings were recorded in the high-dose group at the end of the dosing period in the liver. Prominent lobular architecture was noted in 6/10 high-dose males and 1/10 high-dose females. The low incidence of this finding in the low- and mid-dose males (2/10 and 1/10, respectively) was not considered to be treatment-related due to the low and non-dose-related incidence, and lack of correlation to organ weight or microscopic findings. In the stomach, dark red foci in the glandular mucosa (correlating to acute mucosal haemorrhage) was noted in 1/10, 2/10, 1/10 and 6/10 males in the control, low- mid- and high-dose group, respectively. Irregular surface of the non-glandular stomach (correlated to squamous hyperplasia) was noted in 0/10, 0/10, 0/10 and 1/10 males in the control, low- mid- and high-dose group, respectively. There were no macroscopic findings in the liver and stomach of male or female animals at the end of the recovery period.

Microscopic findings were noted in the thyroid gland, liver, jejunum, and stomach of males and females, and in the kidney of males.

Centrilobular hepatocellular hypertrophy was noted in 6/10 high-dose males (minimal) and 10/10 high-dose females (3/10 minimal, 7/10 mild) following the dosing period. The effect is likely correlated to higher liver weights and to the prominent lobular architecture noted macroscopically. This effect was absent in any other dose group, including the control group. Centrilobular hypertrophy was not observed in males or females after the recovery period, thereby indicating an adaptive response.

A combination of findings was noted in the non-glandular stomach (forestomach) following the dosing period. Squamous mucosal hyperplasia (minimal to moderate) was observed in 0/10, 0/10, 0/10 and 8/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 1/10, 1/10 and 10/10 females in the control, low- mid- and high-dose group, respectively. Minimal hyperkeratosis was noted in 6/10 males and 8/10 females in the high-dose group only. Squamous mucosal edema (minimal to mild) was observed in 3/10, 1/10, 2/10 and 6/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 1/10, 1/10 and 3/10 females in the control, low- mid- and high-dose group, respectively. Minimal vacuolation also occurred in 0/10, 0/10, 1/10 and 1/10 males in the control, low- mid- and high-dose group, respectively. In the non-glandular stomach ulceration (minimal to mild) was observed in 0/10, 0/10, 1/10 and 3/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 0/10 and 1/10 females in the control, low- mid- and high-dose group, respectively. In the glandular stomach, focal acute mucosal haemorrhage was noted in 0/10, 0/10, 1/10 and 3/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 0/10 and 1/10 females in the control, low- mid- and high-dose group, respectively. The few occurrences in the low-, and mid-dose groups were not considered to be clear indications of a treatment-related effect. Following the recovery period, no lesions were noted in the stomach of the control animals. In the high-dose group, minimal squamous mucosal hyperplasia and hyperkeratosis was noted in 2/5 males and 3/5 females, respectively. Minimal vacuolation of the limiting ridge was noted in 2/5 males. Due to the correlated macroscopic and microscopic findings in the forestomach, these are considered toxicologically relevant in the high-dose group.

Vacuolation of the villous lamina propria of the jejunum was observed following the dosing period in 0/10, 0/10, 1/10 and 10/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 0/10 and 10/10 females in the control, low- mid- and high-dose group, respectively. The finding was characterized by discrete small to large clear vacuoles in the lamina propria of the villi, and it is most likely that these vacuoles represent dilated lacteals. In some vacuoles a lining of attenuated endothelial-like cells were observed. At the end of the recovery period, the incidence and severity in males and females was comparable to the end of dosing, suggesting no recovery had occurred. As the effect does not appear to have any influence on the health of the animals, it is not considered toxicologically relevant.

In males, hyaline droplet accumulation was noted in the kidneys following the dosing period. This was observed in 6/10, 6/10, 8/10 and 7/10 males in the control, low- mid- and high-dose group, respectively. The severity increased with dose, with all the control males showing minimal accumulation and the high-dose animals showing mild to moderate accumulation. Following the recovery period 4/5 and 3/5 males in the control and high-dose group, respectively, showed hyaline droplet accumulation. As this is a well-known effect in male rats under similar treatment conditions, this is not considered to be relevant to humans.

In the thyroid gland colloid alteration was noted following the dosing period in 2/10, 3/10, 2/10 and 6/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 0/10 and 4/10 females in the control, low- mid- and high-dose group, respectively. Follicular cell hypertrophy was noted following the dosing period in 2/10, 3/10, 3/10 and 4/10 males in the control, low- mid- and high-dose group, respectively, and in 0/10, 0/10, 0/10 and 5/10 females in the control, low- mid- and high-dose group, respectively. Following the recovery period, minimal colloid alteration was noted in 1/5 and 3/5 males in the control and high-dose group, respectively, and in 0/5 and 1/5 females in the control and high-dose group, respectively. The incidence of hypertrophy was 1/5 and 1/5 males in the control and high-dose group, respectively, and 0/5 and 0/5 females in the control and high-dose group, respectively. This indicates a (partial) recovery of the microscopic thyroid gland effects.

The results of the food consumption measurement, opthalmoscopic examination, urinalysis, estrous cycle determination and neurobehavioural examination were similar between the control and treatment groups. Based on the findings of this study, a No-Observed-Adverse-Effect-Level (NOAEL) =1000 mg/kg bw/day for systemic toxicity and a NOAEL = 300 mg/kg bw/day for local toxicity was determined.

Studies in the AE category

Studies investigating repeated dose toxicity are available for the following AE substances (Table 1):

Table 1

CAS No.	EC No.	Substance	Subgroup	Study protocol	Hazard conclusion
26183-52-8	500-046-6	Decan-1-ol, ethoxylated	Linear	OECD 422	NOAEL systemic = 950 mg/kg bw/day
68439-50-9	500-213-3	Alcohols, C12-14, ethoxylated	Linear	OECD 422	NOAEL systemic = 1000 mg/kg bw/day
9004-95-9	939-518-5	Hexadecan-1-ol, ethoxylated	Linear	OECD 422	NOAEL systemic = 1000 mg/kg bw/day NOAEL local = 300 mg/kg bw/day
68439-49-6	939-518-5	Alcohols, C16-18 (even numbered), ethoxylated, < 2.5 EO	Linear	OECD 422	NOAEL systemic = 1000 mg/kg bw/day
9004-98-2	500-016-2	(Z)-9-Octadecen-1-ol ethoxylated	Linear	OECD 422	NOAEL systemic = 1000 mg/kg bw/day
160901-09-7	500-446-0	Alcohols, C9-11, branched and linear, ethoxylated	Mixed branched & linear	OECD 422	NOAEL systemic = 300 mg/kg bw/day
160901-19-9	500-457-0	Alcohols, C12-13, branched and linear, ethoxylated	Mixed branched & linear	OECD 422	NOAEL systemic = 300 mg/kg bw/day
106232-83-1	500-294-5	Alcohols, C12-15, branched and linear, ethoxylated	Mixed branched & linear	OECD 422	NOAEL systemic = 1000 mg/kg bw/day
68439-50-9	500-213-3	Alcohols, C12-14, ethoxylated	Linear	OECD 408	NOAEL systemic = 1000 mg/kg bw/day
68920-66-1	500-236-9	Alcohols, C16-18 and C18-unsatd., ethoxylated	Linear	OECD 408	NOAEL systemic = 300 mg/kg bw/day
160901-09-7	500-446-0	Alcohols, C9-11, branched and linear, ethoxylated	Mixed branched & linear	OECD 408	NOAEL systemic = 300 mg/kg bw/day NOAEL local = 300 mg/kg bw/day
106232-83-1	500-294-5	Alcohols, C12-15, branched and linear, ethoxylated	Mixed branched & linear	OECD 408	NOAEL systemic = 1000 mg/kg bw/day NOAEL local = 300 mg/kg bw/day

The data available for alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5) is consistent with the overall RDT data for AE substances. For a detailed evaluation of the repeated dose toxicity potential of the substances in the AE category, please refer to the category justification attached to the category object.

Justification for classification or non-classification

The available subacute and subchronic data on repeated dose toxicity obtained with alcohols, C12-15, branched and linear, ethoxylated (CAS No. 106232-83-1, EC No. 500-294-5) and with other members of the Alcohol Ethoxylates (AE) category do not meet the criteria for classification according to the CLP Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.